Masaryk University Faculty of Science Department of Botany and Zoology

Species diversity and phylogenetic relationships among gill-specific monogenean parasites (Platyhelminthes: Dactylogyridae) of (Cichlidae) from

Ph.D. Thesis

by Chahrazed Rahmouni

Supervisor prof. RNDr. Andrea Vetešníková Šimková, Ph.D.

Consultant Maarten Vanhove, Ph.D.

Brno 2021

Bibliographic Entry

Author Chahrazed Rahmouni, MSc.

Masaryk University, Faculty of Science

Department of Botany and Zoology

Title of Thesis: diversity and phylogenetic relationships among gill-specific monogenean parasites (Platyhelminthes: Dactylogyridae) of cichlids (Cichlidae) from Lake Tanganyika

Degree programme: Ecological and Evolutionary Biology

Specialization: Parasitology

Supervisor: prof. RNDr. Andrea Vetešníková Šimková, Ph.D.

Academic Year: 2020/2021

Number of Pages: 296

Keywords: Cichlidae, gill parasites, , , Lake Tanganyika, phylogeny, host-parasite associations, host specificity, host range

Bibliografický záznam

Author Chahrazed Rahmouni, MSc.

Masarykova Univerzita, Přírodovědecká Fakulta

Ústav Botaniky a Zoologie

Název práce: Druhová diverzita a fylogenetické vztahy hostitelsky specifických žaberních monogeneí parazitujícich ryby čeledi Cichlidae v jezeře Tanganyika

Studijní program: Ekologická a Evoluční Biologie

Specializace: Parazitologie

Vedoucí práce: prof. RNDr. Andrea Vetešníková Šimková, Ph.D.

Akademický rok: 2020/2021

Počet stran: 296

Klíčová slova: Cichlidae, žaberní parazité, Monogenea, Cichlidogyrus, jezero Tanganyika, fylogeneze, hostitelsko-parazitické asociace

Abstract

This Ph.D. thesis focuses on the gill-specific monogenean parasites of cichlids inhabiting the oldest of the East African Great Lakes, the Tanganyika. This system is one of the main hotspots of the diversity worldwide counting 241 species of 16 highly diverse lineages (tribes). Lake Tanganyika cichlids are considered as one of the most successful vertebrate adaptive radiations, however, the knowledge on their parasite fauna is still limited. Herein, the gill monogeneans (Platyhelminthes, Dactylogyridae) infecting cichlids from most endemic tribes of Lake Tanganyika were studied using morphometrics, geomorphometrics and phylogenetics. The main objectives of this thesis were to study (i) the species and morphological diversity of the monogeneans belonging to Cichlidogyrus from cichlids inhabiting Lake Tanganyika (study A, B), (ii) the morphological and molecular intraspecific variabilities in selected species of Cichlidogyrus parasitizing cichlid species with contrasting dispersal capacities (study C), and phylogenetically closely related cichlid species (congeneric representatives) versus representatives of phylogenetically distant lineages (study D), and finally (iii) the phylogeny of Cichlidogyrus parasitizing cichlids and the history of host-parasite associations in this system (study E). Cichlid species representing the majority of Lake Tanganyika tribes were collected from various locations along the lake shorelines and nearby smaller freshwater bodies. In total, 31 Cichlidogyrus species were identified, of which 25 species were recognised as new for science. This includes 15 species that were formally described within this doctoral thesis from 11 cichlid hosts of six tribes (Cyprichromini, Cyphotilapiini, , , , and Tylochromini). The remaining species are awaiting formal description. Morphological similarities in Cichlidogyrus species parasitizing closely related hosts along the lake were identified. A checklist of the species of Cichlidogyrus from Lake Tanganyika and outside of the lake highlighted the importance of morphological diversity of the sclerotized part of the vagina, and that of the heel (part of the male copulatory organ), a feature missing in many species. Additionally, it was shown that species of Cichlidogyrus cluster in more than the previously recognised four groups (studies A, B) defined based on the morphology of the hook pairs. In the second chapter (study C), we hypothesised an association between the host dispersal capacity and the intraspecific variability of sclerotized parts of the haptor of their host specific monogeneans. Two Cichlidogyrus species were studied, C. gistelincki parasitizing a cichlid species with good dispersal capacity (‘’ horei, Tropheini) and C.

4 milangelnari parasitizing a cichlid species with poor dispersal capacity ( microlepidotus, Cyprichromini). Populations of C. milangelnari parasitizing the poor dispersers were more differentiated in their anchor shape when compared to populations of C. gistelincki parasitizing well-dispersing hosts. Next study (study D) was focused on C. nshomboi widely reported in Boulengerochromis microlepis representing an ancient tribe in Lake Tanganyika (Boulengerochromini), and surprisingly retrieved also from representatives of a distant lineage, Perissodini ( microlepis, P. straeleni and microlepis). An inheritance from a common ancestor, host switching event or incipient speciation were suggested to explain the presence of C. nshomboi on unrelated hosts. The later scenario was supported by genetic divergence in the nuclear and mitochondrial genes. The intertribal shape and size variations of anchors were attributed to phenotypic plasticity or to adaptation. The lack of morphological and genetic differentiations among the populations of C. nshomboi parasitizing three cichlid species of Perissodini was related to their population genetic structure. Moreover, our results supported the correlation between host body size, intensity of infection and anchors size. In the last chapter of the present Ph.D. thesis (study E), we presented the first comprehensive phylogenetic study of Cichlidogyrus parasitizing members of most cichlid tribes from LT, including dactylogyridean representatives from the rest of African freshwater systems. Cichlidogyrus parasitizing mainly West African cichlid tribes revealed to be paraphyletic with respect to species parasitizing hosts belonging to the East African cichlid radiation, which constituted a well-supported monophylum. Cichlidogyrus belonging to Tylochromini and hosts that have colonised Lake Tanganyika only recently, cluster with their non-Lake Tanganyika relatives. The diversification of Cichlidogyrus in the lake seems to be driven by failure to diverge in old lineages of cichlids, cospeciation in more recently evolved ones, and host switching followed by parasite duplication at various host tribal level. Evaluation of host specificity and structural evolution of haptoral and reproductive organs in Lake Tanganyika Cichlidogyrus revealed that strict specialist species with larval hook size and lacking sclerotized vagina represent the most ancestral states of haptor configuration and vagina sclerotization characters, suggesting that Cichlidogyrus in this system evolved from a very simple form to a more complex one like their West African congeners. Generalist species among Cichlidogyrus with a sclerotized vagina parasitizing ancient Lake Tanganyika host lineages seem to have developed a different hook configuration, most probably to ensure successful colonization of new hosts, so far phylogenetically unrelated.

5

Abstrakt

Předložená disertační práce se zaměřuje na žaberní parazity taxonu Monogenea, které infikují cichlidy obývající nejstarší z východoafrických Velkých jezer, Tanganiku. Tento systém je jedním z hlavních světových hotspotů diversity cichlid, čítá 241 druhů 16 vysoce rozmanitých linií (tribů). Cichlidy jezera Tanganika jsou považovány za jednu z nejúspěšnějších adaptivních radiací obratlovců, avšak znalosti o jejich parazitofauně jsou stále limitovány. Pomocí morfometrie, geomorfometrie a fylogenetiky byli studováni žaberní paraziti taxonu Monogenea (Platyhelminthes, Dactylogyridae) infikující cichlidy z většiny endemických tribů jezera Tanganika. Hlavním cílem dizertační práce bylo studium (i) druhové a morfologické diverzity monogeneí náležících do rodu Cichlidogyrus parazitující cichlidy obývající jezero Tanganika (studie A, B), (ii) morfologické a molekulární vnitrodruhové variability u vybraných druhů rodu Cichlidogyrus parazitující druhy čeledi Cichlidae s kontrastními disperzními schopnostmi (studie C), nebo fylogeneticky blízce příbuzné druhy cichlid (kongeneričtí zástupci) versus druhy fylogeneticky vzdálených linií (studie D), a nakonec (iii) fylogeneze monogeneí rodu Cichlidogyrus parazitující ryby čeledi Cichlidae a evoluční historie parazito-hostitelských vztahů v tomto systému (studie E). Druhy cichlid představující většinu tribů jezera Tanganika byly vzorkovány na různých lokalitách podél břehů jezera a v blízkých menších vodních zdrojích. Celkem bylo identifikováno 31 druhů rodu Cichlidogyrus, z nichž 25 druhů bylo identifikováno jako druhy nové pro vědu. Z tohoto počtu bylo 15 druhů formálně popsaných v rámci předložené disertační práce, jedná se o druhy zaznamenané na 11 hostitelích šesti tribů Cichlidae (Cyprichromini, Cyphotilapiini, Ectodini, Eretmodini, Tropheini a Tylochromini). Zbývající druhy čekají na formální popis. Byly identifikovány morfologické podobnosti u druhů rodu Cichlidogyrus parazitující na blízce příbuzných hostitelích podél jezera. Checklist druhů rodu Cichlidogyrus z jezera Tanganika i mimo jezero naznačuje význam morfologické rozmanitosti sklerotizovaných části samičích a samčích reprodukčních orgánů. Dále bylo prokázáno, že druhy rodu Cichlidogyrus formují více než čtyři dříve uznávané skupiny (studie A, B) definované na základě morfologie párů marginálních háčků. Druhá kapitola (studie C) vycházela z předpokladu souvislosti mezi disperzní kapacitou hostitele a vnitrodruhovou variabilitou sklerotizovaných částí přichycovacího orgánu (haptoru) parazitů taxonu Monogenea. Byly studovány dva druhy rodu Cichlidogyrus, C. gistelincki parazitující na druhu hostitele s dobrou disperzní schopností („Ctenochromis“ horei,

6

Tropheini) a C. milangelnari parazitující na hostitelích s nízkou disperzní kapacitou (Cyprichromis microlepidotus, Cyprichromini). Populace C. milangelnari parazitující na hostiteli s nízkou disperzní kapacitou byly ve srovnání s populacemi C. gistelincki parazitujícími na hostitelích s dobrou disperzní schopností více diferencované ve tvaru středních háčků. Další studie (studie D) byla zaměřena na druh parazita C. nshomboi široce dokumentovaného u hostitele Boulengerochromis microlepis, který představuje starobylý tribus v jezeře Tanganika (Boulengerochromini), a také byl tento parazit překvapivě zaznamenán na zástupcích vzdálené linie tj. v rámci Perissodini (, P. straeleni a ). K vysvětlení prezence C. nshomboi na nepříbuzných hostitelích byla navržena společná historie (společný předek), hostitelský přeskok (host switching) nebo začínající speciace. Poslední z těchto scénářů byl podpořen genetickou divergencí v jaderných a mitochondriálních genech. Variabilita ve tvaru a velikosti středních háčků mezi populacemi C. nshomboi parazitujícími hostitele různých tribů cichlid mohou být přičítány fenotypové plasticitě nebo adaptaci. Nedostatek morfologické a genetické diferenciace mezi populacemi C. nshomboi parazitujícími na třech druzích (zástupcích Perissodini) souvisel s jejich populační genetickou strukturou. Získané výsledky navíc podpořily korelaci mezi velikostí těla hostitele, intenzitou infekce a velikostí středních háčků. Poslední kapitola předložené dizertační práce (studie E) je věnovaná první komplexní fylogenetické studii parazitů rodu Cichlidogyrus infikujících zástupce cichlid většiny tribů z jezera Tanganiky, včetně zástupců rodu Cichlidogyrus z dalších afrických sladkovodních systémů. Cichlidogyrus parazitující hlavně západoafrické zástupce cichlid byl parafyletický s ohledem na pozici druhů parazitujících na hostitelích náležejících k východoafrické radiaci cichlid, Cichlidogyrus této východoafrické radiace byl monofyletický. Druhy rodu Cichlidogyrus parazitující hostitele náležících do tribů Tylochromini a Oreochromini, kteří kolonizovali jezero Tanganika teprve nedávno, formují klastry se svými příbuznými mimo jezero Tanganika. Zdá se, že diverzifikace parazitů rodu Cichlidogyrus v jezeře Tanganika je spojená s absencí divergence parazitů ve starých liniích cichlid (failure to diverge), s kospeciací u evolučně mladších linií, s hostitelským přeskokem (host switching) a následnou duplikací parazitů v rámci různých tribů hostitele. Analýza hostitelské specificity a strukturální evoluce haptoru a reprodukčních orgánů rodu Cichlidogyrus v jezeře Tanganika odhalila, že striktní specialisti s larvální velikostí marginálních háčků a postrádající sklerotizaci v samičích reprodukčních orgánech představují ancestrální formy konfigurace haptoru a sklerotizovných znaků samičích reprodukčních orgánů, co naznačuje, že Cichlidogyrus v systému jezera Tanganika se vyvinul z velmi jednoduché do složitější formy podobně jako v případě jejich

7 západoafrických kongeneričtích zástupců. Výstupy studie naznačují, že druhy rodu Cichlidogyrus, které jsou klasifikovány jako generalisti parazitující na starobylých hostitelských liniích jezera Tanganika, mají sklerotizované samičí orgány a odlišnou konfiguraci středních háčků, která vznikla pravděpodobně k zajištění úspěšné kolonizace nových fylogeneticky nepříbuzných hostitelů.

8

To ‘Djida’, ‘Babacha’, Maria, Maissa and Adam!

For their endless love, support, encouragement and prayers.

9

Acknowledgements

I would like to express my deepest gratitude to my supervisors Andrea Vetešníková Šimková and Maarten Vanhove for the big roles they played during the course of the research, and especially for their patience and help throughout my Ph.D. journey. Their advices on both research and my scientific career have been invaluable. I still remember our first meeting, via skype, when they introduced me to the wonderful world of cichlids and their diverse parasites, which years later, became a Ph.D. thesis. They actually provoked my fascination in this field during the first minutes of our meeting. As I recall the first moments with Andrea and Maarten, who later became my supervisors, I cannot forget to thank Pr. Jean Lou Justine who suggested my name as a potential candidate when he heard about the proposal. Without him, I would never have had the opportunity to meet my supervisors and work with them. I am extremely grateful to my jury of defence: Jean Lou Justine and Serge Morand for their participation in my defence, helpful comments to my thesis and further scientific career. I would also like to extend my deepest appreciation to Milan Gelnar for his daily energy, enthusiasm, great help and constructive discussions about science and life in general. I wish to express my special gratefulness to all my colleagues from the laboratory of parasitology and who became my friends, and even my family, it was a great pleasure to be a member of your group! I deeply indebted to my co-authors as well. I am extremely grateful to Maarten Van Steenberge, who is one of the best specialists of cichlids from Lake Tanganyika, and who opened my mind to another exciting view of science and introduced the ‘geomorphometry’ in my life. He offered me several opportunities during my stays in Belgium to learn the approach that considerably helped me during my studies. Many thanks for your never-ending enthusiasm, ideas, advices, suggestions and hours of interesting discussions. It was a great pleasure for me to cooperate with you! At the same time, I do wish to express my deepest gratitude to Thierry Backeljau, Tom Artois and their entire group from the Royal Belgian Institute of Natural Sciences and Hasselt University, respectively, who welcomed me in the premises of their laboratories during my internships in Belgium. I would like to say thanks to my friends/colleagues from foreign institutions (Democratic , , Switzerland and Austria) for their precious help with the co-

10 organisation of the fieldwork, taxonomical identification of cichlids and for their scientific input on Lake Tanganyika cichlids. I owe special thanks to Nick Wilson and Teddy for the language corrections of my thesis. My research would have been impossible without financial support. The realization of the thesis was fully supported by Czech Grant Agency, projects P505/12/G112—European Centre of Ichtyoparasitology (ECIP). Last but not least, I would like to thank my family and friends from my home country, especially my best friends Biba, Protolyse and M., for overall and unconditional support during my studies abroad. You were, and still you are present in my soul and spirit during all these years that I have spent away from you, I would never have succeeded without you. It is a pity that the space is too limited to list all of you, but I remember you and all your help. A special thank goes to Ella (Racha), my lovely cat, for her companionship and comfort she provided during my research and writing process, especially during the Covid-19 lockdown. Thank you, sweetie!

11

List of publications included in the thesis

The present thesis includes five scientific papers, of which three were already published and two manuscripts are currently under review/ready for submission.

Section I Species and morphological diversity of gill-specific monogeneans of Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts Study A Rahmouni, C., Vanhove, M.P.M. & Šimková, A. (2017). Underexplored diversity of gill monogeneans in cichlids from Lake Tanganyika: eight new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) from the northern basin of the lake, with remarks on the vagina and the heel of the male copulatory organ. Parasites & Vectors; 10, 591. https://doi.org/10.1186/s13071-017-2460-6 [IF: 3.031]. Overall contribution estimated at 70% (parasite microscopic observation, species identification and drawing of parasites, the checklist creation, data analysis, manuscript writing and revision). Study B Rahmouni, C., Vanhove, M.P.M. & Šimková, A. (2018). Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) parasitizing the gills of Congolese cichlids from northern Lake Tanganyika. PeerJ 6:e5604. https://doi.org/10.7717/peerj.5604 [IF: 2.353]. Overall contribution estimated at 70% (processing of and parasitological material, parasite microscopic observation, species identification and drawing of parasites, manuscript writing and revision).

Section II Intraspecific variability of gill-specific monogeneans of Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts Study C Rahmouni, C., Van Steenberge, M., Vanhove, M.P.M. & Šimková, A. (2020). Intraspecific morphological variation in Cichlidogyrus (Monogenea) parasitizing two cichlid hosts from Lake Tanganyika exhibiting different dispersal capacities. Hydrobiologia. https://doi.org/10.1007/s10750-020-04429-1 [IF: 2.385]. Overall contribution estimated at 70% (processing fish and parasitological material, parasite microscopic observation, parasite species identification, geomorphometric and molecular analyses, data analysis, manuscript writing and revision).

12

Study D Rahmouni, C., Vanhove, M.P.M., Šimková, A. & Van Steenberge, M., Conservative divergent evolution in a gill monogenean parasitizing distant cichlid lineages of Lake Tanganyika: Cichlidogyrus nshomboi (Monogenea: Dactylogyridae) from representatives of Boulengerochromini and Perissodini (submitted to Evolutionary Biology) [IF: 2.013]. Overall contribution estimated at 60% (processing fish and parasitological material, parasite microscopic observation, parasite species identification, geomorphometric and molecular analyses, data analysis, manuscript writing and revision).

Section III Phylogeny, cophylogeny and host specificity correlates in species of Cichlidogyrus parasitizing Lake Tanganyika cichlids Study E Rahmouni, C., Vanhove, M.P.M., Koblmüller, S., Mendlová, M. & Šimková A. Molecular phylogeny and speciation patterns in host-specific monogeneans (Cichlidogyrus, Dactylogyridae) parasitizing cichlids in Lake Tanganyika (to be submitted to International Journal for Parasitology) [IF: 3.530]. Overall contribution estimated at 60% (processing parasitological material, microscopic observation, laboratory analyses, data analysis, manuscript writing and revision).

13

Table of contents

1 Introduction 15 2 Research goals 17 3 Literature overview 19 Part 1 – Understanding host diversity 3.1 Biodiversity of freshwater ichthyofauna in the African continent 19 3.2 Cichlid fish 20 3.2.1 Western and Central African cichlids 22 3.2.2 The East African Rift System and its cichlid fauna 26 3.2.3 Speciation mechanisms in East Great African Lakes 28 3.3 Lake Tanganyika 31 3.3.1 Geographical location, hydrogeography and history 31 3.3.2 Diversity of Lake Tanganyika cichlid assemblages 32 Part 2 – Parasite diversity 3.4 Parasitic helminths: general overview 38 3.4.1 Monogenea 39 3.4.2 Taxonomic and phylogenetic status of Monogenea 40 3.4.3 Morphological adaptations 42 3.4.4 Host specificity 43 3.4.5 Speciation in monogeneans 44 3.5 Diversity of monogeneans in African fish fauna 47 3.6 Diversity of monogeneans in African cichlids 47 3.7 Current knowledge on Cichlidogyrus 50 3.7.1 Cichlidogyrus parasitizing Western and Central African cichlids 50 3.7.2 Cichlidogyrus in the Tanganyika system 54 3.7.3 Non-Cichlidogyrus monogeneans from the Tanganyika system 56 4 Material and methods 57 4.1 Collection of fish and monogenean taxa 57 4.2 Morphometric and geomorphometric analyses 59 4.3 DNA-Isolation, amplification, and sequencing 60 4.4 Phylogenetic and cophylogenetic analyses 61 4.5 Host specificity and its relationship to parasite morphology in the 62 Tanganyika system 5 Results and discussion 66 Section I – Species and morphological diversity of gill-specific 67 monogeneans of Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts Section II – Intraspecific variability of gill-specific monogeneans of 72 Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts Section III – Phylogeny, cophylogeny and host specificity correlates in 76 species of Cichlidogyrus parasitizing Lake Tanganyika cichlids Conclusions and future perspectives 80 References 84 Appendix 118

14

1. Introduction

Parasitism is one of the most successful strategies of life on earth as most free-living organisms are potentially infected by one to several species of parasites (Windsor 1998). Whether with a direct life cycle or a very complex one, parasitic organisms are synchronized with their hosts to ensure optimal individual growth, survival and fecundity (Garamszegi 2009). They also interact with their hosts in a dynamic way by continuously evolving over time through gaining and/or loosing hosts. This feature shapes the evolutionary trajectories of both host and parasite lineages (Nylin et al. 2018). The phenomenal pressures that parasites exert further pattern the host population size and influence the host physiological processes and behaviour. This made from parasites a powerful and suitable model to study fundamental questions in evolutionary biology. Research on parasite diversity and host-parasite interactions has received great scientific interest and, over the past decades, it is particularly relevant when investigating a large variety of traits related to their hosts (Williams et al. 1992).

The presented thesis focuses on ectoparasitic monogeneans, which represent a cosmopolitan and species-rich taxon of (platyhelminths) infecting mainly the external surfaces of aquatic vertebrates, especially the gills, fins and skin of freshwater and marine (Roberts and Janovy 2009). Monogeneans are highly diverse in terms of number of species, morphology, and ecology (Cribb et al. 2002; Theisen et al. 2017) with a more or less known phylogeny (or at least attempts to resolve their phylogeny using various genes) (Boeger and Kritsky 1993; Mollaret et al. 1997; Mollaret et al. 2000; Jovelin and Justine 2001; Egger et al. 2015). Furthermore, they are highly host species-specific in term of the number of host species used by a given parasite species (Poulin et al. 2006). They are even more host-specific than other parasitic groups such as acanthocephalans and nematodes (Poulin 1992). This made monogeneans the ideal candidates for addressing fundamental ecological and evolutionary questions related to host-parasite associations (Poulin 2002).

The cichlids living in Lake Tanganyika (LT), the oldest of the East Great African Lakes, are the objective of the parasitological study presented in this thesis. With an estimated 3,000 species (Fricke et al. 2021), cichlids are distributed all over freshwater bodies, mostly. Throughout their distributional range in Africa, they have successfully demonstrated their capacity of generating adaptive radiations resulting in an impressive diversity of body shapes, colour patterns, behaviour, and eco-morphological specializations (Koblmüller et al., 2008; Salzburger et al., 2014). The extraordinary diversification of African cichlids represents a

15 classic example of vicariance, for instance, and offered them an important place within the freshwater ichthyofauna (Cavin 2017). Identified as one of the main hotspots of cichlid diversity (Snoeks 2000), LT became one of the most important and challenging model systems for evolutionary biology research. The presence of representatives of almost all major African cichlid lineages, a high level of endemism in cichlids, and a variety of parental care types and foraging strategies were shown for this system (Meyer et al. 2015b; Takahashi and Sota 2016). From the prime study of Poll (1986) who defined the cichlid tribes in LT, plenty of studies have followed, ranging from taxonomic descriptions and revisions, to genetic characterisations and, very recently, the whole-genome sequencing of 241 species members of 16 tribes inhabiting the lake (Koblmüller et al. 2008b; Ronco et al. 2020; 2021).

African cichlids demonstrated to host an impressive diversity of parasite communities. Six genera of dactylogyridean monogeneans out of 14 were recognised on African cichlids (Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Mendoza-Palmero et al. 2017). Cichlidogyrus Paperna, 1960 showed to be particularly species-rich and a total of 131 species are nowadays known from African and Levantine cichlids (Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Vanhove et al. 2016; Geraerts et al. 2020 and references herein). Mirroring its cichlid flocks, LT contains a unique assemblage of species of Cichlidogyrus that can be classified into several morphological groups. Hitherto, 39 species of these monogeneans have been described from 15 cichlid species from various tribes (Řehulková et al., 2018 and references herein). In term of host specificity, LT species of Cichlidogyrus showed to infect both related and unrelated cichlid tribes (Kmentovà et al., 2016 and references herein), just like their West African relatives through host switching (Mendlová and Šimková 2014). The first taxonomic documentation on the parasite fauna of LT, especially that of monogeneans, was unbalanced as most of the field studies have targeted only a few cichlid tribes like Tropheini and Ectodini, keeping most of the LT tribes underexplored (Muterezi Bukinga et al., 2012; Vanhove et al., 2011a, b). Similarly, only scarce phylogenetic and cophylogenetic studies have been performed based on the West African cichlid-Cichlidogyrus system (Pouyaud et al. 2006; Mendlová et al. 2010; Mendlová et al. 2012; Mendlová and Šimková 2014; Messu Mandeng et al. 2015), and that inhabiting LT (Vanhove et al. 2015). On the other hand, geomorphometric techniques based on data of sclerotized structures of the haptoral apparatus, mostly, combined with genetics were adopted to investigate the population structure of species of Cichlidogyrus and the intraspecific shape variation of their hard parts.

16

However, only few lineages were studied, mainly those inhabiting the deep realms (Kmentová et al., 2016).

2. Research goals

The overall aims of the doctoral thesis are following:

a) To investigate the morphological and species diversity of the gill monogenean flatworms belonging to Cichlidogyrus parasitizing diverse endemic cichlid lineages of LT. b) To study the association between the host dispersal capacity and the intraspecific variability of the attachment organ in terms of shape variation in host-specific monogenean species. c) To investigate whether the genetic and morphological differentiation in Cichlidogyrus mirrors the phylogenetic and ecological distinctness, as well as the geographical structure of their cichlid hosts. d) To use molecular genetics to infer the phylogenetic history of Cichlidogyrus known so far from West African and LT cichlid hosts, and to investigate coevolutionary patterns, morphological adaptation and host specificity correlates in the cichlid-Cichlidogyrus system in LT.

17

“Diversity may be the hardest thing for a society to live without, and perhaps the most

dangerous thing for a society to be without”

William Sloane Coffin Jr.

Bujumbura, 2013; photo by Radim Blažek (Institute of Vertebrate Biology, Czech Academy of Science, Brno)

18

3. Literature overview

Part 1 Understanding host diversity

3.1 Biodiversity of freshwater ichthyofauna in the African continent

Freshwater is an essential resource for human life and has no substitute. Among all freely available water on the Earth’s surface, freshwater bodies represent only 2.5%, of which 0.26% of liquid freshwater are lakes, reservoirs, and rivers (Carpenter et al. 2011). Freshwater ecosystems are home to diverse micro-organisms, plants, invertebrates and fish. The knowledge of the total diversity of freshwaters is still incomplete. However, compared to invertebrates and micro-organisms, fish taxa are better known especially in biodiversity hotspot regions like the tropics (Dudgeon et al. 2006). Freshwater habitats harbour 40% of all fish species, with the remaining diversity found in marine habitats (Bloom et al. 2013). Speciation rates in lineages showed to be substantially faster on average than those of marine lineages. Generally speaking, transitions to freshwaters, however, do not increase the rates of speciation (Rabosky 2020).

The African continent contains several major basins (Fig. 1) sharing a much older history than those in the temperate zones (Thieme et al. 2005). Furthermore, Africa has 87 freshwater ecoregions with ten major habitat types (Fouchy et al. 2019). The large number of ecoregions in Africa is the result of high climatic and hydrological fluctuations. The large lakes, mostly located in the Eastern part of the continent, represent hotspots for species biodiversity and endemism (Darwall et al. 2011) (Fig. 1). The freshwater ecosystems in Africa hold one of the most species rich ichthyofaunas on the planet being home to an estimated 3 300 fish species belonging to 76 families, of which Rafinesque, 1815 and Cichlidae Heckel, 1840 are the most dominant. Compared to South America, the African ichthyofauna has fewer families and species but it is unique in its higher proportion of basal families, of which representatives are found in many fish communities, and in its impressive diversity of species flocks endemic to multiple lakes and rivers (Lavoué et al. 2005; Lévêque et al. 2008).

19

Fig. 1 (A) The ichthyofaunal ecoregions including the major basins, lakes and rivers, as well as the distribution of freshwater species richness of fishes in the African continent (B) (Nyboer et al. 2019).

3.2 Cichlid fish

Cichlids are teleost fish belonging to the class (ray-finned fishes) and the largest order of fish – . Based on molecular data and morphological features, a few taxonomists suggested the order (Betancur et al. 2013) restricted to cichlids as a new suborder, and Cichlomorphae (Betancur et al. 2013) was suggested to accommodate the tropical marine family Pholidichthyidae Jordan, 1896 and the Cichlidae (see Betancur et al. 2017). Moreover, based on mitochondrial genes, cichlids were showed to be closely related to the marine Embiotocidae Agassiz, 1853 (surfperches), Pomacentridae Bonaparte, 1831 (damselfishes), but not, as previously thought, to Labridae Cuvier, 1816 and related families ( and parrotfishes) within the Labroidei suborder (Wainwright et al. 2012). So far, more than 2200 nominal species of cichlids showing particular substrate and mouth-brooding parental care behaviours are known (Fricke et al. 2021).

The cichlids are recognised as a key vertebrate model system for understanding the evolutionary assembly of biodiversity (Kocher 2004). They successfully occur across several southern landmasses in freshwater habitats, brackish and marginal marine environments. They are widely distributed and currently present in southern continental regions including Africa

20

(>1,000 species), Cuba and Hispaniola (4 species), the Middle East (5 species), Madagascar (>18 species), India and Sri Lanka (3 species), South and Middle America (~400 species), Iran (1 species) and the Antilles (5 species) (Chakrabarty 2004; Cavin 2017).

Investigating the deep evolutionary history of cichlids has long been dominated by vicariance models of biogeography considering the current distribution of cichlids, which was assumed to be linked to the Gondwana break-up during the mid to late Mesozoic (135–90 Ma) (Stiassny 1991; Chakrabarty 2004). Indeed, these fishes display a typical Gondwanan distribution; basal representatives occur in Madagascar and India, while lineages with later divergence inhabit Africa and South America. Recent studies, however, have revised this macroevolutionary time scale and reported a cichlid origin post-dating the tectonic fragmentation of Gondwana (Friedman et al. 2013; Matschiner et al. 2020). It has been proposed that parasitological evidence could help distinguish between these two scenarios (Pariselle et al. 2011; Vanhove et al. 2016). Most ancient fossil cichlids belong to Mahengechromis Murray, 2001 from and date back only to the (54–38 Ma) (Murray 2001). Based on molecular clock estimates of divergence, dispersal events across marine environments seem to be at the origin of the current distribution of the family. Indeed, Matschiner et al. (2020) and a few earlier studies (Kumazawa et al. 2000; Murray 2001; Vences et al. 2001; Friedman et al. 2013; Matschiner et al. 2016; Matschiner et al. 2017) suggested post-Gondwana diversification scenarios which could explain the current phylogeography of cichlids by considering these fish at least as old as the initial Gondwanan split (150 Ma) (Matschiner et al. 2020). A long-distance oceanic dispersal was also hypothesised, but in this case, cichlids might be younger than the separation of Gondwana (~70 Ma). An independent colonisation of freshwater habitats was alternatively suggested corresponding to a young age of cichlids. This scenario requires a common marine ancestor that has since either gone extinct or remained undiscovered and from which cichlids on all four landmasses have independently evolved (~45 Ma) (Matschiner et al. 2020) (Fig. 2b). Numerous extrinsic environmental factors and intrinsic species-specific traits such as breeding behaviour, niche partitioning, colour- based sexual selection, and feeding adaptations contributed to the ecological success of cichlids in tropical America and Africa (Curry-Lindahl et al. 1976; Salzburger 2009). It has also been hypothesised that the modest diversity of riverine cichlids throughout the world was driven by range expansion, colonisation by river capture and subsequent geographic isolation as a result of vicariant and geographic speciation (Katongo et al. 2007).

21

Phylogenetic analyses using both nuclear and mitochondrial genes combined with morphological features indicated that the endemic Malagasy and southern Asian subfamily Etroplinae Van Couvering, 1982 is paraphyletic and represents the sister taxon to all other cichlids. In contrast, another lineage restricted to Madagascar, Ptychochrominae Sparks and Smith 2004 (4 genera), is monophyletic and represents the sister group to the monophyletic group including both the African and Middle Eastern Fowler, 1934, plus the Neotropical lineage Cichlinae Bonaparte, 1835 (Farias et al. 2001; Sparks and Smith 2004) (Fig. 2a).

Fig. 2 (a) Map showing cichlid representatives from India, Sri Lanka, and Madagascar forming the most basal lineages of the monophyletic African and American clades as sister-groups (Salzburger and Meyer, 2004, adapted by myself). (b) Three diversification scenarios for the phylogeographic history of cichlid fishes (Matschiner et al. 2020).

3.2.1 Western and Central African cichlids

Among the four cichlid subfamilies (see above), Pseudocrenilabrinae (restricted to Africa and Middle East) forms the largest clade with approximately 150 genera and thousands of species (Fricke et al. 2021). The classification of West and Central African cichlids has long been controversial and cichlid representatives were continuously reassigned based on anatomical characters (mostly) to other genera and tribes (taxonomic rank between family and ) after taxonomical revisions. First, was erected from based on the neurocranial apophysis for articulation of the upper pharyngeal bones (Regan 1920; Regan

22

1922). Later, four ‘types’ of pharyngeal apophysis representing the genera Regan, 1920 (with the simplest structure), Tilapia Smith, 1840, Boulenger, 1898 and Haplochromis Hilgendorf, 1888 were discerned by Greenwood (1978), followed by the pioneering study by Poll (1986) who also defined the tribes inhabiting LT. The monophyletic origin of African cichlids was supported by both molecular data and morphological features, and six major monophyletic lineages were recognised: chromidotilapiines, tylochromines, hemichromines, pelmatochromines, haplotilapiines, plus the monotypic genus Heterochromis Regan, 1922 (Fig. 3 and 4) (Stiassny 1991; Kocher et al. 1995; Mayer et al. 1998; Streelman et al. 1998; Farias et al. 2000; Klett and Meyer 2002; Sparks and Smith 2004). The West African cichlids were shown, however, to be paraphyletic and basal to other African lineages (Zardoya et al. 1996; Farias et al. 2001).

Fig. 3 Cladogram showing relationships within Cichlidae worldwide. Cichlid lineages inhabiting continental Africa are in blue, while the red group comprises most African cichlids including tilapiines and . The collapsed clade represents the East African cichlid flocks as it contains several tribes (Sparks and Smith 2004; Schwarzer et al. 2009; Dunz and Schliewen 2013; Kevrekidis et al. 2019).

The chromidotilapiines belong to the most ancient species-rich lineage that originated in the Oligocene-Eocene (Greenwood 1987; Schwarzer et al. 2014). The monophyletic origin of Tylochromini Poll, 1986, represented by a single genus Tylochromis, was confirmed (Stiassny 1990). The representatives of this tribe are widely distributed in rivers, lakes, and coastal lagoons throughout central and Western Africa, but absent in the Nile and drainage systems (Stiassny 1990). With a pan-African distribution, the haplotilapiines represent the most speciose clade of the pseudocrenilabrines and include nine tribes, in addition to the East African radiation lineage known as austrotilapiines (Schwarzer et al., 2009). The lineages

23 within haplotilapiines number from a single representative (Etiini Dunz and Schliewen, 2013) to hundreds of species (Haplochromini Trewavas, 1983 but are virtually absent in West Africa (Greenwood 1979)) (Schwarzer et al. 2009; Dunz and Schliewen 2013) (Fig. 3). The monophyly of the haplotilapiines was repetitively confirmed by phylogenetic analyses (Schliewen and Stiassny 2003; Schwarzer et al. 2009; Dunz and Schliewen 2013). Furthermore, the recent study of Dunz and Schliewen (2013) revised the tribal level of the haplotilapiines; two clades of the West African boreotilapiines (sensu Schwarzer et al., 2009) were split into the tribes Gobiocichlini, Coelotilapiini, Heterotilapiini and Coptodonini Dunz and Schliewen, 2013, in addition to the tribes Pelmatolapiini, Oreochromini, Steatocranini, Paracoptodonini Dunz and Schliewen, 2013, and Tilapiini Trewavas, 1983.

Oreochromini and Coptodonini harbour most of the generic and species diversity of West African cichlids (Dunz and Schliewen 2010). The former tribe holds both ‘widespread/riverine’ (Trewavas 1983) and ‘lake-endemic’ representatives (Trewavas et al. 1972; Seegers and Tichy 1999). Thys van den Audenaerde (1968) revised the ‘Tilapia’ and assigned T. deckerti Thys van den Audenaerde, 1967 (endemic to Lake Ejagham in (Froese and Pauly 2021)) to the subgenus Coptodon Gervais, 1853 (type species T. zillii (Gervais, 1848)) to accommodate Tilapiini. For a long time, ‘tilapia’ was commonly used for the group of species belonging to Günther, 1889, Rüppell, 1852 and Tilapia Smith, 1840 (Agnèse et al. 1997). Currently, Tilapiini contains only southern African species (Dunz and Schliewen 2013). This group has been receiving attention because of their importance in tropical and subtropical aquaculture, economic growth, and their high adaptabilities to extreme environmental conditions (Beveridge and McAndrew 2000). Moreover, the ‘tilapia’ species exhibit successful introgression in natural conditions and in aquaculture (Macaranas et al. 1986).

24

Fig. 4 Map showing the distribution of most Western and Central African cichlid lineages with representative species for each tribe. (a) coelotilapiines (represented by Coelotilapia joka (Thys van den Audenaerde, 1969)); (b) paracoptodonines (Paracoptodon tholloni (Sauvage, 1884)); (c) heterotilapiines (Heterotilapia butstikoferi (Hubrecht, 1883)); (d) pelmatolapiines (Pelmatolapia mariae (Boulenger, 1899)); (e) Coptodonini (C. zilli); (f) gobiocichlines (‘Tilapia’ brevimanus Boulenger, 1991); and (g) tilapiines (T. sparrmanii Smith, 1840) (adapted from Dunz, 2012).

25

3.2.2 The East African Rift System and its cichlid fauna

The East African Rift System is a region where the Earth's tectonic forces are dynamic and form new plates by splitting old ones apart (Baker and Kalinga 2009) resulting in a series of small and large lakes and rivers (Chorowicz 2005) (Fig. 5). The geological history of the formation of the East African Rift Lakes is highly complex and still debated (Weiss et al. 2015). The exact timing of when the rifting in the western branch started is still undetermined, but palaeontologists suggest it to have begun as early as ∼25 Ma to as recently as ∼12–10 Ma (Nyblade and Brazier 2002; Roberts et al. 2012). The rifting in the western branch is known for its low frequency of volcanic activity, extensive downfaulting, and wet climates; factors that have contributed to form larger, deeper, and more persistent lakes (Salzburger et al. 2014). In contrast, rifting in the eastern branch began ∼30–35 Ma in the Afar and Ethiopian Plateau (Nyblade and Brazier 2002), and is characterised by frequent volcanic activity that formed comparatively small and shallow water bodies like Lake Turkana (Salzburger et al. 2014). The series of climate changes in East Africa had a direct impact on its biodiversity (Haywood et al. 2000; Kingston et al. 2002; Anderson et al. 2011). Moreover, the climate changes during the last 500,000 years have provoked extreme fluctuations in the levels of East African Lakes and have considerably shaped the population structures of their fauna (Cohen et al. 2007; Scholz et al. 2007).

Eastern Africa is the main hotspot of cichlid biodiversity, where thousands of highly diverse species inhabit several freshwater habitats. It is now well known that the cichlid fauna occurring in this region is distinguishable from the West African hydrographical systems. The former group forms so-called ‘species flocks’ with a high degree of endemism in each of the lakes and rivers (Snoeks 2000; Salzburger and Meyer 2004). For such high diversity and endemism rates, intra-lacustrine speciation was suggested as the most common form of diversification in large lakes (Sturmbauer et al. 2011). Boulenger (1898) was the first ichthyologist who reported the diversity of the East African fish fauna at the end of the 19th century. Since then, the cichlid assemblages inhabiting these lakes have been extensively investigated at various levels. East African radiation cichlids show efficient brood care. Several representatives of haplochromines exhibiting behaviour have evolved independently from ancestral substrate-brooding lineages (Stiassny 1991; Salzburger et al. 2002b). Haplochromini includes hundreds of species (Greenwood 1979; Poll 1986b) found in both small and large lakes and connected rivers (Curry-Lindahl et al. 1976). It has long been

26 assumed that lacustrine species flocks have arisen from the riverine species. Thus, taxonomists gave greater attention to lacustrine haplochromines as they underwent explosive adaptive radiations in the three Great Lakes , Victoria and Tanganyika, than to their riverine relatives which are not so easy to identify and are less accessible for sampling (Terai et al. 2004).

The lakes found in the Eastern Rift Valley (Fig. 5), not including Turkana, are characterised by their smaller size compared to those of the Western Rift and constitute several independent inland drainage basins (Baker and Kalinga 2009). These small lakes are often shallow and harbour relatively few species (Salzburger and Meyer 2004). Together with Lake Turkana, the lakes Albert, Edward, George and Rukwa (Fig. 5) are exceptional due to low levels of cichlid diversity and endemism of haplochromines and ‘tilapia’ species (Ricardo 1939; Burgis et al. 1973; Moriarty et al. 1973; Kolding 1993; Reinthal and Keenleyside 1993; Rognon and Guyomard 1997; Snoeks 2000; Chorowicz 2005; Vranken et al. 2019). Similarly, an absence of radiation in Lake Bangweulu (not shown in Fig. 5) was reported. In this system, it was assumed that Zambezian fish lineages, including cichlids, had subsequently colonised Lake Mweru downstream, as the inverse invasion seemed unlikely because of waterfalls and extensive rapids (Meier et al. 2019). Lake Mweru (Fig. 5), in contrast, showed multiple contemporary adaptive radiations of endemic haplochromines (Katongo et al. 2005; Katongo et al. 2017; Meier et al. 2019). Lake Kivu (Fig. 5) is one of the small lakes situated in the western branch of the Albertine Rift in a very active volcanic area (Baker and Kalinga 2009). This lake, with LT, lies within the drainage basin of the (Ofori-Amoah 2019). In terms of cichlid fauna, this habitat has long been considered a species-poor lake. The most recent checklist published by Snoeks et al. (2012) documented 19 cichlid species of tilapiines and haplochromines. Finally, Lake Cohoha (not shown in Fig. 5) located in a depression of the north-eastern part of the Burundese highlands is dominated by haplochromines, as well as introduced , all of economic importance.

27

Fig. 5 (a) Map of the East African Rift system showing nine Great Lakes and the biodiversity of their faunas, and (b) the total number of genera in each lake, and the degree of endemism (Salzburger et al. 2014).

3.2.3 Speciation mechanisms in East Great African Lakes

The spectacular adaptive radiations in East African Lakes provides prime material for evolutionary studies (Lowe-McConnell 2003). However, the total number of species known from these lakes is underestimated as most of the lakes are still underexplored (Snoeks 2001; Salzburger et al. 2014) and some taxa, especially invertebrates, need exhaustive systematic revisions (West et al. 2003). Lakes Malawi, Victoria and Tanganyika are typical for their species-rich flocks and the high degree of their endemism (Fig. 5). Currently, over 2,000 cichlid species (Fricke et al. 2021), subdivided into a single to over ten austrotilapiine tribes, are known in this area, with plenty of species awaiting taxonomic descriptions and/or revisions (Terai et al. 2004; Schedel et al. 2019; Ronco et al. 2021). Most of the species exhibit geographical distribution restricted to a single lake, and some populations may occur in even more restricted ecological niches over a few kilometres only (Baric et al. 2003; Snoeks 2001).

The cichlid diversity in East African Lakes inspired evolutionary biologists to investigate the speciation mechanisms that occurred during the history of the cichlid assemblages and their habitats. In the past, it was thought that species exhibiting highly diversified morphologies would be characteristic for evolutionarily older flocks, whereas

28 morphologically similar species would point to a younger evolutionary age (Larson et al. 1985). Although sympatric speciation driven by natural and sexual selection were suggested as evolutionary processes to explain the cichlid diversity in Lakes Malawi and Victoria (Schliewen et al. 2001; Schliewen and Klee 2004), the former speciation mode has not been evidenced in LT (Sturmbauer 1998). It was assumed that the cichlid radiations in the East African Lakes evolved in a relatively recent evolutionary time (Snoeks 2000; Salzburger and Meyer 2004; Kocher 2004). Evolutionary biologists suggested a scenario of cichlid adaptive radiation for the East African species flocks. The first step consisted on colonising a variety of free habitats by riverine generalist species resistant to seasonal fluctuations. Then, the first speciation events most likely occurred by invasion of major habitats (rocks, sand bottom and pelagic zone) (Danley and Kocher 2001), offering adaptation opportunities to the first emerging species possibly via sympatric mechanisms, and given the great dispersal ability of generalist colonisers (Sturmbauer 1998). The second stage consisted of emerging species within each fundamental niche, which likely exhibited distinguishable trophic structures, i.e., different feeding behaviour. This resulted in a reduction of gene flow and thus, species divergence (Sturmbauer et al. 2003; Koblmüller et al. 2007). The last phase is most likely restricted to haplochromine radiations of Lakes Malawi and Victoria where sexual selection was the mechanism driving the speciation and leading to the divergence of reproductive characters without large eco- morphological changes (Streelman and Danley 2003).

Lake Malawi (Fig. 5) occupies the Southern Rift Valley and drains into the Zambezi River (Baker and Kalinga 2009). With over 400 endemic species (some estimates even reach 1,000 (Turner et al. 2001)), cichlids exhibit typical morphological and behavioural features including body shape, melanin patterning, reproductive behaviours, and habitat preference resulting from ecological adaptations (Fryer 1959; Ribbink et al. 1983). The great majority of Malawian cichlids are endemic haplochromines (Fig. 6) with higher morphological diversity than the much younger cichlid species flock of Lake Victoria, followed by a few ‘tilapia’ representatives like the endemic Oreochromis lidole (Trewavas, 1941) (Danley et al. 2012; Genner and Turner 2012; Kocher 2004; Moran et al. 1994). From a single Astatotilapia-like ancestor, the Malawian cichlid flocks have entered the lake in a relatively short period of time (500,000–2 Ma), before the formation of the lake itself during a late Pleistocene dry-up (Delvaux 1995). This resulted in a variety of trophic niches for pelagic planktivorous, piscivores, detritivores, molluscivorous, paedophages, scale-eaters, and insectivores (Curry- Lindahl et al. 1976; Johnson et al. 1996; Meyer et al. 1990; Sturmbauer et al. 2001). Lake

29

Malawi contains two major haplochromine groups; the rock‐dwelling ‘mbuna’ that live on rocky shores and feed mainly on attached , and the omnivore sand‐dwelling ‘non-mbuna’ clade living in sandy shores or the ‘pelagic zone’, additionally to other oligotypic lineages (Moran et al. 1994; Ribbink et al. 1983; Seehausen et al. 1999).

Lake Victoria (Fig. 5) is situated in a region of erosion surfaces instead of dynamic escarpments and is lined by a shoreline of considerable variety (Baker and Kalinga 2009). This lake is the world’s largest tropical lake and the youngest one of the three Great Lakes (250,000– 750,000 years old) (Johnson et al. 1996). Through the confirmed monophyly of most cichlids inhabiting Lake Victoria and the weak genetic differentiation among its flocks (Mayer et al. 1998; Meyer et al. 1990), the molecular analyses often resulted in different topologies because of persistent polymorphism through the speciation phase (Nagl et al. 1998) and gene flow (Samonte et al. 2007). The Lake Victoria system harbours a unique species-rich ‘super-flock’ (Greenwood 1979) of about 500 endemic haplochromine cichlids (Fig. 6) that have originated from older surrounding freshwaters (Lakes Albert, Edward, George, Kyoga, and Kivu) and derived from two divergent lineages; Lake Victoria’s rock‐dwelling cichlids (mbipi) and the remaining representing all other endemic cichlids from the lake. Each lineage has most likely invaded the lake and rapidly diverged within the last 12,500 years (Nagl et al. 2000).

Fig. 6 (a) Schematic trees from combined phylogenetic analyses of the East African Radiation tribes based on mitochondrial cyt-b and (b) ND2 sequences data, and thus combining several previous studies. Native tribes are indicated with circles, whereas the absent ones are with crosses (see Takahashi and Sota, 2016).

30

3.3 Lake Tanganyika 3.3.1 Geographical location, hydrogeography and history

The cichlid flocks inhabiting LT have been the focus of several molecular evolutionary studies and became an influential model in general evolutionary theory (Koblmüller et al. 2005). Long and narrow in the southwestern branch of the Rift Valley, LT is located at an elevation of 772 m along the boundaries of both Tanzania and Burundi in the East, the Democratic Republic of the Congo (DRC) in the West, and in the South-West (Mccoll 2005; Aryeetey-Attoh 2009). This freshwater body measures 650 km in a north-south direction and has a width of up to 70 km (Degens et al. 1971). Thus, it is the second largest lake in the world, and the second deepest with a depth of 580 m (Mccoll 2005; Aryeetey-Attoh 2009). Lake Tanganyika is connected to several rivers, at least 18 flow into the lake, including the largest rivers Kalambo, Malagarasi, Luichi and Ruzizi. In turn, LT flows into the Congo River system before entering the Atlantic Ocean (Beauchamp 1939).

The Tanganyika rift is formed as a series of half-grabens under the influence of geological processes. It consists of three basins and a number of sub-basins separated by relatively shallow transverse sills (Chorowicz 2005). Cohen et al. (1993) estimated that the northern and southern basins began to form 7 to 8 Ma and 2 to 4 Ma, respectively, whereas the structural basins of central LT were older and started to form between 9 and 12 Ma (or even earlier, see Lezzar et al., 2002; Roller et al., 2010). This coincides with the creation of the Kivu- Ruzizi dome on the North end of modern LT (Cohen et al. 2007). On the other hand, it has been suggested that the cichlid assemblages of LT may be younger than LT basins themselves and aged 5–6 Ma (Genner et al., 2007; Koblmüller et al., 2008). The northern basin has the highest volume and counts three asymmetric sub-basins: the Bujumbura (350 m deep), Rumonge (1,150 m deep), and Kigoma sub-basins (1,310 m deep) (Rolet 1991). Contrariwise, the south basin which lies in the Northern Province of Zambia is the deepest (Verburg et al. 2011). Evidently, the rifting began in the central basin, which was infilled first, followed by an extension northwards, then southwards (Sander and Rosendahl 1989). The current LT seems to be the product of the fusion of three proto-lakes to a single large lake during its long history (Cohen et al., 1997; Cohen et al., 1993). The central basin was most likely infilled by the proto- Malagarasi River inflow and Lukuga River outflow draining after the uplift of the East African Plateau and the initiation of rifting (Cohen et al., 1997; Stankiewicz and de Wit, 2006), followed by the formation of the northern basin by the proto-Rusizi River (Cohen et al., 1997). Due to tectonic and glaciation periods, the lake level experienced several unconformities and

31 dramatically fluctuated. The lake level fluctuations were mostly documented in the northern basin (Cohen et al., 1997), while field studies targeting the central and southern basins are still missing to determine the exact processes that caused the low-stand events in the lake (Danley et al. 2012).

3.3.2 Diversity of Lake Tanganyika cichlid assemblages

Lake Tanganyika harbours a variety of invertebrates, cichlids and non-cichlid fishes. Impressively, most of the species, mainly cichlids, are unique to the lake itself, and do not occur in other regions as a result of a long period of isolation (Worthington and Ricardo 1936). This cichlid fauna is morphologically, ecologically and behaviourally the most diverse of the African lakes (Snoeks 2000). Poll (1986) used meristic and morphometric data, scales, dentition, soft- tissue anatomy, and osteology to define 12 tribes, each of which has from a single to hundreds of species. They are the following: , Cyprichromini, Ectodini, Eretmodini, , , Perissodini, Tilapiini, Trematocarini, Tropheini and Tylochromini Poll, 1986, plus Haplochromini. Later, Takahashi (2003) revised the concept of tribes and proposed a morphologically-based phylogeny. His classification matched Poll’s one, however, he proposed 16 potential tribes in the lake. Molecular studies (Koblmüller et al., 2008; Meyer et al., 2015, 2017; Muschick et al., 2012; Salzburger, 2018; Schwarzer et al., 2009; Weiss et al., 2015) supported, in part, the previous work of Takahashi (2003) i.e., the establishment of the tribes Benthochromini and Boulengerochromini Takahashi, 2003, and Cyphotilapiini Salzburger, Meyer, Baric, Verheyen and Sturmbauer, 2002. In addition, Koblmüller et al. (2008) suggested splitting the members of the Bathybatini Poll, 1986 into Bathybatini sensu stricto and Hemibatini Koblmüller, Duftner, Katongo, Phiri and Sturmbauer, 2005.

While most LT tribes are endemic, a few of them show a distributional range including Central and Western African freshwaters habitats. The endemic O. tanganicae (Günther, 1893) is the sole native representative of Oreochromini in the lake (Takahashi 2003; Dunz and Schliewen 2013). This is also the case for the single tylochromine Tylochromis polylepis (Boulanger, 1900), a species widespread in Western Africa (Stiassny 1990). Phylogenetic studies showed that members of these tribes have colonised the lake fairly recently (Klett and Meyer 2002; Koch et al. 2007). Several cichlid representatives have been misplaced and are still awaiting large-scale taxonomic revisions. For instance the congeners permaxillaris (David, 1936) and G. pfefferi (Boulenger, 1898) belong to two tribes,

32

Limnochromini and Tropheini, respectively, based on molecular data (Salzburger et al., 2002; Takahashi, 2003). ‘Ctenochromis’ horei (Günther, 1894) belongs to Tropheini based on DNA sequence data (Sturmbauer et al. 2003), while its congener C. benthicola (Matthes, 1962) clusters within Cyphotilapiini (Muschick et al. 2012).

The phylogenetic relationships of LT cichlids have long been controversial and constantly debated. Nishida (1991) investigated the relationship among the representatives of different tribes using molecular data (allozymes) and defined the so-called ‘H-lineage’ comprising Eretmodini, Limnochromini, Benthochromini, Ectodini, Perissodini, Tropheini, Cyphotilapiini, and Haplochromini. Later, this clade was rejected based on mitochondrial genes (Koblmüller et al., 2005; Salzburger et al., 2002). Clabaut et al. (2005) defined the so-called ‘C-lineage’, an equivalent to the ‘H-lineage’ of Nishida (1991) but without Eretmodini. However, recent phylogenetic studies of Meyer et al. (2015) and Irisarri et al. (2018) supported the position of Eretmodini within the ‘H-lineage’. This tribe was also shown to be closely related to Orthochromini Clabaut, Salzburger et Meyer, 2005 and Haplochromini (including Tropheini) (Irisarri et al. 2018). Takahashi et al. (2001), based on short interspersed elements insertions (SINE), supported the monophyly of the so-called ‘MVhL-clade’ composed of species flocks from out of LT (Malawi and Victoria), the ‘H-lineage’ and Lamprologini. Later, phylogenetic analyses based on mitochondrial genes (Klett and Meyer 2002; Salzburger et al. 2002b; Koblmüller et al. 2005), as well as nuclear sequences (Takahashi and Okada 2002; Terai et al. 2003) allowed the recognition of Bathybatini, Hemibatini and Trematocarini as the ancestral lineages of the ‘MVhL-clade’. The individual composition of each tribe is well established and the monophyly of each tribe is well supported in phylogenetic analyses (see for instance Takahashi and Sota, 2016), while the relationships among some tribes were uncertain, due to rapid diversification, hybridisation, and adaptation to particular ecological niches at the onset of the radiation (Takahashi and Koblmüller 2011). This was the case of the relationships between Bathybatini, Boulengerochromini and Trematocarini, whose ancestors are one of the seeding lineages of the radiation together with Hemibatini, Eretmodini, Lamprologini, and the ‘C-lineage’ (Salzburger et al., 2002). These early divergent lineages colonised the lake at an early stage of its formation (proto-lakes) and, at the same time, most likely underwent major diversification through successive divergence events when the majority of the species was not formed yet. Thus, they contributed to the so-called ‘primary lacustrine radiation’ which probably occurred during the onset of deep-water conditions about 5–6 Ma (Koblmüller et al., 2008; Salzburger et al., 2002; Sturmbauer et al., 2010). On the other hand, Irisarri et al. (2018)

33 and Meyer et al. (2017) suggested complex intertribal introgression involving either Steatocranini Dunz and Schliewen, 2013 (represented by Steatocranus casuarius Poll, 1939), which is the ancestor of Bathybathini and Boulengerochromini, and the remaining 'modern' lineages, or a second hybridisation between Benthochromini and Perissodini. Nevertheless, the study of Ronco et al. (2021) based on whole-genome phylogenetic analyses combined with morphological measurements of ecologically relevant trait complexes (body shape, upper oral jaw morphology and lower pharyngeal jaw shape) finally allowed the establishment of the phylogenetic context of cichlid evolution in LT and to provide the most recent tree illustrating the relationships among tribes (Fig. 7).

Fig. 7 The most recent time-calibrated species tree using whole-genome nuclear sequence data of the cichlid fishes of African LT (Ronco et al. 2021).

34

In terms of taxonomic diversity, a total of 241 valid cichlid species subdivided into 16 tribes are recognised in the Tanganyika system based on the most recent studies of Ronco et al. (2020; 2021). Boulengerochromini represents the poorest tribe in terms of species number in the lake, while Lamprologini is the most species-rich (Fig. 8). In contrast to the monotypic substrate-brooder boulengerochromine Boulengerochromis microlepis (Boulenger, 1899), Bathybatini clusters eight ecologically divergent (Kirchberger et al. 2012). Boulengerochromis microlepis is the world’s largest cichlid and one of the top-predators in LT. Highly mobile, B. microlepis disperses over long distances without habitat restrictions, resulting in a lack of phylogeographic structure (Koblmüller et al. 2014). Members of Bathybatini show a lake-wide distribution and occur mainly in the deep-water habitats (Kirchberger et al. 2012; Konings 2019). Trematocarini is represented by a single endemic genus, Boulenger, 1899 including nine species (Takahashi 2002; Froese and Pauly 2021). Widely distributed throughout the lake, species of this tribe are specialised in alternating between feeding in the dark and migrating to shallower water when the sun sets (Konings 2019). There are at least 80 valid species within Lamprologini in LT subdivided into eight genera (Konings 2019; Froese and Pauly 2021). Few lamprologine representatives have secondarily colonised the lower Congo and Malagarasi River systems (Salzburger et al., 2002; Schelly et al., 2003; Sturmbauer et al., 2010). Hybridisation is known to have driven the speciation of East African cichlid assemblages (Kornfield and Smith 2000; Seehausen 2015). marunguensis Büscher, 1989 is a result of hybridisation between two ancient and genetically distinct species (olivaceous‐clade and helianthus‐clade) (Salzburger et al., 2002). Contrariwise, despite being the most species-rich tribe of cichlids overall and widespread across Africa, Haplochromini (excluding Tropheini) in LT is represented by a single genus of two species only (Konings 2019; Froese and Pauly 2021). Astatotilapia burtoni (Günther, 1894) is a member of the ‘modern haplochromines’ of LT (Salzburger et al. 2005; Meyer et al. 2015a). Haplochromini in the lake showed to be the sister group of the Lake Victoria super-flock of Astatotilapia Pellegrin, 1904 and they inhabit river mouths where they secondly diversified, rather than the lake itself (Koblmüller et al., 2008; Wagner et al., 2012). This was further supported by molecular data which revealed a LT origin for this tribe to further colonise large parts of African freshwater systems, and later re-colonise the lake and again diversify (Sturmbauer and Meyer 1993; Salzburger et al. 2002b). Moreover, introgressive hybridisation linked to lake‐level fluctuations and large‐scale migration events was suggested to explain the phylogeographic pattern and shared haplotypes between populations of A. burtoni along a north-south axis (Pauquet et al. 2018). Similarly, genetically divergent populations, and

35 morphological and ecological differentiation along the lake–stream gradient were evidenced in this species (Theis et al. 2014). Phylogenetically, Tropheini were repetitively nested to Haplochromini (Salzburger et al. 2002b; Schwarzer et al. 2012; Ronco et al. 2021). The former is one of the most species-rich cichlid tribes endemic to the lake with nine genera including approximately 24 mouthbrooders (Froese and Pauly 2021; Konings 2019; Takahashi and Koblmüller 2014). Poll (1956) estimated that more than 10% of Lake Tanganyika's cichlid species exhibit obvious sexual colour dimorphism. This is the case for Cyprichromini, Benthochromini, and a few species of Ectodini, Tropheini, and Bathybatini. An evolutionary trend towards progressive adaptation to the pelagic zone was evidenced in representatives of Cyprichromini (Brandstätter et al. 2005). Perissodini, a closely related lineage to Cyprichromini, contains nine species that are all mouthbrooders, but that belong to two groups with different ecologies (Koblmüller et al. 2007; Takahashi et al. 2007). The first group includes open-water predators that feed on juveniles of pelagic fishes and on zooplankton, more specifically Haplotaxodon microlepis Boulenger, 1906 and Haplotaxodon trifasciatus Takahashi and Nakaya, 1999 (considered synonyms by Konings (2019)). The second group includes six lepidophagous (scale-eating) representatives of Perissodus Boulenger, 1898 and hecqui Boulenger, 1899. The scale-eating diet is a feeding mode which is not recognised in any other LT tribes. Most members of this scale-eating group occur in deeper- waters, thus little is known about their ecology and behaviour. Perissodus microlepis Boulenger, 1898 and, to a lesser degree, Perissodus straeleni (Poll, 1948), however, are found in shallower waters (Konings 2019). These sister species are relatively recent offshoots of the perissodine radiation supporting the deep benthic or pelagic habitat origin of the scale-eating in LT (Koblmüller et al. 2007). A complex past hybridisation between ancestors of Boulengerochromini and Perissodini has been hypothesised (Meyer et al. 2017; Irisarri et al. 2018). Benthochromini is a species-poor lineage comprising a single endemic genus Benthochromis Poll, 1986 including two streamlined mouthbrooding species in the deep- water realms (Takahashi 2003; Takahashi 2008; Konings 2019; Froese and Pauly 2021). Although taxonomic reassessment is necessary, three genera grouping five species cluster within Eretmodini (Takahashi 2003; Konings 2019). Genetic data evidenced the presence of six lineages, suggesting the existence of cryptic species (Rüber et al. 1999). Further, the significant differentiation found between sympatric populations of Tanganicodus irsacae Poll, 1960 and cyanostictus Boulenger, 1898 suggested a hybridisation after a secondary contact (Rüber et al. 2001).

36

A total of 34 valid cichlid species belong to the endemic tribe Ectodini which includes ten genera (Konings 2019; Froese and Pauly 2021). Ectodine species are known for their diversity in habitat preference and social behaviours, providing one of the best examples to study the evolution of vertebrate social organization (Barlow 2000). Limnochromini includes six genera of ten mouthbrooder species, mostly benthic deep-water invertebrate feeders and mud dwellers. Finally, Cyphotilapiini includes three endemic representatives: Cyphotilapia frontosa (Boulenger, 1906), C. gibberosa (Takahashi and Nakaya, 2003), and Trematochromis benthicola (Matthes, 1962) (Takahashi and Hori 2006; Muschick et al. 2012; Meyer et al. 2015b). The former species has a lake-wide distribution in each habitat with rocks, while its congener is restricted to the southern half of the lake (Konings 2019).

Numerous fish species (non-cichlids) from various fish families occur in the lake such as the spotted African Lungfish Protopterus dolloi Boulenger, 1900 (Protopteridae Peters, 1855), cornish jack anguilloides (Linnaeus, 1758) (), torpedo robber Alestes macrophthalmus Günther, 1867 (Alestidae Cockerell 1910), smoothhead catfish Clarias liocephalus Boulenger, 1898 (Clariidae Bonaparte, 1846), Acapoeta tanganicae (Boulenger, 1900) (Cyprinidae) and a few other representatives like Nile perches and sardines (Worthington and Ricardo 1936; Plisnier et al. 2009).

Fig. 8 Taxonomic diversity of LT cichlids per tribe. Coloured partitions in the bar plot indicate the number of described species, different hatchings are used to highlight ‘species needing revisions’ and ‘museum species’. White partitions refer to so far undescribed species of the two categories ‘description in preparation’ and ‘potential species’ (Ronco et al. 2020).

37

Part 2 Parasite diversity

3.4 Parasitic helminths: general overview

Whereas the estimation of the total number of some parasitic taxa such as Protozoa remains a hard task, studies documented between 75,000 and 300,000 helminth species parasitizing the vertebrates, of which between 3% and 5% are threatened and will most likely become extinct in the next 50 to 100 years (Dobson et al. 2008). Helminths count three major phyla (i) Platyhelminthes Claus, 1887, which include Neodermata (Nordmann, 1832) composed of three classes i.e., Monogenea van Beneden, 1858, Trematoda Rudolphi, 1808 (flukes) and Cestoda Rudolphi, 1808 (tapeworms), (ii) Nematoda Diesing, 1861 (roundworms) and (iii) Acanthocephala Kölreuter, 1771 (thorny-headed worms). Although nearly all groups of flatworms harbour symbiotic representatives, Neodermata count exclusively obligate parasites of all main groups of vertebrates with an estimated number of 60,000 species (Appeltans et al. 2012; Caira and Littlewood 2013). Highly important for their parasitic life-style, obligate parasites develop an unciliated, syncytical epidermis (the ‘neodermis’) designed to facilitate nutrient absorption and immune system evasion instead of the ciliated one present in juveniles (free-living stage) (Hooge 2001; Mulvenna et al. 2010).

The phylogeny and interrelationships of flatworms have long been unresolved despite numerous morphological and molecular phylogenetic studies (for instance Brooks, 1982; Ehlers, 1984; Zamparo et al., 2001). Although still not accurately, true flatworms (Platyhelminthes) showed to be nested amongst a major group of invertebrates, Lophotrochozoa (Dunn et al. 2008). Moreover, recent transcriptomic and genomic data allowed the identification of Bothrioplanida, a group of freshwater ‘microturbellarians’ as the putative closest ancestor to Neodermata (Laumer and Giribet 2014; Egger et al. 2015). In the same study, Cestoda was shown to be closer to Trematoda than to Monogenea, rejecting, therefore, Cercomeromorpha (Egger et al. 2015) (Fig. 9). Morphological studies based on ultrastructural characters have long been valuable in assessing the phylogenetic relationships among flatworms (Rohde 1991; Hertel 1993). However, the recent use of the ultrastructure of the excretory ducts did not provide any support for the close relationship between Bothrioplanida and basal Neodermata evidenced by sequences data (Poddubnaya et al. 2020).

Most flatworms are active feeders and, they are hermaphrodites (Monogenea, Cestoda and most representatives of Trematoda) (Baron 1996; Kennedy 2006; Roberts and Janovy 2009). Unless progenetic i.e., sexually mature at larval stage, the groups of trematodes,

38 cestodes, as well as all acanthocephalans and most nematodes (few are parthenogenetic with a mixture of simple and complex life cycles) show complex (heteroxenous) life cycles (Streit 2008). However, monoxenous worms, represented mostly by Monogenea and a few nematodes (-parasitic groups, see Roberts and Janovy, 2009), live on/in a single host species during their whole life cycle (Roberts and Janovy 2009; Chubb et al. 2010). Life cycles in different groups of helminths evolved by upward or downward routes allowing a single-host cycle to become complex (see Parker et al., 2015). The first ancestral invertebrate intermediate host for trematodes and cestodes was incorporated later (Littlewood et al., 1999), making the transition from a direct to an indirect life cycle in a single evolutionary event. The direct life cycle of monogeneans represent the ancestral state in Neodermata (Park et al. 2007; Hahn et al. 2014).

Fig. 9 Tree of relationships among 11 platyhelminth classes based on genomic data (Egger et al. 2015).

3.4.1 Monogenea

Monogenea is a diverse parasite group and currently contains over 5,500 known species (Řehulková et al. 2018). In recent years, it was assumed that every major group of fishlike vertebrates (i.e., sharing some characteristics of a fish), are parasitized by monogeneans (Kearn 2004). If this prediction is accurate, there may be around 25,000 species of monogeneans worldwide (Whittington 1998). The group of monogeneans is diverse not only by number of species but also with respect to their morphology and ecology. Indeed, from a parasitic ancestor of skin of early vertebrates, monogeneans have expanded to infect different organs of a range

39 of aquatic organisms (Whittington et al. 2000). The majority of monogeneans are attached to the gills and external surfaces (skin, fins and nostrils) of fish, however, some of them infect the external surfaces (Llewellyn 1984) and internal organs of vertebrates (Euzet and Combes 1998; Woo 2006) with one species even infecting the eyes of mammals (Thurston and Laws 1965). The biodiversity of fish and their monogenean parasites appeared to be unbalanced in the different systems. Ecological studies revealed that, compared to fish hosts in the temperate zones, tropical fish species harbour more monogenean species per host species (Rohde 2002). In contrast, tropical fish species exhibit less diverse parasite communities of internal monogenean species than do their temperate counterparts (Choudhury and Dick 2000; Poulin 2001).

3.4.2 Taxonomic and phylogenetic status of Monogenea

Despite the persistent doubts regarding their monophyly (Justine 1998; Perkins et al. 2010; Littlewood and Waeschenbach 2015), monogeneans classically comprise two subclasses, either the (i) (Fig. 10a-d) and Justine, 1991 (Fig. 10e-g) based on both the spermiogenesis and spermatozoon ultrastructure and the attachment organs in the adult stage, or (ii) Polyonchoinea Bychowsky, 1937 and Heteronchoinea Boeger and Kritsky (1993) based on the larval attachment organ (Justine 1998). The paraphyly of mono- and polyopisthocotyleans was suggested to support the fact that endoparasitism and the origin of complex life cycles was a singular and later innovation during the radiation of parasitic forms (Littlewood 2006). Molecular phylogenies proved, contrariwise, the monophyly of each of the two lineages as members of Neodermata, together with Cestoda and Trematoda (Egger et al., 2015; Littlewood et al., 1999; Mollaret et al., 2000, 1997). The phylogenetic study of Mollaret et al. (2000) supported Boeger and Kritsky's (1993) suggestion to include both Polystomatoinea Lebedev, 1986 (tetrapod monogeneans) and Oligonchoinea Bychowsky, 1937 (Polyopisthocotylea) within Heteronchoinea. Based on the WoRMS database (2021), Monopisthocotylea currently includes five orders: Capsalidea Baird, 1853, Gyrodactylidea Beneden and Hesse, 1864, Bychowski, 1933, Monocotylidea Taschenberg, 1879 and Montchadskyellidea Bychowsky, Korotajeva and Nagibina, 1970 (endoparasisitc flatworms, not shown in Fig. 10). Polyopisthocotylea comprises three orders only: Chimaericolidea Brinkmann, 1942, Diclybothriidea Bykhovskii and Gusev, 1950, and Price, 1936 (see Fig. 10). The apomorphies that unite all monogeneans include the oncomiracidium larvae with three ciliated zones, two pairs of pigmented eyes in larval and

40 adult stages, one pair of ventral anchors (or spines) and a single filament (Marks and Maule 2006).

Fig. 10 A variety of ectoparasitic monogenean families showing the diversity of the attachment organ in Monopisthocotylea (a-d) and Polyopisthocotylea (e-g) subclasses. (a) Entobdella whittingtoni Kearn, Karlsbakk, Evans- Gowing and Gerasev, 2015 (Capsalidae); (b) bicorniculus Nitta and Nagasawa, 2016 (Dactylogyridae); (c) Gyrodactylus lamothei Mendoza-Palmero, Sereno-Uribe and Salgado-Maldonado, 2009 (Gyrodactylidae); (d) Monocotyle luquei Chero, Cruces, Iannacone, Sanchez, Minaya, Sáez and Alvariño, 2016 (Monocotylidae); (e) Chimaericola colliei Beverley-Burton, Chisholm and Last, 1991 (Chimaericolidae); (f) Narcinecotyle longifilamentus Torres-Carrera, Ruiz- Escobar, García-Prieto and Oceguera-Figueroa, 2020 (Hexabothriidae); and (g) Paradiplozoon iraqensi Al-Nasiri and Balbuena, 2016 ().

41

3.4.3 Morphological adaptations

Monogeneans resemble endoparasitic digeneans in showing a leech-like looping locomotion (Kearn and Evans-Gowing 1998; Whittington and Cribb 2001). Adult specimens possess a prohaptor located in the anterior part of their body. This structure has attaching capacities in the form of adhesive pads and cephalic openings (Buchmann and Bresciani 2006). The hind part is formed by an attachment structure (opisthaptor) that is highly differentiated in terms of shape and size. It bears an adhesive apparatus equipped with various sclerotized structures (Fig. 10), ranging from hooks, anchors, and connective bars, to clamps, suckers, and squamodiscs (Roberts and Janovy 2009; Řehulková et al. 2018). The haptoral structures are considered to be taxonomically important parts for identification of monogeneans. Furthermore, haptoral structures in many monogenean groups are characteristic for major phylogenetic lineages, whereas the sclerites in the reproductive organs are important for species-level identification (Pouyaud et al. 2006; Messu Mandeng et al. 2015). In addition, haptoral structures, especially the anchors, were shown to be more appropriate for geomorphometric studies as they are more resistant to deformation when mounted on a slide. This allowed their use when investigating associations between monogenean morphology and various host traits such as morphology, ecology, phylogeny and population structure (Khang et al., 2016; Kmentová et al., 2016, 2019; Rodríguez-González et al., 2017; Vignon et al., 2011).

The evolutionary success of monogeneans is related to the diversity of their attachment organ and its adaptation to hosts and infection sites (Whittington and Chisholm 2008). Indeed, it has been assumed that the ancestors of the monogeneans were opportunistic browsers able to switch hosts (Kearn 1994). The acquisition of hooks was the most successful achievement of early monogeneans. The attachment was most likely achieved by section of adhesives serving for temporary attachment to substrates, then turned into hooks by assembled internal oncoblast cells for permanent parasitism. The further evolutionary feature concerned the ways in which the primary attachment organs (marginal hooks) have been supplemented by secondary structures (anchors or hamuli) or by tertiary structures (friction pads, muscular loculi, clamps, etc) (Kearn 1994). The high diversity of the attachment organs in monogeneans combined with their low population density resulted in a high specificity to specific microhabitats within hosts to ensure optimal mating contacts (Rohde 1979; Rohde and Hobbs 1986; Matějusová et al. 2003). The microhabitat specificity is not usually related to interspecific competition in monogeneans (Šimková et al. 2000; 2002b), however, it was suggested that the gill

42 monogeneans living in high population densities may modify their niches due to interspecific competition (e.g. Kadlec et al. 2003).

3.4.4 Host specificity

Ectoparasites exhibit direct transmission. This means that monogeneans can be transmitted either by physical contact between hosts, or through the oncomiracidium infectious stage, which makes them more host-specific when compared to endoparasites (Poulin 1992). The specificity measures the restriction or the exclusivity of a given parasite species to occur in/on one or more host species (host range) (Rohde, 1982; Whittington et al., 2000; Whittington, 1997). So far, four levels of specificity were described i.e., basic, structural, phylogenetic specificity and specificity in geographical space (Poulin et al. 2011). While structural specificity considers the number of host species and the prevalence or abundance of parasites on individual host species (Rohde and Rohde 2008; Marques et al. 2011), phylogenetic specificity takes into account the phylogenetic relationship of the hosts (Whitfield 1979; Humphery-Smith 1989; Poulin and Mouillot 2003; Kvach and Sasal 2010; Cooper et al. 2012). Specificity in geographical space includes the geographical distribution of host species used by a given parasite species (Krasnov et al. 2011a; Krasnov et al. 2011b). At geographical scale, widespread host species are parasitized by a larger number of parasite species than hosts with limited distribution (Esch and Fernández 1993). Generally speaking, the host-specificity of parasites is the result of interactions of a number of ecological, physiological, behavioural, and evolutionary parameters (Desdevises et al., 2002; Morand et al., 2002; Sasal et al., 1999). Fish parasites with a direct life cycle and free-living larval stages are characterised by a narrower spectrum of host species compared to other groups with complex life cycles involving intermediate hosts (Sasal et al. 1998).

Monogeneans may occur on a single host species (specialists), or on two or more non- congeneric hosts of the same terminal clade (intermediate generalist), or on closely related hosts i.e., congeneric host species, phylogenetically closely related non-congeneric hosts (intermediate specialists), or even phylogenetically unrelated host species (generalists) (Desdevises et al., 2002; Franceschini et al., 2018; Šimková et al., 2006; Thatcher, 2006). In monogeneans, several factors related to the host were shown to contribute to host specificity. Ward (1992) introduced the hypothesis of specialisation on predictable resources, i.e., organisms tend to specialise on stable resources (i.e., those not subjected to sudden changes), which minimises their risks of . A larger host provides parasites with a larger area for infection and, thus, more suitable niches for colonisation (Poulin 1995). This was repetitively

43 evidenced i.e., for monogeneans infecting Adriatic fish hosts (Sasal et al. 1999), members of Lamellodiscus Johnston and Tiegs, 1922 (Diplectanidae Monticelli, 1903) parasitizing sympatric Mediterranean sparids (Desdevises et al., 2002), and the highly host-specific species of Dactylogyrus Diesing, 1850 parasitizing European freshwater cyprinids (Šimková et al. 2001; 2006). Moreover, larger fish live longer and, therefore, represent a more stable resource for their parasite. This was suggested to explain the presence of specialist species of Lamellodiscus on larger hosts, while generalists parasitized relatively smaller fish (Desdevises et al., 2002). In monogeneans belonging to Dactylogyrus, haptor morphometry (anchor size) of specialists correlated with host predictability (longevity), however, such a pattern was not found for generalists (Šimková et al. 2001). The enemy-free space hypothesis (i.e., ways of living that reduce or eliminate a species' vulnerability to one or more species of natural enemies) (Jefferies and Lawton 1984) was not confirmed in the case of Dactylogyrus as species-rich communities were composed by both specialists and generalists, whereas species-poor communities included mainly generalists (Šimková et al. 2001). Ecological specialisation is another hypothesis related to host-specificity in ecological studies. The basic assumption is that species exploiting more resources are more widespread and locally more abundant in nature than species restricted to a narrow range of resources (Brown 1984; Gaston and Lawton 1990). In other words, host specificity may correlate with parasite distribution. While this assumption was not evidenced in endoparasitic nematodes parasitizing terrestrial mammals (Morand and Guégan 2000), the study of Dactylogyrus is in line with the hypothesis of ecological specialisation. Generalist species parasitized a wider range of cyprinid hosts with higher local abundance and prevalence compared to specialists (Šimková et al., 2002; 2006).

3.4.5 Speciation in monogeneans

Monogeneans appear to be suitable candidates for studying host-parasite coevolutionary relationships due to their life cycle and fast infection ability (Poulin and Morand 2000). Complete cospeciation (Fig. 11a) at the macroevolutionary level seems rare in parasitic flatworms and was shown to be restricted to higher taxonomical levels as a result of geographical isolation of particular host and parasite lineages (Boeger and Kritsky 1997; De Vienne et al. 2013). The numerous studies investigating the speciation and diversification of congeneric monogeneans revealed either ‘host switching’ or ‘duplication’ (intra-host speciation) events (Fig. 11) rather than cospeciation. Brooks and McLennan (1991) hypothesised an association between host-specificity and cospeciation and duplication events. Formally, cospeciation was shown as a main speciation mode in the highly host specific

44 chewing lice parasitizing pocket gophers (Hafner and Nadler 1988; Hafner et al. 1994). Generally speaking, sympatric speciation occurs either on phylogenetically distant hosts by host switching or on the same host species by parasite duplication, whereas allopatric speciation can take place on two geographically or behaviourally isolated host species (or populations of the same host species) (Brooks 1979). Failure to diverge occurs when parasite diversification does not follow that of the host lineage (Brooks 1979; Johnson et al. 2003). In Lamellodiscus, host- parasite associations were driven by ecological factors, which facilitated host switching rather than cospeciation (Desdevises et al., 2002). Host switching seems to be common in the evolutionary history of the viviparous Gyrodactylus von Nordmann, 1832 (Hahn et al. 2015) as previously predicted by Brooks and McLennan (1993), Rohde (1996), Poulin (2002) and Cribb et al. (2002). Species of Gyrodactylus display the widest host range among monogeneans (Harris et al. 2004). Sympatric speciation by host switching on closely related hosts was also evidenced in species of Gyrodactylus parasitizing marine gobiids (Huyse and Volckaert 2002; Huyse et al. 2003). Moreover, Huyse and Volckaert (2005) reported cospeciation in Gyrodactylus in several host-parasite complexes, but this was not associated with high host- specificity. Furthermore, speciation by geographic isolation (allopatric mode), host switching and instant isolation by host-specificity were evidenced in Gyrodactylus from European salmonids (Meinilä et al. 2004). For this genus, the key innovations needed for switching such as orientation to a new type of host as adult, mechanical endurance on it, and tolerance against host defence systems, seem readily exploitable on the new host family (Ziętara and Lumme 2002). Contrariwise, a combination of host switching and duplication events accounted for the diversification of Ligophorus Euzet et Suriano, 1977, a gill monogenean parasitizing Mediterranean mugilids (Blasco-Costa et al. 2012). Intra-host duplication was shown to be the main speciation event in members of Dactylogyrus parasitizing central European and Mediterranean cyprinids (Šimková et al. 2004; Benovics et al. 2020a), Thaparocleidus Jain, 1952 parasitizing Asian pangasiid catfishes (Šimková et al. 2013), and Anacanthorus Mizelle and Price, 1965 parasitizing neotropical characiform hosts. In the latter case, host-sharing was also identified but only between congeneric host species (Da Graça et al. 2018).

Known as the Fahrenholz rule, on-going host-parasite cospeciation is assumed to correlate with host-specificity of given parasite taxa, resulting in congruent phylogenetic trees of hosts and parasites (Fahrenholz 1913; Brooks et al. 1993). Generally speaking however, host switching between either phylogenetically close host lineages, or unrelated sympatric hosts results in incongruences between host and parasite phylogenies (Brooks and McLennan 1991;

45

Page 1993). When parasite speciation follows multiple host-switches, the tree topologies of hosts and their parasites may be, in contrast, congruent (De Vienne et al. 2007). The phylogenetic incongruences caused by host switching and/or duplication events were previously evidenced in diplectanid monogeneans (Desdevises et al., 2002; Šimková et al., 2013). These events are hardly identifiable in a phylogeny. Moreover, Page (1993) suggested that the presence of one or more parasite lineages on the same host results in incongruent host and parasite phylogenies. In parasites with a complex life cycle, incongruence between host and parasite phylogenies is also influenced by the presence of intermediate hosts in the life cycle, which most likely serve as a vector providing parasite dispersion (Wickström et al. 2003; Criscione et al. 2006). Finally, the extinction (lineage sorting) of a parasite in a host lineage during evolutionary time is one of the evolutionary scenarios known to cause phylogenetic incongruences. However, this event received less attention when investigating coevolutionary patterns in monogeneans, but was documented for instance in chewing lice and their bird hosts (MacLeod et al. 2010).

Fig. 11 Host–parasite co-evolutionary scenarios (a) co-speciation, (b) duplication (intra-host speciation), (c) extinction (lineage sorting), (d) “missing the boat”, (e) failure to diverge, and (f) host switching. The solid green region shows the lineage of the host, and the black line within shows the lineage of the parasite (Beer et al., 2019 adapted from Page, 1993).

46

3.5 Diversity of monogeneans in African fish fauna

The African ichthyofauna has been revealed to host a diverse fauna of parasitic flatworms. Considering the fish diversity in African freshwater habitats, the species diversity of their parasites is assumed to be considerably underexplored. The recent systematic checklist of Řehulková et al. (2018) documented over 450 parasite species infecting external surfaces or internal organs of all fish families. African monogeneans are represented by three families of Polyonchoinea comprising 33 genera, and two families of Oligonchoinea with three genera only. African Dactylogyridae currently count 26 genera including approximately 423 species, whereas Gyrodactylidae harbours 51 species subdivided into six genera (Řehulková et al., 2018; WoRMS, 2021). The representatives of Gyrodactylidae and Dactylogyridae were described from the gills of various fish host species representatives of Characiformes, Cypriniformes, Siluriformes, Anabantiformes Britz, 1995, Berg, 1940 and Perciformes (Řehulková et al. 2018; Fricke et al. 2021). Representatives of Diplectanidae, usually known from marine perciform fishes (Domingues and Boeger 2008), occur in the gills of freshwater lakes perches and only a single species is known so far (Paperna and Thurston 1969; Ergens 1981; Kmentová et al. 2019).

3.6 Diversity of monogeneans in African cichlids

A few years ago, Pariselle et al. (2011) provided a complete overview of the monogenean genera known from cichlids worldwide and their biogeographical distribution. Since, a new ectoparasitic genus described from Neotropical cichlids was added to the list (Mendoza-Palmero et al. 2017). Table 1 summarizes the ecto- and endoparasitic monogenean genera known from cichlids worldwide so far. Different ectoparasitic genera of monogeneans are distributed in cichlids from the different continents (Pariselle et al. 2011), whereas the same endoparasitic genus ( Paperna, 1963) was found in Africa and Asia. The geographical pattern of cichlid parasite distribution was explained by the marine dispersal of hosts between continents (one of the possible scenarios of cichlid distribution, see above). Indeed, cichlids have most likely lost their ectoparasitic fauna because of a lack of tolerance to salinity changes and, at the same time, conserved their endoparasitic monogeneans as they are protected from physicochemical environmental variation (Pariselle et al. 2011).

47

Table 1 List of ecto- and endoparasitic monogenean genera known from cichlids worldwide and their endemic distribution (Pariselle et al. 2011; Mendoza-Palmero et al. 2017). (1) Madagascar; (2) Asia; (3) West Africa; (4) East Africa; (5) Levant; (6) Iran; and (7) South America.

(1) (2) (3) (4) (5) (6) (7) Ectoparasitic genera

Insulacleidus Rakotofiringa and Euzet 1983

Ceylonotrema Gussev, 1963

Sclerocleidoides Agarwal, Yadav and Kritsky, 2011

Cichlidogyrus Paperna, 1960

Onchobdella Paperna 1968

Scutogyrus Pariselle and Euzet, 1995

Gussevia Kohn and Paperna, 1964 Sciadicleithrum Kritsky, Thatcher and Boeger, 1989 Trinidactylus Hanek, Molnar and Fernando, 1974 Tucuranella Mendoza-Franco, Scholz and Rozkošná, 2010 Parasciadicleithrum Mendoza-Palmero et al., 2017 Gyrodactylus von Nordmann, 1832 Endoparasitic genera Enterogyrus Paperna, 1963 Urogyrus Bilong Bilong, Birgi and Euzet, 1994

Currently, a single gyrodactylid genus against seven dactylogyridean genera are commonly recognised on cichlids inhabiting the Levant, Madagascar and Africa. Eighteen species of Gyrodactylus were described, mostly from coptodonine and oreochromine hosts (see overview in Dos Santos et al., 2019). The hard parts of the haptor in Gyrodactylus comprise 16 marginal hooks and a pair of anchors that are connected by superficial and deep bars (Prenter et al. 2004). Using mitochondrial genome data, G. nyanzae Paperna, 1973 parasitizing African coptodonines and oreochromines revealed to be paraphyletic and closely related to the representatives of Macrogyrodactylus Malmberg, 1957 and Paragyrodactylus Gvosdev and Martechov, 1953, two gyrodactylid genera reported from African and Asian hosts, respectively (Vanhove et al. 2018). Of dactylogyrideans (Table 1), there are two endoparasitic (mesoparasitic) and five ectoparasitic genera of monogeneans. They are endoparasitic Enterogyrus, and Urogyrus Bilong Bilong, Birgi and Euzet, 1994, and ectoparasitic Insulacleidus Rakotofiringa and Euzet, 1983, Onchobdella Paperna, 1968, Scutogyrus Pariselle and Euzet 1995 and Cichlidogyrus Paperna, 1960 (Fig. 12). It should be noted, however, that under artificial conditions the American dactylogyridean genus Sciadicleithrum was identified

48 on African cichlid hosts (Jiménez-García et al., 2001; see Mendoza-Palmero et al., 2017; Pariselle et al., 2011 and Table 1 for monogenean genera of neotropical cichlids). Western Africa was extensively investigated for cichlid parasites, especially for monogeneans. This revealed a high monogenean diversity in cichlids in this area with a total of 157 species (Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Rahmouni et al. 2017; Řehulková et al. 2018). Within Dactylogyridae, Enterogyrus comprises eight species found in the stomach and Urogyrus counts a single species infecting the urinary bladder (Pariselle and Euzet 2009). Species of Enterogyrus possess seven pairs of hooks, two pairs of middle hooks, and a single ventral connective bar, while the haptor in Urogyrus is characterised by the presence of a single pair of each of the middle hooks and a single connective bar, plus seven pairs of hooks (Paperna 1963; Bilong Bilong et al. 1994; Pariselle and Euzet 2009) (Fig. 12). Members of Enterogyrus were recorded from West African hemichromines and haplotilapiines, among which members of Oreochromini and Coptodonini. Similarly, member of this genus showed to parasitize East, Central and South African haplochromines (Paperna 1963; Bilong Bilong 1988; Bilong Bilong et al. 1989; Pariselle et al. 1991; Bilong Bilong et al. 1996; Madanire-Moyo and Avenant-Oldewage 2014). However, the type species E. cichlidarum Paperna, 1963 was described from C. zillii (Gervais, 1848) from the Levant (Paperna 1963). Also, few species were successfully introduced to Europe, America and Asia through commercial cichlids (see review in Madanire-Moyo and Avenant-Oldewage, 2014). Adaptive evolution in two mitochondrial genes was evidenced in mesoparasitic E. malmbergi Bilong Bilong, 1988 known from Cameroonian O. niloticus (Linnaeus, 1758) (Zhang et al., 2019). Urogyrus mostly parasitizes West African chromidotilapiines (Bilong Bilong et al. 1994; Raeymaekers et al. 2013) (Fig. 12).

In terms of gill monogeneans, cichlids native to Madagascar have been shown to be parasitized by members of Insulacleidus, a genus restricted to this region with only three species described up to date (Rakotofiringa and Euzet 1983). Morphologically, species of Insulacleidus exhibit two pairs of anchors, one dorsal and one ventral transverse bar, and 14 marginal hooks (Rakotofiringa and Euzet 1983; Pariselle and Euzet 2009) (Fig. 12). Onchobdella, with nine known species, was reported from West African chromidotilapiines, hemichromines and pelmatochromines (Bilong Bilong and Euzet, 1995; Jorissen et al., 2018; Paperna, 1968; Pariselle and Euzet, 2004; Pariselle and Euzet, 1995). The haptor in species of Onchobdella contains five to six pairs of short marginal hooks, a pair of large dorsal anchors that are arranged distal-laterally, one pair of small ventral anchors associated with two ventral bars and one large

49 horseshoe-shaped or straight dorsal bar (Paperna 1968b; Pariselle and Euzet 2004) (Fig. 12). Scutogyrus harbouring seven species was recognised on pelmatotilapiines and oreochromines (Dossou, 1982; Douëllou, 1993; Paperna and Thurston, 1969; Pariselle et al., 2013; Pariselle and Euzet, 1995). Ecological conditions in West African crater lakes were hypothesised to facilitate lateral transfer (host switching) of representatives of Scutogyrus from oreochromine to tilapiine hosts (Pariselle et al. 2013). Species of Scutogyrus were also reported out of Africa, from cultured tilapias in the Neotropics for instance (Aguirre-Fey et al. 2015). Very recently, Caña-Bozada et al. (2021) provided the first complete mitochondrial genome of S. longicornis (Paperna and Thurston, 1969) known to parasitize a range of African oreochromine hosts, as well as alien tilapias in Asia and the Neotropics (le Roux and Avenant-Oldewage, 2010; Zhang et al., 2019).

Fig. 12 Species and morphological diversity of ecto- (red) and endoparasitic (blue) dactylogyridean monogeneans known so far from African cichlids. The distribution of cichlids in Africa is shown above. Abbreviations: (DA) dorsal anchor; (VA) ventral anchor; (VB) ventral bar and (DB) dorsal bar (marginal hooks not shown)) (adapted after Pariselle and Euzet 2009).

3.7 Current knowledge on Cichlidogyrus 3.7.1 Cichlidogyrus parasitizing Western and Central African cichlids

Compared to other African dactylogyridean monogeneans parasitizing cichlid fish, Cichlidogyrus is the most speciose genus currently counting 131 species described from approximately 112 host species (Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Jorissen et al. 2018; Řehulková et al. 2018; Geraerts et al. 2020 and references herein). West Africa harbour most of the known species diversity of Cichlidogyrus where, so far, over 80 species are known to infect about 80 cichlid hosts (Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Rahmouni et al. 2017; Geraerts et al. 2020). Overall, West African

50 species of Cichlidogyrus show a relatively narrow host range i.e., most species are intermediate specialists (Mendlová and Šimková 2014). Cichlidogyrus arthracanthus Paperna, (1960) was the first monogenean species described from C. zillii inhabiting the Levant region (Paperna 1960). This species was shown to widely parasitize oreochromines and coptodonines throughout the African continent, additionally to representatives of the genus Tristramella Trewavas, 1942 native to the Jordan system (Pariselle et al., 2013; Pariselle and Euzet, 1995, 2009; Řehulková et al., 2018). This is also the case of C. halli (Price and Kirk, 1967), a species described from O. shiranus Boulenger, 1897, widely parasitizing West and East African oreochromines and haplochromines (Price and Kirk 1967; Paperna 1979; Douëllou 1993; Pariselle and Euzet 1997; Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Jorissen et al. 2017). This species was reported on alien tilapias in Asia as well (Maneepitaksanti and Nagasawa 2012). Cichlidogyrus tiberianus showed similar patterns of host-specificity and geographical distribution i.e., infecting various cichlid lineages across the African continent and Levant region (Paperna 1960; Paperna 1964; Paperna and Thurston 1969; Ergens 1981; Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010; Pariselle et al. 2013; Vanhove et al. 2013b), and was also reported from alien Asian cichlids (see for instance Bondad-Reantaso and Arthur, 1990). Contrariwise, all Cichlidogyrus species from tylochromines known so far are considered intermediate specialists restricted to sympatric congeneric species (Pariselle and Euzet 1994; Pariselle et al. 2014; Jorissen et al. 2018). Cichlidogyrus zambezensis Douëllou, 1993 described from the haplochromine Serranochromis macrocephalus (Boulenger, 1899) is an intermediate specialist in Bangweulu-Mweru ecoregion as its occurrence is limited to species of Serranochromis (Jorissen et al. 2017), whereas it is a generalist in Lake Kariba also parasitizing distantly related lineages (Haplochromini and Oreochromini) (Douëllou 1993). Similarly, its congener C. sclerosus Paperna and Thurston 1969 described from East African O. mossambicus (Peters, 1852) occurs on the gills of East African representatives of Haplochromini and Oreochromini (Paperna 1979; Douëllou 1993; Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010). Cichlidogyrus haplochromii Paperna and Thurston, 1969 described from Haplochromis guiarti (Pellegrin, 1904) from Lake Victoria is, however, limited to East African haplochromines (Paperna 1979; Douëllou 1993; Pariselle and Euzet 2009; le Roux and Avenant-Oldewage 2010). Freshwater habitats in Central Africa like those located in the Lower Congo ecoregion harbour a variety of cichlid lineages, which are showed to host an important species diversity of Cichlidogyrus resulting from biogeographical barriers that have most likely facilitated speciation (Jorissen et al. 2018). Cichlidogyrus papernastrema Price, Peebles and Bamford 1969 is mostly restricted to South

51

African T. sparrmanii Smith, 1840 (Tilapiini) (Price et al. 1969; Jorissen et al. 2018). All the above-mentioned Cichlidogyrus species are among a long list of Cichlidogyrus species illustrating the variation in the host range and geographical distribution of this genus across Africa. Host-specificity of West African species of Cichlidogyrus and Scutogyrus seems to be independent of parasite phylogeny, while it is linked to host phylogeny (Mendlová and Šimková 2014). Intermediate specialists were recognised as the ancestral state of host-specificity for the lineages of Cichlidogyrus and Scutogyrus parasitizing West African cichlids (Mendlová and Šimková 2014). The morphometry of their haptoral sclerites further showed a negligible correlation to host-specificity (Mendlová and Šimková 2014).

The haptor of Cichlidogyrus is equipped with dorsal and ventral transverse bars, two pairs of anchors and seven pairs of marginal hooks (Fig. 12) (Paperna 1960; Pariselle and Euzet 2009). This arrangement is similar to that of Scutogyrus, just in the latter the ventral bar is supported by a wide oval plate and the dorsal one shows typical long outgrowths (Paperna, 1960; Pariselle and Euzet, 1995; Pariselle and Euzet, 2009). Furthermore, some morphological similarities between species of Cichlidogyrus and Onchobdella parasitizing West African hosts were reported (Jorissen et al. 2018). Species of Cichlidogyrus are commonly classified within four potential morphological groups (Fig. 13) based on the size of hook pairs and presence/absence of the sclerotized vagina (Pariselle et al. 2003; Pariselle and Euzet 2009; Vignon et al. 2011 and references herin). The first group harbours species showing short hook pairs I-VII, a copulatory tube without a swollen proximal portion and a non-sclerotized vagina. The second group is characterised by the presence of long hook pair I (pair V always with larval size), and short pairs II-IV, VI and VII, plus a copulatory tube without a swollen portion, and a sclerotized vagina. The last group clusters species with shorter hook pair I and longer pairs II- IV, VI and VII, a copulatory tube without a swollen portion, and a non-sclerotized vagina (Pariselle and Euzet 2003; Vignon et al. 2011). Meanwhile, C. arthracanthus and few other species display a unique organisation with massive pairs I and III compared to pair V (larval shape) (Price et al. 1969; Bilong Bilong et al. 1996; Vignon et al. 2011).

52

4

Fig. 13 Morphological groups of species of Cichlidogyrus based on size and shape of hook pairs. (A) short hook pairs I-VII; (B) long hook pair I (pair V with larval size) and short pairs II-IV, VI and VII; (C) shorter hook pair I (pair V with larval size) and longer pairs II-IV, VI and VII; (D) long hook pairs II-IV, VI and VII, with pair I more or less massive. For each group, a single hook is represented (adapted after Vignon et al. 2011).

The phylogenetic relationships among West African species of Cichlidogyrus including species of Scutogyrus were inferred from the sequence data of ribosomal nuclear markers. The study of Pouyaud et al. (2006) was the first one to focus on West African species of Cichlidogyrus parasitizing ‘tilapias’ representatives and their phylogenetic relationships. In their study, the haptoral morphology was shown to be more suitable to infer phylogenetic relationships among Cichlidogyrus species than the morphology of the reproductive organs. Further, C. tilapiae (Paperna 1960), C. arthracanthus and C. halli appeared to belong to distinct phylogenetic lineages, which was not supported by morphological features. The morphological and genetic proximity found between C. tiberianus and C. aegypticus Ergens, 1981 parasitizing oreochromines and coptodonines suggested a colonisation, followed by a host switching event on their common ancestor (Pouyaud et al. 2006). Pouyaud et al. (2006) further reported the monophyly of Scutogyrus and distinct genetic groups of C. halli. Mendlová et al. (2012) supported the monophyly of Scutogyrus as well, while Cichlidogyrus was shown to be paraphyletic. It should be noted that C. mbirizei Muterezi Bukinga, Vanhove, Van Steenberge and Pariselle, 2012, C. halli and C. sclerosus are the first species of Cichlidogyrus that were targeted for mitochondrial genome sequencing (Vanhove et al. 2018; Zhang et al. 2019a). The mitogenome tree further supported the monophyly of the Cichlidogyrus-Scutogyrus group (Caña-Bozada et al. 2021). Cichlidogyrus species parasitizing West African tylochromines were shown to be more early divergent than other species of the Cichlidogyrus-Scutogyrus lineage (Mendlová et al. 2010; Mendlová et al. 2012; Mendlová and Šimková 2014). This fully

53 matches the cichlid phylogeny as cichlid representatives belonging to this lineage were ancestral to other African cichlids (Stiassny 1989; Dunz and Schliewen 2013).

Host switching in monogeneans, also in species of Cichlidogyrus, between their cichlid hosts seems to be far from unusual and can be considered as one of the main mechanisms that contributed considerably to the diversification of this genus in Africa. Within the coptodonine hosts inhabiting the Lower Congo Basin, a spillback effect was reported for C. cubitus Dossou, 1982 known so far only from the representatives of this tribe (Dossou 1982; Pariselle and Euzet 2009; Jorissen et al. 2018). Moreover, there are some reported cases of host switch of Cichlidogyrus to non-cichlid hosts inhabiting Cameroonian freshwater habitats. It is the case of C. amieti Birgi and Euzet, 1983 known from nothobranchiid (non-cichlids) hosts Aphyosemion cameronense (Boulenger, 1903) and A. obscurum (Boulenger, 1911) (Birgi and Euzet 1983), and C. inconsultans Birgi and Lambert, 1987 and C. Birgi and Lambert, 1986, both from nandid (non-cichlids) hosts Polycentropsis abbreviata Boulenger, 1901 (Birgi and Lambert 1986; Birgi and Lambert 1987). Phylogenetic analyses confirmed the recent host switching event of Cichlidogyrus from a cichlid to nothobranchiid hosts (Messu Mandeng et al. 2015). In Malagasy cichlids, spillover events between cichlids of different geographical origin are also known, e.g. species of Cichlidogyrus found on endemic cichlids and non-cichlids in the island were probably transferred from introduced African tilapias (Šimková et al. 2019).

3.7.2 Cichlidogyrus in the Tanganyika system

The knowledge on the parasite fauna of LT cichlids is still limited. Nevertheless, LT seems to harbour a unique assemblage of Cichlidogyrus species that reflect its cichlid diversity. Prior to this thesis, a total of 24 species of Cichlidogyrus were known to parasitize 22 host species belonging to Bathybatini, Benthochromini, Boulengerochromini, Ectodini, Haplochromini, Limnochromini, Oreochromini, Trematocarini, Tropheini, and Tylochromini (Gillardin et al., 2012; Kmentová et al., 2016; Muterezi Bukinga et al., 2012; Pariselle et al., 2015b, 2015a; Van Steenberge et al., 2015; Vanhove et al., 2011). Of 16 LT tribes, Ectodini was the first one to be investigated for Cichlidogyrus and four species were identified of boops (Boulenger, 1901), O. nasuta (Poll and Matthes, 1962) and O. ventralis (Boulenger, 1898) (Vanhove et al., 2011). The two latter species demonstrated to host more species of Cichlidogyrus i.e., C. centesimus, C. makasai, C. sturmbaueri and C. vandekerkhovei Vanhove, Volckaert and Pariselle, 2011 compared to its congener. Further, Raeymaekers et al. (2013) evidenced different parasite communities among allopatric cichlid populations of Tropheus. Still with Tropheini, the phylogeny of Cichlidogyrus demonstrated to

54 be congruent with that of their cichlid hosts and geographically-dependent diversification was documented (Vanhove et al. 2015).

In terms of host-specificity, Kmentová et al. (2016) reported based on the available literature that 17 of 24 Cichlidogyrus species known at that time in LT cichlids are strict specialists. Their study reported only five species classified as intermediate specialists, and a single intermediate generalist species. The intermediate specialists include C. centesimus, C. makasai, C. sturmbaueri and C. vandekerkhovei Vanhove, Volckaert and Pariselle, 2011 parasitizing Ectodini and C. franswittei Pariselle and Vanhove, 2015 parasitizing members of Tropheini, more specifically Pseudosimochromis curvifrons (Poll, 1942) and P. marginatus (Poll, 1956), and C. mbirizei parasitizing O. tanganicae, and O. niloticus and O. mossambicus (Muterezi Bukinga et al., 2012; Van Steenberge et al., 2015; Vanhove et al., 2011). Deep-water realms revealed reduced host-specificity of monogeneans probably as a result of low cichlid density in these habitats (Kmentová et al., 2016). Indeed, a range of representatives from Bathybatini showed to host the intermediate generalist C. casuarinus Pariselle, Muterezi Bukinga and Vanhove, 2015 known so far from minor Boulenger, 1906; B. fasciatus Boulenger, 1901; B. vittatus Boulenger, 1914, plus B. leo Poll, 1956 and (Boulenger, 1901) (Kmentová et al., 2016; Pariselle et al., 2015a).

Morphologically, LT assemblage of Cichlidogyrus can be classified into several lineages, which mirrors the cichlid hosts’ phylogenetic affinities (Pariselle et al. 2015b). Likewise, using species of Cichlidogyrus, historical relationships between riverine and lacustrine African tylochromines and oreochromines were illustrated (Muterezi Bukinga et al. 2012). Further, intraspecific size variation was observed in the MCO of Cichlidogyrus species parasitizing Ectodini (Vanhove et al., 2011). From an ecological perspective, Tropheini representatives with low dispersal capacities revealed to be infected by a large number of Cichlidogyrus species compared to hosts from same tribe with a high dispersal capacity (Grégoir et al. 2015). Furthermore, the differentiation of Cichlidogyrus communities parasitizing Tropheini seemed to be independent of host evolution and dispersal (Hablützel et al. 2016).

55

3.7.3 Non-Cichlidogyrus monogeneans from the Tanganyika system

Vanhove et al. (2011) provided the first documentation on monogeneans parasitizing cichlids of the LT system. The study comprised the description of three gyrodactylid species, namely G. sturmbaueri, G. thysi and G. zimbae Vanhove, Snoeks, Volckaert and Huyse, 2011 from the gills of the tropheine type host diagramma (Günther, 1894). Since, no other species of Gyrodactylus were described from the lake. Lake Tanganyika gyrodactylids showed a close evolutionary relationship to a few relatives from West African oreochromines. Moreover, the diversity of Gyrodactylus was attributed to possible parasite colonisation from outside the lake, as well as migration of the highly mobile, but less stenotypic tropheine hosts, and parasite exchange between lacustrine and riverine habitats (Vanhove et al., 2011). Further, the study of parasite communities of distinct colour morphs of the rock-dwelling populations of Tropheus (Tropehini) inhabiting southern LT revealed significant variation across populations for infection by Gyrodactylus, and various endoparasites (Raeymaekers et al. 2013). On the other hand, monogeneans parasitizing LT clupeids did not support the ‘magnifying glass’ hypothesis which states that parasites are successful candidates used to evaluate a host’s history at high resolution (Nieberding and Olivieri 2007). Indeed, various monogenean populations belonging to the recently described genus Kapentagyrus Kmentová, Gelnar and Vanhove, 2018 did not show any clear pattern of geographical structure (Kmentová et al. 2020).

56

4 Materials and methods 4.1 Collection of fish and monogenean taxa

The parasitological material used for the present thesis was sampled between 2008 and 2016. Fish samples were collected along LT shorelines and neighbouring freshwater systems (Table 2). Gill samples from H. microlepis stem from historical specimens stored in the ichthyological collections of the Royal Museum for Central Africa (RMCA, Belgium). In total, 260 cichlid specimens were targeted in the present study for the presence of Cichlidogyrus. This includes 25 cichlid species belonging to 10 tribes endemic to LT (Fig. 14A-Y). Cichlid hosts were identified in situ and dissected based on standard protocol described by Ergens and Lom (1970). Fin clips from freshly collected cichlid specimens were preserved in 96% ethanol and carcasses of most investigated cichlids were deposited in the RMCA. Overall, 2168 specimens of Cichlidogyrus were studied belonging to 34 species, of which nine species were already known. For freshly collected host specimens, both gill chambers were examined for monogeneans, whereas only one gill chamber was examined for the historical samples. Gill arches were separated via dorsal and ventral section using standard parasitological procedures and transferred into a Petri dish containing water. Adult specimens of Cichlidogyrus were detached from the gill arches and isolated according to Musilová et al. (2009) using an MST130 stereoscopic microscope. Freshly collected monogenean specimens were mounted on slides with glycerine ammonium picrate mixture (GAP) (Malmberg 1957), while specimens isolated from the museum samples were mounted on a slide under a coverslip in Hoyer’s medium (Humason 1979). Monogenean specimens used for DNA analyses were cut in half using fine needles under a dissecting microscope, the haptoral part of the body was conserved in 96% ethanol for genomic DNA extraction. The remaining part containing the reproductive organs was completely flattened under a coverslip and fixed in GAP. Basic epidemiological data, i.e., prevalence, mean abundance, minimum and maximum intensity of infection, were calculated for each monogenean species according to Bush et al. (1997). Host nomenclature follows FishBase (Froese and Pauly 2021) and Catalogue of fishes (Fricke et al. 2021).

57

Fig. 14 Cichlid host species sampled in LT and neighbouring freshwater systems investigated in the present study. Photos are ordered by cichlid tribes. (A) Boulengerochromini (Boulengerochromis microlepis); (B) Cyphotilapiini (Cyphotilapia frontosa); (C) Cyprichromini (Cyprichromis microlepidotus); (D-H) Ectodini ((D) dewindti; (E) Cardiopharynx schoutedeni; (F) melanostigma; (G) ; (H) flavipinnis); (I- J) Eretmodini ((I) Eretmodus marksmithi; (J) Tanganicodus irsacae); (K-L) Haplochromini ((K) Astatotilapia burtoni; (L) Astatotilapia stappersii); (M-N) Lamprologini ((M) callipterus; (N) Neolamprologus fasciatus); (O-Q) Perissodini ((O) Perissodus microlepis; (P) Haplotaxodon microlepis; (Q) Perissodus straeleni; (R-X) Tropheini ((R) ‘Ctenochromis’ horei; (S) Interochromis loocki; (T) Petrochromis orthognathus; (U) Petrochromis trewavasae; (V) Pseudosimochromis curvifrons; (W) Pseudosimochromis marginatus; (X) Simochromis diagramma); (Y) Tylochromini (Tylochromis polylepis). Photos by Radim Blažek, Maarten Vanhove and Chahrazed Rahmouni (2013-2016). Photos (Q,V) were retrieved from www.Tanganyika.is.

58

4.2 Morphometric and geomorphometric analyses

Parasite identification was conducted using original descriptions and the systematic revision of dactylogyridean parasites of African cichlids by Pariselle and Euzet (2009). Measurements and photographs were taken using an Olympus BX51 phase-contrast microscope and Olympus Stream Image Analysis v. 1.9.3 software. The measurements were expressed in micrometres, and given as the mean followed by the range and the number of measurements (n) in parentheses (measurements of some undescribed species were expressed as the length of the structure in question followed by the number of measurements in parentheses). Drawings of the haptoral sclerotized parts and copulatory organs were made on flattened specimens using an Olympus BX51 microscope equipped with a drawing tube and edited with a graphic tablet compatible with Adobe Illustrator CS6 v. 16.0.0 and Adobe Photoshop v. 13.0. In the present study, the terminology of haptoral sclerotized parts (anchors and hooks; also termed gripi and uncinuli, respectively) follows Gussev (1983), while the numbering of hook pairs (Roman letters I–VII) is that recommended by Mizelle (1936). Indeed, this method is preferred in adult specimens because it takes into consideration both antero-posterior and dorso–ventral positions of hooks (Řehulková et al. 2013). The terminology used for the hooks (short or long) and the classification of haptoral groups follow Pariselle et al. (2003) and Vignon et al. (2011), respectively. To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) (International Commission on Zoological Nomenclature, 2012), details of each species have been submitted to ZooBank. Type material was deposited in the MRAC, the Finnish Museum of Natural History (MZH, Finland), and the Muséum National d’Histoire Naturelle (MNHN, France).

A landmark-based technique using nine landmarks (LMs) (Rodríguez-González et al. 2015) was employed to investigate the intraspecific morphological variation in the DA and VA between groups of Cichlidogyrus at (i) taxonomic (host tribes and species), (ii) ecological (hosts with contrasting dispersal capacities), and geographical scale (north-south axis). Landmarks are discrete anatomical loci observed at the same place in all specimens. For this, slides were photographed, and landmarks were placed on DA and VA which were analysed separately. Anchors located at the right side of the worm were retrieved from specimens for which the whole body was mounted on a slide. The software tps-Util v.1.76, tpsDig2 (Rohlf 2006), and MorphoJ v. 1.06 (Klingenberg 2011) were used to input, digitalize photos, and import coordinate files, respectively. The latter software was also used to visualize the shape variation by means of principal component analyses (PCA), to maximize the differentiation in anchor

59 shape between groups using canonical variate analyses (CVA), and to investigate the effect of the size on the shape of the anchors (allometry) (permutation test (Good 2001)). Statistical software packages like PAST (Hammer et al. 2001) and Statistica v. 13.5 (TIBCO Inc., 2018) were used to build Neighbour-Joining (NJ) trees based on Mahalanobis distances, to compare between the monogenean groups, and to visualize the distribution of the anchor size variation. Voucher material was deposited in the same institutions.

4.3 DNA-Isolation, amplification, and sequencing Fin samples were sequenced for the partial cytochrome b (cyt-b) mitochondrial gene to confirm the identity of the investigated cichlids. Cichlid DNA was extracted from fin clips preserved in ethanol using the DNeasy Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The partial cyt-b gene was amplified following Mendlová et al. (2012). The identification of cichlid species based on the sequence similarity approach was carried out using NCBI BLAST (Altschul et al. 1990).

In addition to morphological features, the conspecificity of monogeneans infecting the respective host species was confirmed using ribosomal fragments of the partial small and large subunits (18S and 28S rDNA), plus the entire first internal transcribed spacer (ITS1). Monogenean individuals were removed from ethanol and dried using an Eppendorf 5301 concentrator under vacuum conditions at 30 °C. Genomic DNA was extracted using similar Kit. The partial 28S fragment (D1–D2) was amplified using forward primer C1 and reverse D2 (Hassouna et al., 1984), or alternatively using forward ANCY55 (Plaisance et al. 2005) and reverse D2 if unsuccessful with the first combination of primers. The partial 18S rRNA gene and the entire ITS1 region were amplified using the forward primer S1 (Sinnappah et al. 2001) and reverse primer IR8 (Šimková et al. 2003), or alternatively using forward S1 and reverse LIG5.8 (Blasco-Costa et al. 2012) if unsuccessful with the first combination of primers. We further obtained sequences of the partial mitochondrial COI gene for a selection of samples using ASmit1 and ASmit2 (Littlewood et al. 1997), with Schisto3 as internal primers for the nested PCR (Plaisance et al. 2008). For DNA amplification, we followed the protocols published by Plaisance et al. (2008), Mendlová et al. (2012) and Benovics et al. (2020). The PCR products were electrophoresed on a 1% agarose gel and then purified by either High Pure PCR product purification kit™ (Roche, Mannheim, Germany), or treated with ExoSAP-IT (Ecoli, Bratislava, SK) according to the manufacturer’s instructions. For cichlid hosts and their species of Cichlidogyrus, PCR amplicons were sequenced directly from both strands using the same primers as in the initial amplification PCR. Sequencing was performed on an ABI 3130

60

Genetic Analyzer (Applied Biosystems) using BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems by Thermo Fisher Scientific, Prague, Czech Republic).

4.4 Phylogenetic and cophylogenetic analyses Sequence data was checked manually for sequencing errors, edited using Sequencher® v.5.0 (Gene Codes Corporation, Ann Arbor, MI USA), and aligned using ClustalW (Thompson et al. 1994) implemented in MEGA X (Kumar et al. 2018). The .FAS format of the sequence fragments was subjected to BLAST program to confirm whether in order to check for contamination and compare them with all previously submitted sequences of Cichlidogyrus. The CLC Sequence Viewer v. 8.0 (CLC Bio-Qiagen, 2016) was used to visualize the nucleotide substitutions in the nuclear and the mitochondrial alignments. Uncorrected p- distances were calculated between specimens and groups using MEGA X. For nuclear and mitochondrial gene sequences, DnaSP v. 5.10.01 (Librado and Rozas 2009) was used to study the genetic diversity by calculating the number of polymorphic sites, nucleotide diversity, number of haplotypes, sequence conservation, and average number of nucleotide differences, as well as to determine the numbers of deletions in the sequences. Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Species of Cichlidogyrus parasitizing African cichlids from outside LT were also included in the analyses to determine the position of Cichlidogyrus spp. from LT cichlids in relation to all African congeners of which molecular data are available. The incongruence length difference test (ILD) was performed on the concatenated genes in the WinClada program (Farris 1995). The dataset was partitioned and the optimal DNA evolutionary model was selected for each marker individually, using jModelTest v. 2.1.10 (Guindon et al. 2010; Darriba et al. 2012). Likelihood mapping analysis based on quartet puzzling (Strimmer and Von Haeseler 1997) was performed on TREE-PUZZLE v. 5.2 (Schmidt et al. 2002). GBlocks v. 0.91b (Talavera and Castresana 2007) was used to remove unreliably aligned sequences (unwanted gaps and ambiguously aligned regions). IQ-TREE v. 1.5.5 (Nguyen et al. 2015) and MrBayes v. 3.2.1 (Ronquist et al. 2012) were used to infer phylogenetic trees by means of ML and BI methods, respectively. FigTree v. 1.4.4 software (Rambaut 2009) and PhotoShop CS6 were used for editing and visualizing final trees. All newly obtained sequences of nuclear and mitochondrial genes were deposited in GenBank.

The recent phylogeny of cichlids published by Ronco et al. (2021) was used for mapping the host lineages onto the parasite phylogeny using Mesquite v. 3.5 (Maddison and Maddison 2019) and for cophylogenetic analyses. The tanglegram illustrating the associations between

61 the investigated LT cichlids and their species of Cichlidogyrus was reconstructed using TreeMap v. 3.0b (Charleston 2012). TreeGraph v. 2.15 (Müller and Müller 2004) was used to remove extra taxa of fish as well as of monogeneans. The distance-based method ParaFit (Legendre et al. 2002) implemented in CopyCat (Meier-Kolthoff et al. 2007) was used for each host and parasite phylogeny. The event‐based method was performed with Jane v. 4.0 software (Conow et al. 2010) by attributing ten different cost schemes to assess the importance of each coevolutionary event in the host-parasite system investigated.

4.5 Host specificity and its relationship to parasite morphology in the Tanganyika system

The host specificity was expressed at the local level i.e. by considering species of Cichlidogyrus based on previous records from LT cichlids (Kmentová et al., 2016) and data used in study E. Host range was expressed as the total number of LT cichlid species parasitized by a given species of Cichlidogyrus. The index of host specificity (IS) was defined as follows: (i) strict specialists parasitizing a single cichlid host species, (ii) intermediate specialists parasitizing two or more congeneric host species, (iii) intermediate generalists from heterogeneric host species from the same tribe, and (iv) true generalists from phylogenetically more distantly related cichlid species. The mapping of haptoral groups based on hooks configuration following Vignon et al. (2011) and Rahmouni et al. (2017), and sclerotization in the vagina (present or absent) onto the parasite phylogeny was performed in Mesquite using the ML tree. The delimitation of Vignon et al. (2011) is as follows: group A for species of Cichlidogyrus with short hook pairs I-IV, VI and VII (pair V with larval size); group B for species of Cichlidogyrus exhibiting long hook pair I (pair V with larval size) and short pairs II- IV, VI and VII; group C for species of Cichlidogyrus with short hook pair I (pair V with larval size) and longer pairs II-IV, VI and VII; and group D for species of Cichlidogyrus showing long hook pairs I-VII, except larval-sized pair V.

62

Table 2 Cichlid hosts and their tribes sampled along the Lake Tanganyika shoreline and in neighbouring freshwater habitats investigated in the present study, with the date and locality of sampling, number of monogenean specimens collected and their corresponding species of Cichlidogyrus and their authors. NF: number of collected cichlid specimens; NP: number of collected monogenean specimens. Dashes indicate localities where targeted cichlids were sampled, and which showed to not host monogeneans.

Cichlid Tribe Cichlid species NF NP Date of Locality od sampling Country Coordinates Cichlidogyrus spp. Reference sampling Boulengerochromini Boulengerochromis microlepis 02 98 05/09/2013 Bujumbura fish market Burundi 3°23'S, 29°22'E C. nshomboi (Muterezi Bukinga et al. 2012; Takahashi, 2003 (Boulenger, 1899) Rahmouni et al., submitted) Cyphotilapiini Cyphotilapia frontosa 02 50 07/08/2016 Makabola village DRC 3°32′S, 29°09′E C. adkoningsi (Rahmouni et al. 2018) Salzburger, et al., 2002 (Boulenger, 1906) C. habluetzeli (Rahmouni et al. 2018) Cyprichromini Cyprichromis microlepidotus 03 78 23/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E C. milangelnari (Rahmouni et al. 2017) Poll, 1986 (Poll, 1956) 12 35 07/08/2016 Kalundo DRC 3°26′S, 29°07′E (Rahmouni et al. 2020) Ectodini Aulonocranus dewindti 03 20 07/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E C. discophonum (Rahmouni et al. 2017) Poll, 1986 (Boulenger, 1899) C. pseudoaspiralis (Rahmouni et al. 2017) Cardiopharynx schoutedeni 06 32 09/09/2013 Mulongwe fish market DRC 3°22′S, 29°06′E C. koblmuelleri (Rahmouni et al. 2018) Poll, 1942 C. habluetzeli (Rahmouni et al. 2018) Callochromis melanostigma 06 02 08/08/2016 Kilomoni beach DRC 3°20′S, 29°10′E C. sp. ‘C. melanostigma’ (Rahmouni et al. 2018) (Boulenger, 1906) Ophthalmotilapia nasuta 04 42 09/09/2013 Magara Burundi 3°44′S, 29°19′E C. aspiralis (Rahmouni et al. 2017) (Poll and Matthes, 1962) C. glacicremoratus (Rahmouni et al. 2017) C. rectangulus (Rahmouni et al. 2017) Xenotilapia flavipinnis 04 05 08/08/2016 Pemba DRC 3°37′S, 29°09′E C. sp. ‘X. flavipinnis 1’ (Rahmouni et al. 2018) Poll, 1985 Eretmodini Eretmodus marksmithi Burgess, 12 11 04/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E C. jeanloujustinei (Rahmouni et al. 2017) Poll, 1986 2012 10 - 03/09/2013 Magara Burundi 3°44′S, 29°19′E 13 - 23/09/2013 Mukuruka Burundi 4°14′S, 29°33′E Tanganicodus irsacae 07 13 20/09/2013 Mukuruka Burundi 4°14′S, 29°33′E C. evikae (Rahmouni et al. 2017) Poll, 1950 Haplochromini Astatotilapia burtoni 09 19 25/09/2013 Lake Cohoha Burundi - Cichlidogyrus sp. 1 Rahmouni et al., unpublished Trewavas, 1983 (Günther, 1893) 11 137 16/09/2013 Kilomoni beach DRC 3°20'S, 29°10'E C. gillardinae (Muterezi Bukinga et al. 2012) 01 - 25/09/2013 Bujumbura fish market Burundi 3°23'S, 29°22'E 02 - 09/09/2013 Mulongwe fish market DRC 3°22′S, 29°06′E 08 09 2015 Chitili system Zambia 8°36′S, 31°11′E Cichlidogyrus sp. 2 Rahmouni et al., unpublished 08 04 2015 Chitili system Zambia 8°38′S, 31°12′E

63

08 30 2015 Kalmbo system Zambia 8°38′S, 31°11′E 08 - 2015 Kalmbo system Zambia 8°35′S, 31°11′E Astatotilapia stappersii 01 19 16/09/2013 Kilomoni beach DRC 3°20'S, 29°10'E C. gillardinae Rahmouni et al., unpublished Poll, 1943 Lamprologini Lamprologus callipterus 06 262 10/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E Cichlidogyrus sp. 3-8 Unpublished data Poll, 1986 Boulenger, 1906 02 61 04/09/2013 Magara Burundi 3°44′S, 29°19′E 06 456 19/09/2013 Mukuruka Burundi 4°14′S, 29°33′E 04 101 20/09/2013 Mvugo Burundi 4°15'S, 29°34'E Neolamprologus fasciatus 03 42 23/08/2011 Wonzye Point Zambia 8°43'S, 31°08'E Cichlidogyrus sp. 9 and 10 Unpublished data (Boulenger, 1898) - - 18/04/2008 Kalambo Lodge Zambia 8°37’S, 31°12'E Perissodini Perissodus microlepis 03 08 09/09/2013 Magara Burundi 3°44′S, 29°19′E C. nshomboi (Muterezi Bukinga et al. 2012; Poll, 1986 Boulenger, 1898 Rahmouni et al., submitted) 05 39 09/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E 11 09 - Mtosi Zambia 7°35′S, 30°38′E 01 07 11/05/2010 Mugayo DRC 6°46′S, 29°33′E 06 02 25/03/2010 Bulumba Island DRC 3°46′S, 29°07′E Perissodus straeleni 07 05 14/05/2010 Mukumba DRC 6°56′S, 29°42′E C. nshomboi Rahmouni et al., submitted Poll, 1948 02 02 15/04/2010 Mtoto DRC 6°58′S, 29°43′E 01 02 - Mtosi Zambia 7°35′S, 30°38′E Haplotaxodon microlepis 03 03 1957 Luhanga DRC 3°31′S, 29°08′E C. nshomboi Rahmouni et al., submitted Boulenger, 1906 02 - 24/04/2010 Murega DRC 5°38′S, 29°23′E 02 - 1997 Makumba DRC 6°56′S, 29°42′E Tropheini ‘Ctenochromis’ horei 07 133 7/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E C. gistelincki (Gillardin et al. 2012; Poll, 1986 Günther, 1893 Rahmouni et al. 2020) 01 37 17/09/2013 Mukuruka Burundi 4°14′S, 29°33′E 01 - 26/09/2013 Bujumbura fish market Burundi 3°23'S, 29°22'E 04 69 06/09/2013 Magara Burundi 3°44′S, 29°19′E 05 158 25/09/2013 Mvugo Burundi 4°15'S, 29°34'E Interochromis loocki 02 12 08/08/2016 Pemba DRC 3°37′S, 29°09′E C. antoineparisellei (Rahmouni et al. 2018) (Poll, 1949) C. sp. ‘I. loocki 5’ (Rahmouni et al. 2018) Petrochromis orthognathus 04 46 08/08/2016 Pemba DRC 3°37′S, 29°09′E C. masilyai (Rahmouni et al. 2018) Matthes, 1959 C. sp. ‘P. orthognathus 2’ (Rahmouni et al. 2018)

64

C. sp. ‘P. orthognathus 3’ (Rahmouni et al. 2018) Petrochromis trewavasae 01 05 08/08/2016 Pemba DRC 3°37′S, 29°09′E C. salzburgeri (Rahmouni et al. 2018) Poll, 1948 Pseudosimochromis curvifrons 01 12 08/08/2016 Pemba DRC 3°37′S, 29°09′E C. franswittei (Van Steenberge et al. 2015; Poll, 1942 Rahmouni et al., unpublished) Pseudosimochromis marginatus 01 03 08/08/2013 Pemba 3°37′S, 29°09′E C. franswittei (Van Steenberge et al. 2015; (Poll, 1956) Rahmouni et al., unpublished)

Simochromis diagramma 10 15 05/09/2013 Magara Burundi 3°44′S, 29°19′E C. banyankimbonai (Van Steenberge et al. 2015; (Günther, 1893) Rahmouni et al., unpublished) 10 61 06/09/2013 Nyaruhongoka Burundi 3°41′S, 29°20′E C. muterezii (Van Steenberge et al. 2015; Rahmouni et al., unpublished) C. raeymaekersi (Van Steenberge et al. 2015; Rahmouni et al., unpublished) Tylochromini Tylochromis polylepis 01 14 09/09/2013 Mulongwe fish market DRC 3°22′S, 29°06′E C. mulimbwai (Muterezi Bukinga et al. 2012; Poll, 1986 (Boulenger, 1900) Rahmouni et al., unpublished) C. muzumanii (Muterezi Bukinga et al. 2012; Rahmouni et al., unpublished) C. sergemorandi (Rahmouni et al. 2018)

65

5 Results and discussion

This chapter presents a compilation of already published papers and submitted manuscripts to scientific journals. Prior the submission of Ph.D. thesis, three papers were published in 2017, 2018 and 2020, in addition to two papers which were submitted and are currently under review. The results in this chapter are divided into three main sections with several papers and/or manuscripts. Two papers are comprised in the first section, which focused on the species and morphological diversity of gill-specific flatworms of Cichlidogyrus parasitizing cichlid hosts from opposite lakeshores of LT. The second section includes one paper and one manuscript dealing with intraspecific variability of the genus Cichlidogyrus parasitizing Tanganyika cichlids showing contrasting dispersal capacities, congeneric species and phylogenetically distantly related host tribes. The last section comprises a single manuscript focused on (co)phylogeny and host-specificity correlates in members of Cichlidogyrus parasitizing Lake Tanganyika cichlid tribes. The full text of all papers is included as the appendices.

66

Section I Species and morphological diversity of gill-specific monogeneans of Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts

Study A Rahmouni, C., Vanhove, M.P.M. and Šimková, A. (2017). Underexplored diversity of gill monogeneans in cichlids from Lake Tanganyika: eight new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) from the northern basin of the lake, with remarks on the vagina and the heel of the male copulatory organ Parasites & Vectors; 10, 591. https://doi.org/10.1186/s13071-017-2460-6

Study B Rahmouni, C., Vanhove, M.P.M. and Šimková, A. (2018). Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) parasitizing the gills of Congolese cichlids from northern Lake Tanganyika PeerJ 6:e5604. https://doi.org/10.7717/peerj.5604

We studied cichlids representing northern littoral fish communities along Burundese (study A) and opposite Congolese (study B) lakeshores of LT for the presence of gill monogeneans belonging to Cichlidogyrus. Overall, five cichlid species members of Cyprichromini, Eretmodini and Ectodini were investigated for the first study, and eight cichlid species belonging to Cyphotilapiini, Ectodini, Tropheini and Tylochromini, were investigated in study B (see Table 2). Original descriptions were used to check the conspecificity of the newly described species of monogeneans in both studies, whereas fish samples were sequenced to confirm the identity of the investigated cichlid hosts in study B.

The cyprichromine and eretmodine cichlid hosts showed to host a single and two species of Cichlidogyrus, respectively. These were identified and described as new species for science. Cichlids of these tribes from LT had never been investigated for parasites earlier. The newly described species are: C. milangelnari from Cyprichromis microlepidotus; C. jeanloujustinei from Eretmodus marksmithi and C. evikae from Tanganicodus irsacae. New species of Cichlidogyrus were also identified on cichlids belonging to Ectodini. They are: C. aspiralis, C. glacicremoratus and C. rectangulus from Ophtahlmotilapia nasuta, C. pseudoaspiralis and C. discophonum from Aulonocranus dewindti (study A), C. koblmuelleri and C. habluetzeli on C. schoutedeni (study B). Lake Tanganyika Cyphotilapia frontosa from Cyphotilapiini revealed to host two new species i.e., C. adkoningsi and C. habluetzeli, the latter also found to parasitize Cardiopharynx schoutedeni. Three species of Cichlidogyrus were found on the gills of

67 examined cichlid species of Tropheini i.e., C. antoineparisellei on Interochromis loocki, C. masilyai on Petrochromis orthognathus and C. salzburgeri on P. trewavasae (study B). From the most ancestral tribe, Tylochromini and its sole representative in the lake Tylochromis polylepis, the species assigned to Cichlidogyrus sp. ‘T. polylepis 3’ for lack of material in a previous study of Muterezi Bukinga et al. (2012) was formally described in study B as C. sergemorandi. Likewise, this study further includes a morphological characterisation of the MCO of six species of Cichlidogyrus found on the gills of the tropheines I. loocki and P. orthognathus, and on those of the ectodines Callochromis melanostigma and Xenotilapia flavipinnis. These species are still undescribed because of a lack of material.

Similarity in haptoral and reproductive features in the newly described species reflected the phylogenetic relatedness of their cichlid hosts from LT, as well as with their congeners inhabiting outside of the lake as observed with the monogenean communities from riverine and lacustrine cichlids (see Pariselle and Euzet 1994). This was evidenced through the similar morphotype of haptoral sclerites like dorsal bars with their typical short and straight auricles, and MCO with straight heel and a copulatory tube ending distally in a spirally coiled thickening in species of Cichlidogyrus infecting ectodines i.e., in C. centesimus Vanhove, Volckaert & Pariselle, 2011 and the newly described species C. aspiralis, C. pseudoaspiralis (study A) and C. habluetzeli (study B). These morphological similarities are also shared between species parasitizing cichlids of some ancient and closely related LT tribes i.e., C. nshomboi from the world’s biggest cichlid i.e., B. microlepis of the Boulengerochromini (Muterezi Bukinga et al. 2012) and C. casuarinus from bathybatine hosts (Pariselle et al. 2015a). Moreover, morphological resemblances between species of Cichlidogyrus parasitizing LT T. polylepis i.e., C. mulimbwai Muterezi Bukinga, Vanhove, Van Steenberge & Pariselle, 2012, C. muzumanii and the newly described C. sergemorandi and their West African congeners parasitizing tylochromines (Pariselle and Euzet 1994; Muterezi Bukinga et al. 2012; Pariselle et al. 2014; Jorissen et al. 2018) recall the history of Tylochromini in riverine environments prior to the invasion of LT (Klett and Meyer 2002; Koch et al. 2007). Within Ectodini and Tropheini, geographical variation in their monogenean fauna throughout the lake was found as previous studies reported different species of Cichlidogyrus from O. nasuta, I. loocki and Petrochromis spp. (Vanhove et al. 2011b; Pariselle et al. 2015b). This was suggested to be related to high morphological morphs among geographically separated cichlids along LT (Vanhove 2012; Konings 2019).

68

Beside the formal descriptions of the new species, the haptor configuration in LT species of Cichlidogyrus and their congeners inhabiting outside of the lake was discussed. Following previous taxonomic studies focused on Cichlidogyrus, mostly from West African cichlids, LT monogeneans showed to belong to distinct morphological groups regarding the size and shape of the hook pairs (Vignon et al. (2011) and vagina (sclerotized or not) (see Pariselle and Euzet 1996; Pariselle and Euzet 2003). It should be noted that there are no species of Cichlidogyrus from LT showing a swollen portion at the proximal part of MCO and this feature is restricted to only few species parasitizing West African species so far like C. amphoratus Pariselle & Euzet, 1996 (Pariselle and Euzet 1996). The newly described species C. adkoningsi, C. discophonum, C. evikae, C. glacicremoratus, C. jeanloujustinei, C. koblmuelleri, C. masilyai and C. milangelnari from LT exhibited morphological similarities with West African species in possessing short hook pairs I-IV, VI and VII (group A defined in Vignon et al. (2011)) and a non-sclerotized vagina. It should be noted that C. evikae described in our study displays a characteristic shape of the hooks with broad thumb and a proximal protrusion. Up to date, 12 species of Cichlidogyrus from West Africa are known with a similar combination of haptoral (short hooks) and reproductive structures (no sclerotized vagina) (Paperna 1960; Paperna and Thurston 1969; Pariselle and Euzet 1997; Pariselle et al. 2003; Pariselle et al. 2013; Řehulková et al. 2013; Pariselle et al. 2014; Jorissen et al. 2017), and 17 species from the Tanganyika system (Gillardin et al. 2012; Muterezi Bukinga et al. 2012; Pariselle et al. 2015b; Van Steenberge et al. 2015; Kmentová et al. 2016a). Twenty-three other species of Cichlidogyrus, of which only three from LT, belong also to the group of species possessing larval hook pairs (group A), but they do have a sclerotized vagina. Prior to our investigations, there was only C. mbirizei Muterezi Bukinga, Vanhove, Van Steenberge & Pariselle, 2012 know from LT Oreochromini with this configuration (Muterezi Bukinga et al. 2012). Our gill samples from north-western LT (study B) allowed to add C. antoineparisellei and C. sergemorandi to this morphological group. The second haptoral group harbours species characterised by the presence of a long and relatively thick hook pair I (pair V with larval size) and short pairs II-IV, VI and VII (defined as group B in Vignon et al. (2011)). We find 11 Cichlidogyrus species from West Africa and a single species, namely, C. aspiralis from LT described in study A belonging to this haptoral group and also possessing a sclerotized vagina (Paperna 1965; Dossou 1982; Dossou and Birgi 1984; Pariselle and Euzet 1995c; Pariselle and Euzet 1998; Pariselle and Euzet 2004; Řehulková et al. 2013; Pariselle et al. 2014). Of this group, nine species outside of LT show a sclerotized vagina (Paperna 1968a; Paperna 1969; Price et al. 1969; Douëllou 1993; Pariselle and Euzet 1996; Pariselle and Euzet

69

1998; Pariselle and Euzet 2003; Jorissen et al. 2018), unlike LT species C. muzumanii Muterezi Bukinga, Vanhove, Van Steenberge & Pariselle, 2012 from LT T. polylepis (Muterezi Bukinga et al. 2012) and C. pseudoaspiralis described herein from Ectodini (study A). The third haptoral group (described as group C in Vignon et al. (2011)) includes species with short hook pair I (pair V with larval size) and longer pairs II-IV, VI and VII. With regard to the vagina, 27 species of Cichlidogyrus from outside LT appeared to show a similar hook configuration (group C) and a sclerotized vagina (Paperna 1960; Price and Kirk 1967; Ergens 1981; Dossou 1982; Douëllou 1993; Pariselle and Euzet 1995a; N’Douba et al. 1997; Pariselle and Euzet 1997; Pariselle and Euzet 1998; Pariselle et al. 2003; Geraerts et al. 2020). From LT, all species with similar hook configuration (group C) i.e., C. rectangulus and C. salzburgeri lack the sclerotization in their vagina. Based on molecular data and geomorphometry, the type-species C. arthracanthus showed to not belong to none of the above-listed groups in view of its typical haptoral configuration i.e., massive hook pairs I and VI in comparison to hooks V (larval size) (Vignon et al. 2011). Likewise, a few species of Cichlidogyrus from LT, as well as from out of the lake with their hook pairs, “escape” from the classification based on the hook configuration. Two other monogenean species i.e., C. inconsultans and C. nandidae both parasitizing non-cichlid hosts (see literature overview part) exhibit a particular hook configuration with long (not massive) hook pairs I-VII, except the larval one, plus a sclerotized vagina (Birgi and Lambert 1986; Birgi and Lambert 1987). Similarly, some recently described monogenean species from cichlid hosts inhabiting outside LT i.e., C. calycinus, C. polyenso and C. kmentovae Jorissen, Pariselle & Vanhove, 2018 known from hemichromine cichlids and C. maeander Geraerts & Muterezi Bukinga, 2020 parasitizing tilapiines have a typical configuration with a long and thick pair I, and long pairs II-VII (pair V with larval size), all lacking a sclerotized vagina (Jorissen et al. 2018; Geraerts et al. 2020). Lake Tanganyika species C. centesimus, C. casuarinus, C. nshomboi, and C. habluetzeli described in study B do not belong to the defined haptoral groups based on genetic and geomorphometric data due to their typical hook configuration, while all possess a sclerotized vagina. These exceptional observations emphasized the necessity to re-investigate the structural diversity of the hook pairs in Cichlidogyrus and identify the correct “boundaries” between the haptoral groups. This is discussed in study C (see below).

Finally, study A further highlighted the importance of the heel, a sclerotized portion of MCO close to the basal bulb of the copulatory tube, which is relevant to species identification in Cichlidogyrus. This structure, because of sclerotization, was often (but not always),

70 considered to be associated to the accessory piece of the copulatory organ (Řehulková et al. 2013). A few monogenean species, however, lack this structure. It is the case for eight species known from a range of cichlid lineages i.e., C. arfii Pariselle & Euzet, 1995, C. haplochromii Paperna & Thurston, 1969, C. karibae Douëllou, 1993, C. longicirrus Paperna, 1965, C. longipenis Paperna & Thurston, 1969, C. sanseoi Pariselle & Euzet, 2004, C. tilapiae Paperna, 1960, the recently described species C. bixlerzavalai Jorissen, Pariselle & Vanhove, 2018 (Paperna 1960; Paperna 1965; Paperna and Thurston 1969; Pariselle and Euzet 1995c; Pariselle and Euzet 2004; Jorissen et al. 2018). Prior to our investigations, C. attenboroughi was the first record from LT lacking a heel (Kmentová et al. 2016a), followed by C. discophonum and C. milangelnari described herein.

71

Section II Intraspecific variability of gill-specific monogeneans of Cichlidogyrus parasitizing Lake Tanganyika cichlid hosts

Study C Rahmouni, C., Van Steenberge, M., Vanhove, M.P.M. and Šimková, A. (2020). Intraspecific morphological variation in Cichlidogyrus (Monogenea) parasitizing two cichlid hosts from Lake Tanganyika exhibiting different dispersal capacities Hydrobiologia. https://doi.org/10.1007/s10750-020-04429-1

Study D Rahmouni, C., Vanhove, M.P.M., Šimková, A. and Van Steenberge, M., Conservative divergent evolution in a gill monogenean parasitizing distant cichlid lineages of Lake Tanganyika: Cichlidogyrus nshomboi (Monogenea: Dactylogyridae) from representatives of Boulengerochromini and Perissodini (submitted to Evolutionary Biology journal)

In study C, we hypothesised a link between host dispersal capacity in the Tanganyika system and intraspecific variability of their host-specific monogeneans belonging to Cichlidogyrus. Cichlidogyrus gistelincki parasitizing ‘Ctenochromis’ horei (Tropheini) and C. milangelnari parasitizing C. microlepidotus (Cyprichromini) were selected, two cichlid species that show contrasting dispersal abilities as a result of different ecology and behaviour. Cichlidogyrus species were morphologically identified, and identification based on sclerotized structures was confirmed using sequences of the 28S rDNA gene. A landmark-based approach was used to assess the shape variability in dorsal and ventral anchors exhibited by specimens of C. gistelincki and C. milangelnari sampled from various locations. The anchors were favoured instead of hooks or transverse bars as these latter already showed important modifications from the flattening and/or the fixation processes leading to distortion during mounting (Vignon et al. 2011). Previously, a geomorphometric approach was applied for members of Cichlidogyrus from LT parasitizing bathybatine cichlids (Kmentová et al. 2016c).

Based on DNA sequences, there was no intraspecific variability for C. gistelincki, whereas C. milangelnari showed a single nucleotide differentiation among the populations from localities on opposite lakeshores in the northern part of LT. Geomorphometric analyses indicated that populations of C. milangelnari parasitizing poorly dispersing cichlids were more differentiated in terms of shape of the anchors than populations of C. gistelincki infecting well- dispersing hosts. Meanwhile, anchor shape in C. gistelincki was overlapping between the

72 populations inhabiting nearby locations. The lack of a clear geographical trend in the shape of haptoral structures of C. gistelincki could be due to historical and local environmental factors like seasonal fluctuations that might influence morphological diversification in monogeneans (Ergens and Gelnar 1985; Dávidová et al. 2005; Bueno-Silva et al. 2011). Geomorphometry indicated that the fifth landmark, corresponding to the inner roots in anchors, was the most variable in all analyses. These changes, mostly related to anchors shape in this part, are potentially related to morphological variations in other haptoral structures like the transversal bars located closely to the anchors in Cichlidogyrus (Paperna 1960; Pariselle and Euzet 2009) as previously suggested for dactylogyrideans (Rodríguez-González et al. 2015). The use of a single nuclear maker known to be highly conservative among monogeneans (Mendlová et al. 2012; Vanhove et al. 2013a; Mendlová and Šimková 2014; Messu Mandeng et al. 2015) limited our interpretation of the relationship between the pattern of morphological variation in anchors of C. gistelincki and C. milangelnari and that in their genomes.

Study D aimed to investigate to which degree the diversification of C. nshomboi has followed that of its host species belonging to phylogenetically distant tribes in LT. This monogenean species was hitherto known from the boulengerochromine B. microlepis and, surprisingly in our study, it was retrieved from the gills of cichlid species belonging to representatives of Perissodini i.e., P. microlepis, P. straeleni and H. microlepis inhabiting various locations along the lake. Nuclear genes, namely the 18S and 28S rDNA genes, and the sclerotized structures of haptoral and reproductive organs, confirmed the conspecifity of the monogenean specimens. This makes C. nshomboi a true generalist parasitizing phylogenetically unrelated cichlid species belonging to different tribes following the classification of host specificity by Šimková et al. (2006) and Mendlová and Šimková (2014). The presence of C. nshomboi in the gills of hosts belonging to Boulengerochromini and Perissodini could be the result of either an inheritance from a common ancestor or a host-switching event. The former scenario seems, however, unlikely given the phylogenetic distances between the host tribes and the genetic similarity between their monogenean populations. Yet, hybridisation events between ancestors of these tribes could have allowed for a parental inheritance of the monogenean species into the hybrid lineage. The anchored phylogenomic approach performed by Irisarri et al. (2018) suggested, contrariwise, a complex scenario in the case of Boulengerochromini and Perissodini. Regarding the host-switching hypothesis, this is also unlikely as it is well known that this event decreases with increasing phylogenetic distance between donor and recipient hosts (Charleston and Robertson 2002). However, cases of host-

73 switching from a cichlid to a non-cichlid host are already known in Cichlidogyrus (Messu Mandeng et al. 2015). Geomorphometry of haptoral anchors and morphometrical characterisation of MCO parts as well as molecular data were used for this study. Significant shape and size variation of the anchors was observed between specimens of C. nshomboi infecting different host tribes. Anchors of monogenean specimens infecting B. microlepis were larger compared to those of specimens from all perissodines. This was explained by the larger body size of B. microlepis, which is considered as the largest cichlid inhabiting LT (Ochi and Yanagisawa 1998), while all perissodines are known for their smaller size (Konings 2019). Such correlation was previously documented by Mendlová and Šimková (2014) in Cichlidogyrus. In terms of reproductive organs, MCO in C. nshomboi infecting B. microlepis was the smallest in comparison to those in monogeneans from the perissodines. Moreover, specimens of B. microlepis were the most heavily infected by C. nshomboi (the highest number of specimens of C. nshomboi was found on the gills of a specimen of B. microlepis, followed by P. microlepis and P. straeleni). Morphological differentiation of anchors (shape and size) at host tribal level revealed by geomorphometric analysis was explained by phenotypic plasticity or adaptation. While no inter- or intrapopulation genetic variability was found in the 18S and 28S rDNA, the variability in ITS1 rDNA (1.1%) and COI (11.7% to 12.2%) genes, makes it hard to say to what degree the morphological variation reflects these conditions. The genetic differentiation in nuclear and mitochondrial genes reflected incipient speciation as found for other dactylogyrideans from LT clupeids (Kmentová et al. 2020) and gyrodactylids from sympatric callichthyids (Bueno-Silva et al. 2011), a phenomenon never documented for Cichlidogyrus. Moreover, although the relatively high variability found in the COI gene corresponded to previous findings in members of Cichlidogyrus parasitizing LT tropheines (Vanhove et al. 2015), the lack of mitochondrial genetic data for monogenean specimens from several sampling sites in our study did not allow to deeply investigate the population structure of C. nshomboi as performed by Kmentová et al. (2016b) using C. casuarinus infecting bathybatines. This could have supported the morphological differentiation present only between specimens of C. nshomboi infecting congeneric members of Perissodini at geographical scale, which was attributed to phylogenetic relatedness of the hosts, their co-occurrence at the rocky shores, ecomorphological specialization, and population structure along the rocky littoral zone. A lack of differentiation in host colonisation resulting in a failure to diverge was hypothesised to explain the morphological similarity in monogeneans infecting heterospecific perissodines. It should be noted that the monogenean specimens isolated from H. microlepis, unfortunately, could not be

74 used to assess to which degree the intraspecific variability extends in monogenean specimens parasitizing the perissodine hosts as this cichlid host was preserved using a different methodology and their monogeneans were mounted in a different medium. The morphological relationship between C. nshomboi and its congeners from distinct tribes is supported by molecular data, at least for C. nshomboi and C. casuarinus which showed to be genetically closely related based on nucleotide sequences (99.87% and 96.45% of similarity on BLAST for the 28S and ITS1 rDNA, respectively (no differentiation in the 18S rDNA sequences)). The weak host specificity expressed by C. nshomboi parasitizing phylogenetically unrelated hosts could be related to habitat preferences of its hosts as most representatives of Perissodini are deep-water specialists (Konings 2019), just like B. microlepis occurring in benthopelagic environments. Considering this host specificity, our results rejected the hypothesis of an irreversible ‘dead-end’ of the evolution of specificity in LT as already documented by Mendlová and Šimková (2014) for West African cichlid-Cichlidogyrus system. This is detailed in the next section based on study E. Furthermore, results obtained in study D provided motivation to deeply investigate the evolutionary scenarios involved in the speciation of Cichlidogyrus in LT. This is presented and discussed in the next section.

75

Section III Phylogeny, cophylogeny and host specificity correlates in species of Cichlidogyrus parasitizing Lake Tanganyika cichlids Study E Rahmouni, C., Vanhove, M.P.M., Koblmüller, S., Mendlová, M. and Šimková A. Molecular phylogeny and speciation patterns in host-specific monogeneans (Cichlidogyrus, Dactylogyridae) parasitizing cichlids in Lake Tanganyika. (to be submitted to International Journal for Parasitology)

Despite the well-established of Cichlidogyrus (Pariselle and Euzet 2009), still little is known about the relationships between West African monogenean lineages and those occupying the East African Great Lakes (including LT), and about the evolutionary history and diversification of monogeneans in the African Great Lakes. Study E represents the first comprehensive one using molecular data to investigate the phylogeny of Cichlidogyrus from LT cichlid species representing 14 out of 16 cichlid tribes. In this study, we, firstly, investigated the phylogenetic position of species of Cichlidogyrus parasitizing LT cichlid hosts in relation to those from host representatives outside of the lake. Phylogenetic reconstruction included a total of 63 species of Cichlidogyrus representing 25 Cichlidogyrus species and three Scutogyrus species from West African cichlid representatives, a single species obtained from each of Southern African haplochromine cichlid and nothobranchiid (non-cichlid) hosts, and 36 species of Cichlidogyrus from LT itself. The latter group comprises two species which were retrieved from haplochromine representatives inhabiting East African freshwater systems (i.e., outside of LT). Overall, phylogenetic reconstruction revealed that Cichlidogyrus parasitizing mainly West African cichlid tribes are paraphyletic with respect to species parasitizing hosts belonging to the East African cichlid radiation, which constitute a well-supported monophylum. West African Cichlidogyrus and Scutogyrus species included in our analysis formed a monophyletic group in accordance with previous studies of Mendlová et al. (2012) and Caña-Bozada et al. (2021), the last one using mitogenomic data from various representatives of Cichlidogyrus (from LT and outside). Species of Cichlidogyrus parasitizing cichlids belonging to West African hemichromines, coptodonines and oreochromines, as well as a South African representative of Haplochromini, were sister to all Cichlidogyrus species from the East African radiation (LT), consistent with the common ancestry and the phylogenetic relationships among the hosts (Irisarri et al., 2018; Schwarzer et al., 2009). The position of C. amieti from a non- cichlid host within the monogenean species of hemichromines is as previously shown by Messu Mandeng et al. (205). Cichlidogyrus from tylochromine and oreochromine hosts that colonised

76

LT only recently, cluster with their non-LT relatives, indicating that they colonized LT with their current host species, and did not jump over from any of the many cichlid species already present in the lake. Cichlidogyrus mulimbwai known from T. polylepis distributed in LT and its tributaries was early divergent within Cichlidogyrus. This species was sister to C. pouyaudi parasitizing tylochromines inhabiting West Africa, which was previously shown to be basal in the Cichlidogyrus phylogeny (Mendlová et al., 2012). The mapping of cichlid tribes (lineages) onto the phylogenetic reconstruction of Cichlidogyrus showed that Tylochromini was the most plesiomorphic host group for Cichlidogyrus. This is reflected in the African cichlid phylogeny where Tylochromini was repetitively resolved as the most ancestral split, suggesting the longest independent evolutionary history (Salzburger et al. 2002b; Ronco et al. 2021). The molecular phylogeny of Cichlidogyrus in our study provides support for the close relationship between West African and LT tylochromine hosts, and suggests a ‘fingerprint’ of the non-Lake Tanganyika origin of Tylochromini (Salzburger et al. 2002b; Koch et al. 2007). Similarly, the position of C. mbirizei within Cichlidogyrus species parasitizing West African oreochromines represents an evidence of secondary colonization of LT by the oreochromines, of which O. tanganicae (Klett and Meyer 2002). Cichlidogyrus amphoratus and C. sclerosus collected from coptodonine and oreochromine cichlid hosts were sister to the well supported large LT clade of Cichlidogyrus.

Species of Cichlidogyrus infecting distinct tribes in LT clustered in three main clades reflecting the evolutionary history of LT cichlids. Ancestral state reconstruction analysis revealed Cichlidogyrus parasitizing distinct ancient and modern LT tribes included in a few different lineages. Two clades grouped monogeneans parasitizing cichlid hosts representing lineages that have contributed to the ‘primary lacustrine radiation’ in the lake (Salzburger et al. 2002b; Clabaut et al. 2005), and are occupying particular ecological niches ranging from semi- pelagic (Cyprichromini, a few members of Ectodini and Lamprologini) to deep and benthopelagic habitats (Bathybatini, Benthochromini, Boulengerochromini, Cyphotilapiini, Perissodini and Trematocarini) (Konings 2019). The past evolutionary history of these hosts with hybridisation events and ongoing gene flow in the lake (Meyer et al. 2017; Irisarri et al. 2018) showed to be supported by morphological similarities in the haptoral apparatus and MCO features. Particularly, it is the case for C. casuarinus parasitizing members of Bathybatini (Pariselle et al. 2015a; Kmentová et al. 2016c), C. nshomboi parasitizing Boulengerochromini and Perissodini (Muterezi Bukinga et al. 2012 and study D), C. aspiralis and C. pseudoaspiralis parasitizing hosts belonging to Ectodini, and finally C. habluetzeli from species of

77

Cyphotilapiini and Ectodini (studies A and B). The last clade recovered by phylogenetic analysis harboured species of Cichlidogyrus from closely related modern tribes i.e., Haplochromini, Eretmodini and Tropheini. Following the LT cichlid phylogeny, Cichlidogyrus parasitizing tropheine representatives showed to be the most recent offshoot of the Tanganyika radiation (Ronco et al. 2021).

The second part of study E was focussed on the investigation of the role of various speciation processes in the diversification of Cichlidogyrus in LT. A cophylogenetic approach revealed a low contribution of cospeciation to the diversity of Cichlidogyrus in LT, whereas host switch and duplication were more common as previously found by Mendlová et al. (2012) for West African members of Cichlidogyrus. More specifically, most species of Cichlidogyrus from ancient cichlid lineages in LT i.e., C. casuarinus, C. habluetzeli and C. nshomboi showed a failure to diverge, with the exception for C. mulimbwai and C. mbirizei parasitizing Tylochromini and Oreochromini, respectively, which have successfully cospeciated. Lack of speciation observed for C. nshomboi was evidenced using DNA sequence data and geomorphometry of haptoral apparatus (Study D). At a lesser degree, failure to diverge was also recovered in few Cichlidogyrus from more recent LT tribes i.e., C. gillardinae and C. franswittei, each parasitizing congeneric hosts from Haplochromini (Muterezi Bukinga et al. 2012 and study E), and Tropheini (Van Steenberge et al. 2015 and study E), respectively. Overall, host dispersal and sympatric distribution that commonly facilitate ongoing gene flow among monogenean populations was suggested to support lack of species divergence observed in some Cichlidogyrus lineages in LT. Furthermore, incipient speciation as previously hypothesised for C. nshomboi based on nuclear and mitochondrial divergence (study D) or cryptic diversity can be suggested to explain the lack of Cichlidogyrus speciation in LT system. The latter phenomenon was rejected for the case of C. nshomboi as morphological differences in the size and shape of the sclerotized structures were evidenced (study D). In line with the findings of Vanhove et al. (2015) for Cichlidogyrus from Tropheini, few species parasitizing Ectodini, both monogenean species from Eretmodini and most species from Tropheini have cospeciated with their hosts, and then have undergone a diversification by duplications followed by host switching. Sympatric speciation and species flock case as a result of a small-scale radiation event can also be hypothesised to explain the occurrence of multiple congeneric species of Cichlidogyrus in the tropheine S. diagramma and congeneric ectodines and lamprologines belonging to two genera. For Cichlidogyrus parasitizing species of Haplochromini inhabiting East African freshwater systems, ancestral duplication in allopatric

78 conditions favoured by host dispersal throughout the lake, followed by a second parasite duplication have most likely contributed to the diversification of this genus.

In study E, we further investigated whether there is a relationship between parasite phylogeny and morphological adaptation based on haptor (hook pairs) and reproductive organs (sclerotization in the vagina). Following the delimitation of Vignon et al. (2011) for the haptoral groups of species outside of LT and the overview provided in study A regarding the hook configuration and sclerotization in the vagina, most species of Cichlidogyrus showed to belong to the haptoral group A characterised by the presence of larval (short) hooks and lacking sclerotization in their vagina. The haptoral configuration A was also recognised as being ancestral in West African species of Cichlidogyrus by Mendlová et al. (2012). Morphological adaptation to specific hosts (C. nshomboi parasitizing larger host B. microlepis) in LT was suggested for species of Cichlidogyrus representing remaining haptoral groups i.e., species with thicker and/or longer hooks (group D) with a sclerotized vagina, features rarely reported in LT Cichlidogyrus so far.

We discussed in the last part of study E local host specificity in Cichlidogyrus in the Tanganyika system and its potential link to selected morphological features of parasites i.e., hooks and vagina. In line with the study of Kmentová et al. (2016b), species of Cichlidogyrus in LT investigated in this study show narrow host specificity as the majority of species known so far represented strict specialists, whilst presence of generalists seems to be rare, mainly documented in benthopelagic and deep-water realms so far. This partially contradicts the pattern shown for West African species of Cichlidogyrus, which mostly parasitize congeneric hosts, thus the majority of West African Cichlidogyrus species exhibit intermediate host specificity (Mendlová et al., 2012). Our analyses suggest that strict specialists with larval hooks (group A) and a non-sclerotized vagina represent the ancestral character states in LT. This might indicate that decreasing host specificity is at least in some species related to morphological adaptation related to parasite attachment and reproduction i.e., there seems to be an evolutionary trend towards thicker and/or elongated hooks and a sclerotized vagina. This adaptation is apparently not restricted to LT as a few generalist species of Cichlidogyrus from West African non-cichlid hosts with their hooks configuration ‘escape’ from the haptoral delimitation of Vignon et al. (2011), and possess a sclerotized vagina (see overview in study A).

79

4. Conclusions and future perspectives

The presented thesis was focused on gill-specific dactylogyrideans of Cichlidogyrus, which represents the most specious genus known from African and Levantine cichlid fish (Pariselle and Euzet 2009). The main aims were (i) to evaluate the taxonomic diversity of Cichlidogyrus infecting the highly diverse cichlid flocks, endemic to the LT system, (ii) to investigate the intraspecific variability in monogenean species parasitizing LT cichlids with different ecologies, congeneric and phylogenetically unrelated cichlids, (ii) to infer Cichlidogyrus phylogeny and investigate the relationships between its members in LT and outside of LT, and finally (iii) to assess the evolutionary history and speciation patterns of Cichlidogyrus in LT.

Prior our research, LT cichlids were targeted for parasitological investigations, but only a limited number of host species representing a few tribes were studied. This limited the known species diversity of Cichlidogyrus in this environment. Prior to this thesis, only 24 species of Cichlidogyrus were described from only 20 cichlid hosts (Kmentová et al. 2016c), however, a total of 240 highly diverse cichlid species are known from LT (Ronco et al. 2020), and the representatives of at least eight cichlid tribes out of 16 (Ronco et al. 2021) were never investigated for gill flatworms. Gill samples belonging to 25 cichlid species inspected for monogenean flatworms showed to host 34 species of Cichlidogyrus. This has contributed to increase the knowledge on the morphological species richness of Cichlidogyrus by over 30%. Our studies A and B have contributed to enrich the list of Cichlidogyrus in this area by describing 15 new species from 13 cichlid species, members of seven cichlid tribes. Reinvestigation of the gills of some cichlid species like C. melanostigma, I. loocki, P. orthognathus and X. flavipinnis inhabiting the north-western LT lakeshores will allow in the future to formally describe additional species of Cichlidogyrus which we could characterise based on limited sclerotized structures features for lack of sufficient material collected during the fieldtrips (study B). Similarly, the undescribed species of Cichlidogyrus from members of Haplochromini (2 species) and Lamprologini (8 species) included in the phylogenetic and cophylogenetic analyses (study E) will be formally described soon. Further, it should be noted that Coptodonini with its sole non-endemic representative C. rendalli (Boulenger, 1896) in LT is awaiting to be investigated for the presence of parasites as it represents the only tribe whose members have never been sampled and studied for parasites. Similarly, cichlid species belonging to Limnochromini like G. permaxillaris (David, 1936) and auritus (Boulenger, 1901) need to be re-investigated for monogeneans, to formally describe the

80 species reported as new for science in Kmentová et al. (2016b). Unexpectedly, the LT assemblage of Cichlidogyrus from cichlid representatives of Ectodini and Tropheini revealed remarkable biogeographical diversity along the lake, most probably due to limited gene flow among populations. Of Ectodini, the endemic O. heterodonta (Poll & Matthes, 1962) widely distributed in the northern and central parts of LT is the sole species of Ophthalmotilapia Pellegrin, 1904 that has never been investigated for its parasite fauna. A study of gill ectoparasites on this cichlid host would provide additional data on the lake’s parasite species diversity. In contrast, few other cichlid hosts, mostly from Tropheini, have showed to host similar communities of Cichlidogyrus due to their lake-wide distribution resulting in ongoing gene flow between parasites. Therefore, first and new host and locality records including additional morphometrical data for Cichlidogyrus species will be published in the near future. It is about species of Cichlidogyrus parasitizing Boulengerochromini (Muterezi Bukinga et al. 2012), Haplochromini (Muterezi Bukinga et al. 2012), Perissodini, Tropheini (Gillardin et al. 2012; Van Steenberge et al. 2015), and finally Tylochromini (Muterezi Bukinga et al. 2012).

As evidenced for Cichlidogyrus parasitizing cichlids with contrasting dispersal abilities i.e., with or without dispersal ability (study C), or parasitizing congeners versus phylogenetically distinct cichlid tribes (study D), combining geomorphometrics and molecular data is an efficient method in highlighting population processes, phenotypic plasticity and morphological adaptation of sclerotized structures of Cichlidogyrus, mainly haptoral ones. With such approaches, the use of a single molecular marker which is highly conserved in monogenean species, as applied in study C, limited us to evaluate real patterns of variations in DNA of the investigated species. Additional parasite sampling including a wide range of LT localities, supplemented by the molecular analyses of multi-loci data would help us in the future to investigate the population structure of each of the studied cichlid species and their monogeneans across geographical scales. The employment of next-generation data analyses like RADseq, a method already used for parasitic organisms (Scarparo et al. 2021), seems necessary in the case of Cichlidogyrus to well estimate genetic differentiation among populations. While incipient speciation for Cichlidogyrus from boulengerochromine and perissodine hosts was supported by morphological and genetic differentiation evidenced by geomorphometric and DNA sequence data (nuclear and mitochondrial) in study D, monogenean specimens from Cyphotilapiini and Ectodini should also be subjected to such combined analyses in order to deeply explore potential hidden patterns of differentiation.

81

Furthermore, the application of molecular approaches provided the first comprehensive analysis of the phylogenetic position of LT species of Cichlidogyrus in relation to their relatives inhabiting distinct freshwater habitats in Africa, and their relationships within LT cichlid radiation. This allowed us to reveal the polyphyly of Cichlidogyrus from LT assemblage, just like it was found for their congeners in West Africa, and to re-evaluate the paraphyly of this genus at larger scale by including monogenean species known from more geographically and phylogenetically distant cichlid lineages. Using nuclear genes, our phylogenetic outputs highlighted the basal position of non-Lake Tanganyika species of Cichlidogyrus relative to their congeners inhabiting the lake itself and confirmed the earliest diverging position of Cichlidogyrus from tylochromine hosts. In addition, species of Cichlidogyrus reflected phylogenetic relatedness of their eretmodine, haplochromine and tropheine cichlid hosts, with Cichlidogyrus from tropheines as the most recent offshoot of the LT radiation, which was also shown for tropheine cichlids (see for instnace Salzburger et al. 2002b; Sturmbauer et al. 2003). Additionally, we demonstrated that the common evolutionary history of LT cichlids has left a ‘fingerprint’ in their gill flatworms i.e., host-specific monogeneans, as species of Cichlidogyrus parasitizing representatives belonging to most tribes that have seeded the radiation in the lake formed a monophyletic group. This was further supported by morphological similarities in haptoral and reproductive parts exhibited by Cichlidogyrus parasitizing cichlids from a range of tribes (Muterezi Bukinga et al. 2012; Pariselle et al. 2015a; Kmentová et al. 2016b, and references herein).

Vanhove et al. (2015) reported cospeciation as the main diversification event in LT cichlids-Cichlidogyrus but their study was focussed only on the monogenean diversity of tropheine cichlids. Using representatives of Cichlidogyrus from all cichlid tribes occurring in LT, except Limnochromini and Coptodonini, strong coevolutionary structure was found in this system. We revealed that monogeneans from ancient cichlid lineages have mostly failed to diverge due to continuous gene flow among parasite populations in their specific ecological niches, while sympatric and allopatric speciation by duplication and/or host switching have occurred in almost all lineages, whilst cospeciation has the lowest contribution in the diversification of Cichlidogyrus in LT. The cospeciation was mostly evidenced in Cichlidogyrus communities from Tropheini as reported by Vanhove et al. (2015). The deep investigation of coevolutionary patterns of the cichlid-Cichlidogyrus system on a larger phylogenetic coverage requires more host taxa representing the distinct cichlid tribes. Indeed, considerable molecular data for Cichlidogyrus within a single tribe are still missing, as is the

82 case of Lamprologini. We included in our phylogenetic and cophylogenetic analyses the monogenean species recovered from only two lamprologine species, however, this tribe comprises about 40% of the lake’s species i.e., Lamprologini includes the most endemic species-rich and dominant cichlid fauna (Koblmüller et al. 2008b; Konings 2019). As data on the mitochondrial genome for some species of Cichlidogyrus are available (Vanhove et al. 2018), the use of phylogenomic approaches might provide better resolution for the phylogenetic relationships among species of Cichlidogyrus and their evolutionary history.

Species of Cichlidogyrus parasitizing phylogenetically closely related Lake Tanganyika cichlid hosts were morphologically highly similar as observed for their West African congeners (Pariselle and Euzet 2009). Mapping of haptoral morphology in terms of hooks and sclerotization in the vagina onto parasite phylogeny has shown that larval hook size and presence of a non-sclerotized vagina were the ancestral character states, and acquisition of thicker/longer hooks and the sclerotization in the female reproductive organ represented the derived character states. From a host specificity perspective, however, LT species of Cichlidogyrus in contrast to West African species exhibited narrow host specificity i.e., strict specialist lifestyle as an ancestral state, and a decrease of specificity toward generalist states was derived. Exhaustive taxon sampling with wider geographic coverage is necessary to confirm the non-‘dead-end’ hypothesis in LT Cichlidogyrus as previously shown for West African Cichlidogyrus (Mendlová and Šimková 2014). In a similar context, the existence of mostly strict specialist species of Cichlidogyrus in LT and the weak number of representatives exhibiting lower degrees of specificity did not allow us to map this parameter onto parasite phylogeny to avoid biased results. Finally, we reported in study E that there could be an evolutionary importance of the specialisation in Cichlidogyrus (related to parasite attachment and potentially also to reproduction), together with morphological adaptation as generalist species have revealed to possess different hooks configuration and a sclerotized vagina, two features never grouped in a specialist species. The structural diversity in Cichlidogyrus remains to be deeply investigated to perhaps accurately identify the ‘boundaries’ between haptoral (four groups in terms of hooks) and reproductive (two types of vagina) groups.

83

References Agnèse, J. F., Adépo-Gourène, B., Abban, E.K. & Fermon, Y. (1997). Genetic differentiation among natural populations of the Nile tilapia Oreochromis niloticus (Teleostei, Cichlidae). Heredity, 79(Pt 1), 88–96. Aguirre-Fey, D., Benítez-Villa, G.E., Pérez-Ponce de León, G. & Rubio-Godoy, M. (2015). Population dynamics of Cichlidogyrus spp. and Scutogyrus spp. (Monogenea) infecting farmed tilapia in Veracruz, México. Aquaculture, 443, 11–15. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. Anderson, J.B., Warny, S., Askin, R.A., et al. (2011). Progressive Cenozoic cooling and the demise of Antarctica’s last refugium. Proceedings of the National Academy of Sciences of the United States of America, 108(28), 11356–11360. Anderson, T.J,C., Haubold, B., Williams, J.T. et al. (2000). Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Molecular Biology and Evolution, 17(10), 1467–148. Appeltans, W., Ahyong, S.T., Anderson, G. et al. (2012). The magnitude of global marine species diversity. Current Biology, 22(23), 2189–2202. Aryeetey-Attoh, S. (2009). Geography of Sub-Saharan Africa. 3rd edition. Prentice Hall. Pearson. Baker, S. J. K. & Kalinga, O. J. (2009). East African lakes. In: Encycl. Br. https://www.britannica.com/place/East-African-lakes. Accessed 7 Mar 2020. Baric, S., Salzburger, W. & Sturmbauer, C. (2003). Phylogeography and evolution of the Tanganyikan cichlid genus Tropheus based upon mitochondrial DNA sequences. Journal of Molecular Evolution, 56, 54–68. Barlow, G. W. (2000). The cichlid fishes: nature’s grand experiment in evolution. New Edition. Basic Books, New-York. Baron, S. (1996). Medical Microbiology. 4th edition. Galveston (TX). University of Texas Medical Branch at Galveston. Beauchamp, R. S. A. (1939). Hydrology of Lake Tanganyika. Internationale Revue der gesamten Hydrobiologie und Hydrographie, 39(3-4), 316–353. Beer, A., Ingram, T. & Randhawa, H. S. (2019). Role of ecology and phylogeny in determining tapeworm assemblages in skates (Rajiformes). Journal of Helminthology, 93(6), 738–751. Benovics, M., Desdevises, Y., Šanda, R., Vukić, J. & Šimková, A. (2020a). Cophylogenetic

84

relationships between Dactylogyrus (Monogenea) ectoparasites and endemic cyprinoids of the north-eastern European peri-Mediterranean region. Journal of Zoological Systematics and Evolutionary Research, 58(1), 1–21. Benovics, M., Desdevises, Y., Šanda R, Vukić, J. & Šimková, A. (2020b). High diversity of fish ectoparasitic monogeneans (Dactylogyrus) in the Iberian Peninsula: a case of adaptive radiation? Parasitology, 147(4), 418–430. Betancur, R. R., Broughton, R. E., Wiley, E. O., et al. (2013). The tree of life and a new classification of bony fishes. PLoS Currents, ecurrents.tol.53ba26640df0ccaee75bb165c8c26288. Betancur, R. R., Wiley, E.O., Arratia, G. et al. (2017). Phylogenetic classification of bony fishes. BMC Evolutionary Biology, 17: 162. Beveridge, M. C. M.& Mc Andrew, B. J. (2000). Tilapias: Biology and Exploitation. Chapman and Hall. Springer, Dordrecht. Bilong Bilong, C. F. (1988). Enterogyrus malmbergi n. sp. (Monogenea-) parasite de l’estomac du Cichlidae Tilapia nilotica Linné, 1757 au Sud- Cameroun. Annales de la Faculté des Sciences de Yaoundé, Biologie-Biochimie, 5, 51–58. Bilong Bilong, C. F., Birgi, E. & Euzet, L. (1994). Urogyrus cichlidarum gen. nov., sp. nov., Urogyridae fam. nov., monogène parasite de la vessie urinaire de poissons cichlidés au Cameroun. Canadian Journal of Zoology, 72, 561–566. Bilong Bilong, C. F., Birgi, E. & Lambert, A. (1989). Enterogyrus melenensis n. sp. (Monogenea, Ancyrocephalidae), parasite stomacal de Hemichromis fasciatus Peters, 1857 (Téléostéen, Cichlidae) du Sud-Cameroun. Revue de Zoologie Africaine, 103, 99– 105. Bilong Bilong, C. F. & Euzet, L. (1995). Onchobdella bopeleti n. sp. (Monogenea, Ancyrocephalidae) parasite branchial de Hemichromis fasciatus (Peters, 1857) (Cichlidae). Journal Africain de Zoologie, 109, 253–258. Bilong Bilong, C. F., Euzet, L. & Birgi, E. (1996). Monogenean stomach parasites of cichlid fishes from Cameroon: two new species of the genus Enterogyrus Paperna, 1963 (Ancyrocephalidae). Systematic Parasitology. 34, 37–42. Birgi, E. & Euzet, L. (1983). Monogènes parasites des poissons des eaux douces du Cameroun. Présence des genres Cichlidogyrus et Dactylogyrus chez Aphyosemion (Cyprinodontidae). Bulletin de la Société Zoologique de France, 108, 101–106. Birgi. E. & Lambert, A. (1987). À propos de Cichlidogyrus euzeti Birgi & Lambert, 1986 décrit au Cameroun chez un Nandidae Polycentropsis abbreviata Boulenger, 1901. Annales de

85

Parasitologie Humaine et Comparée, 62, 103. Birgi, E. & Lambert, A. (1986). Présence chez un Nandidae (Téléostéen), Polycentropsis abbreviata Boulenger, 1901, du genre Cichlidogyrus (Monogenea, Monopisthocotylea, Ancyrocephalidae). description de Cichlidogyrus nandidae n. sp. Annales de Parasitologie Humaine et Comparée, 61, 521–528. Blasco-Costa, I., Míguez-Lozano, R., Sarabeev, V. & Balbuena, J. A. (2012). Molecular phylogeny of species of Ligophorus (Monogenea: Dactylogyridae) and their affinities within the Dactylogyridae. Parasitology International, 61(4), 619–627. Bloom, D. D., Weir, J. T., Piller, K. R. & Lovejoy, N. R. (2013). Do freshwater fishes diversify faster than marine fishes? A Test using state-dependent diversification analyses and molecular phylogenetics of new world silversides (Atherinopsidae). Evolution, 67(7), 2040–2057. Boeger, W. A. & Kritsky, D. C. (1997). Coevolution of the Monogenoidea (Platyhelminthes) based on a revised hypothesis of parasite phylogeny. International Journal of Parasitology, 27, 1495–1511. Boeger, W. A. & Kritsky, D. C. (1993). Phylogeny and a revised classification of the Monogenoidea Bychowsky, 1937 (Platyhelminthes). Systematic Parasitology, 26, 1–32. Bondad-Reantaso, M. & Arthur, J. (1990). The parasites of Nile Tilapia Oreochromis niloticus (L.) in the Philippines, including an analysis of changes in the parasite fauna of cultured tilapia from fry to marketable size. In: Hirano, R., Hanyu, I. (eds). Proceedings of the Second Asian Fisheries Forum. Asian Fisheries Society, Manila. pp. 729–734 Boulenger, G. A. (1898). A revision of the African and Syrian fishes of the family Cichlida.– Part I. Proceedings of the Zoological Society of London, 66(2), 132–152. Brandstätter, A., Salzburger, W. & Sturmbauer, C. (2005). Mitochondrial phylogeny of the Cyprichromini, a lineage of open-water cichlid fishes endemic to Lake Tanganyika, East Africa. Molecular Phylogenetics and Evolution 34, 382–391. Brooks, D. R. (1982). Higher level classification of parasitic platyhelminths and fundamentals of cestode classification. In: Mettrick, D. F. & Desser, S. S. (ed.) Parasites — Their world and ours, Elsevier Biomedical Press, Amsterdam. pp. 189–193 Brooks, D. R. (1979). Testing the context and extent of host-parasite coevolution. Systematic Zoology, 28(3), 299–307. Brooks, D. R. & McLennan, D. A. (1991). Phylogeny, ecology and behavior: a research program in comparative biology, The Univer. London. Brooks, D. R. & McLennan, D. A. (1993). Parascript: parasites and the language of evolution.

86

1st edition. Washington, DC: Smithsonian Institution Press. Brown, J. H. (1984). On the relationship beween abundance and distribution of species. The American Naturalist, 124(2), 255–279. Buchmann, K. & Bresciani, J. (2006). Monogenea (Phylum Platyhelminthes). In: Fish Diseases and Disorders, Volume 1, 2nd edition. CABI, Oxford, UK. pp. 297-344. Bueno-Silva, M., Boeger, W. A. & Pie, M. R. (2011). Choice matters: incipient speciation in Gyrodactylus corydori (Monogenoidea: Gyrodactylidae). International Journal of Parasitology, 41(6), 657–667. Burgis, M. J., Darlington, J. P., Dunn, I. G. et al. (1973). The biomass and distribution of organisms in Lake George, Uganda. Proceedings of the Royal Society, 184, 271–298. Bush, A. O., Lafferty, K. D., Lotz, J. M. & Shostak, A. W. (1997). Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology, 83, 575–583. Cable, J. & Harris, P. D. (2002). Gyrodactylid developmental biology: historical review, current status and future trends. Internatioanl Journal of Parasitology, 32:255–280. Caira, J. N. & Littlewood, D. T. J. (2013). Worms, Platyhelminthes. In: Encyclopedia of Biodiversity. 2nd edition. Academic Press, Elsevier, Amsterdam. pp. 863–899. Caña-Bozada, V., Llera-Herrera, R., Fajer-Ávila, E. J. & Morales-Serna, F. N. (2021). Mitochondrial genome of Scutogyrus longicornis (Monogenea: Dactylogyridea), a parasite of Nile tilapia Oreochromis niloticus. Parasitology International, 81, 102281. Carpenter, S. R., Stanley, E. H. & Vander Zanden, M. J. (2011). State of the world’s freshwater ecosystems: physical, chemical, and biological changes. Annual Review of Environment and Resources, 36, 75–99. Cavin, L. (2017). Evolutionary histories of freshwater fishes. In: Cavin L. (ed.), 250 Million Years of evolutionary History. Elsevier, pp. 53–125. Chakrabarty, P. (2004). Cichlid biogeography: comment and review. Fish and Fisheries, 5, 97– 119 Charleston, M. A. (2012). TreeMap 3b. A Java program for cophylogeny mapping. Available from: http://sydney.edu.au/engineering/it/_mcharles/. Charleston, M. A. & Robertson, D. L. (2002). Preferential host switching by primate lentiviruses can account for phylogenetic similarity with the primate phylogeny. Systematic Biololgy, 51, 528–535. Chorowicz, J. (2005). The East African rift system. Journal of African Earth Sciences, 43(1-2), 379–410. Choudhury, A. & Dick, T. A. (2000). Richness and diversity of helminth communities in

87

tropical freshwater fishes: empirical evidence. Journal of Biogeography, 27(4), 935–956. Chubb, J. C., Ball, M. A. & Parker, G. A. (2010). Living in intermediate hosts: evolutionary adaptations in larval helminths. Trends in Parasitology, 26(2), 93–102. Clabaut, C., Bunje, P. M. E., Salzburger, W. & Meyer, A. (2007). Geometric morphometric analyses provide evidence for the adaptive character of the Tanganyikan cichlid fish radiations. Evolution, 61(3), 560–78. Clabaut, C., Salzburger, W. & Meyer, A. (2005). Comparative phylogenetic analyses of the adaptive radiation of Lake Tanganyika cichlid fish: nuclear sequences are less homoplasious but also less informative than mitochondrial DNA. Journal of Molecular Evolution, 61, 666–681. Cohen, A. S., Lezzar, K. E., Tiercelin, A. J. J. & Soreghan, M. (1997a) New palaeogeographic and lake-level reconstructions of Lake Tanganyika: implications for tectonic, climatic and biological evolution in a rift lake. Basin Reseach, 9, 107–132. Cohen, A. S., Soreghan, M. J. & Scholz, C. A. (1993). Estimating the age of formation of lakes - an example from Lake Tanganyika, East-African Rift System. Geology, 21, 511–514. Cohen, A. S., Stone, J. R., Beuning, K. R. M., et al. (2007). Ecological consequences of early late Pleistocene megadroughts in tropical Africa. Proceedings of the National Academy of Sciences of the United States of America, 104(42), 16422–16427. Cohen, A. S., Talbot, M. R., Awramik, S. M., Dettman, D. L. & Abell, P. (1997b). Lake level and paleoenvironmental history of Lake Tanganyika, Africa, as inferred from late Holocene and modern stromatolites. Geological Society of America Bulletin, 109, 444– 460. Conow, C., Fielder, D., Ovadia, Y. & Libeskind-Hadas, R. (2010). Jane: a new tool for the cophylogeny reconstruction problem. Algorithms for Molecular Biology 5, 16. Cooper, N., Griffin, R., Franz, M., Omotayo, M., Nunn, C. L. & Fryxell, J. (2012). Phylogenetic host specificity and understanding parasite sharing in primates. Ecological Letters, 15, 1370– 1377. Cribb, T. H., Chisholm, L. A. & Bray, R. A. (2002). Diversity in the Monogenea and Digenea: does lifestyle matter? International Journal of Parasitology, 32, 321–328. Criscione, C. D., Cooper, B. & Blouin, M. S, (2006). Parasite genotypes identify source populations of migratory fish more accurately than fish genotypes. Ecology. 87, 823–828. Curry-Lindahl, K., Fryer, G. & Iles, T. D. (1976). The cichlid fishes of the Great Lakes of Africa: their biology and evolution. TFH Publications. Da Graça, R. J., Fabrin, T. M. C., Gasques, L. S. et al. (2018). Topological congruence between

88

phylogenies of Anacanthorus spp. (Monogenea: Dactylogyridae) and their Characiformes (Actinopterygii) hosts: a case of host-parasite cospeciation. PLoS ONE, 13(3): e0193408. Danley, P. D., Husemann, M., Ding, B. et al. (2012). The impact of the geologic history and paleoclimate on the diversification of East African Cichlids. International Journal of Evolutionary Biology, 2012:20. Danley, P. D. & Kocher, T. D. (2001). Speciation in rapidly diverging systems: lessons from . Molecular Ecology, 10(5), 1075–1086. Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. (2012). JModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9, 772. Darwall, W. R., Smith, K., Allen, D. et al. (2011). The diversity of life in African freshwaters: underwater, under threat. An analysis of the status and distribution of freshwater species throughout mainland Africa. 4th edition. World Conservation Union. Dávidová, M., Jarkovský, J., Matějusován I. & Gelnar, M. (2005). Seasonal occurrence and metrical variability of Gyrodactylus rhodei Žitňan 1964 (Monogenea, Gyrodactylidae). Parasitology Research, 95(6), 398–405. De Vienne, D. M., Giraud, T. & Shykoff, J. A. (2007). When can host shifts produce congruent host and parasite phylogenies? A simulation approach. Journal of Evolutionary Biology, 20, 1428–1438. De Vienne, D. M., Refrégiern G., López-Villavicencion M. et al. (2013). Cospeciation vs host- shift speciation: methods for testing, evidence from natural associations and relation to coevolution. New Phytologist, 198, 347–385. Degens, E. T., Von Herzen, R. P. & Wong, H. K. (1971). Lake tanganyika: water chemistry, sediments, geological structure. Naturwissenschaften, 58, 229–241. Delvaux, D. (1995). Age of Lake Malawi (Nyasa) and water level fluctuations. Musée Royal d'Afrique Centrale, Belgique, Tervuren, 1994, 99–108. Desdevises, Y., Morand, S., Jousson, O. & Legendre, P. (2002a). Coevolution between Lamellodiscus (Monogenea: Diplectanidae) and Sparidae (Teleostei): the Study of a complex host‐parasite system. Evolution, 56, 2459–2471. Desdevises, Y., Morand, S. & Legendre, P. (2002b). Evolution and determinants of host specificity in the genus Lamellodiscus (Monogenea). Biological Journal of the Linnean Society, 77(4), 431–443. Dobson, A., Lafferty, K. D. & Kuris, A. M., Hechinger, R. F. & Jetz, W. (2008). Homage to Linnaeus: how many parasites? How many hosts? Proceedings of the National Academy of Sciences of the United States of America, 105, 11482–11489.

89

Domingues, M. V. & Boeger, W. A. (2008). Phylogeny and revision of Diplectanidae Monticelli, 1903 (Platyhelminthes: Monogenoidea). Zootaxa, 1698, 1–40. Dos Santos, Q. M., Maina, J. N. & Avenant-Oldewage, A. (2019). Gyrodactylus magadiensis n. sp. (Monogenea, Gyrodactylidae) parasitising the gills of (Perciformes, Cichlidae), a fish inhabiting the extreme environment of , Kenya. Parasite, 26, 76. Dossou, C. (1982). Parasites de poissons d’eau douce du Bénin. III– Espèces nouvelles du genre Cichlidogyrus (Monogenea) parasites de Cichlidae. Bulletin de l'Institut Fondamental d'Afrique Noire, 44, 295–322. Dossou, C & Birgi, E. (1984). Monogènes parasites d’Hemichromis fasciatus Peters, 1857 (Teleostei, Cichlidae). Annales des Sciences Naturelles et Zoologiques, 6, 101–109. Douëllou, L. (1993). Monogeneans of the genus Cichlidogyrus Paperna, 1960 (Dactylogyridae: Ancyrocephalinae) from cichlid fishes of Lake Kariba (Zimbabwe) with descriptions of five new species. Systematic Parasitology, 25, 159–186. Dudgeon, D., Arthington, A. H., Gessner, M. O. et al. (2006). Freshwater biodiversity: importance, threats, status and conservation challenges. Biological reviews of the Cambridge Philosophical Society, 81(2), 163–82. Duftner, N., Koblmüller, S. & Sturmbauer, C. (2005). Evolutionary relationships of the Limnochromini, a tribe of benthic deepwater cichlid fish endemic to Lake Tanganyika, East Africa. Journal of Molecular Evolution, 60, 277–289. Dunn, C. W., Hejnol, A., Matus, D. Q. et al. (2008). Broad phylogenomic sampling improves resolution of the animal tree of life. Nature, 452, 745–749. Dunz, A. R. (2012). Revision of the substrate brooding “Tilapia” (Tilapia Smith, 1840 and related taxa), (Teleostei: Perciformes: Cichlidae). Ph.D. dissertation. Ludwig- Maximilians-University, Munich. Dunz, A. R. & Schliewen, U. K. (2010). Description of a Tilapia (Coptodon) species flock of Lake Ejagham (Cameroon), including a redescription of Tilapia deckerti Thys van den Audenaerde, 1967 (Perciformes, Cichlidae). Spixiana, 33(2), 251–280. Dunz, A. R. & Schliewen, U. K. (2013). Molecular phylogeny and revised classification of the haplotilapiine cichlid fishes formerly referred to as “Tilapia”. Molecular Phylogenetics and Evolution, 68, 64–80. Egger, B., Lapraz, F., Tomiczek, B., et al. (2015). A transcriptomic-phylogenomic analysis of the evolutionary relationships of flatworms. Current Biology, 25(10), 1347–1353. Ehlers, U. (1984), Das Phylogenetische System der Plathelminthes. Transactions of the

90

American Microscopical Society, 105(4), 347. Ergens, R. (1981). Nine species of the genus Cichlidogyrus Paperna, 1960 (Monogenea: Ancyrocephalinae) from Egyptian fishes. Folia Parasitologica, 28, 205–214. Ergens, R. & Gelnar, M. (1985). Experimental verification of the effect of temperature on the size of hard parts of opisthaptor of Gyrodactylus katharineri Malmberg, 1964. Folia Parasitologica, 32, 377–380. Ergens, R. & Lom, J. (1970). Causative agents of fish diseases, Academia. Prague. Esch GW, Fernández, C. (1993). A functional biology of parasitism. Chapman and Hall. Euzet, L. & Combes, C. (1998). The selection of habitats among the monogenea. International Journal for Parasitology, 28(10), 1645–1652. Fahrenholz, H. (1913). Ectoparasiten und abstammungslehre Zoologischer Anzeiger, 41, 371– 374. Farias, I. P., Ortí, G. & Meyer, A. (2000). Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes. Journal of Experimental Zoology, 288, 76–92. Farias, I. P., Ortí, G. Sampaio, I. et al. (2001). The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes. Journal of Molecular Evolution, 53, 89–103. Farris, J. (1995). Testing significance of incongruence. Cladistics, 10, 315–319. Fouchy, K., McClain, M. E., Conallin, J. & O’Brien, G. (2019) .Multiple stressors in African freshwater systems. In: Sabater, S., Elosegi A. and Ludwig, R. (eds.), Multiple stressors in river ecosystems: status, impacts and prospects for the future. Elsevier. pp. 179–191. Franceschini, L., Zago, A.C., Müller, M. I. et al. (2018). Morphology and molecular characterization of Demidospermus spirophallus n. sp., D. prolixus n. sp. (Monogenea: Dactylogyridae) and a redescription of D. anus in siluriform catfish from Brazil. Journal of Helminthology, 92(2), 228–243. Fricke, R., Eschmeyer, W. & Fong, J. D. (2021). Catalogue of Fishes: Genera, Species, References. In: http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.as p. Electronic version accessed on 13 January 2021. Friedman, M., Keck, B. P., Dornburg, A., et al. (2013). Molecular and fossil evidence place the origin of cichlid fishes long after Gondwanan rifting. Proceedings of the Royal Society, 280, 20131733–20131733. Froese, R & Pauly, D. (2021). FishBase. World Wide Web electronic publication. http://www.fishbase.org/search.php. Version 01/2021. Accessed Jan 2021.

91

Fryer, G. (1959). The trophic interrelationships and ecology of some littoral communities of Lake Nyasa with especial reference to the fishes, and a discussion of the evolution of a group of rock‐frequenting Cichlidae. Proceedings of the Zoological Society of London, 132(2), 153–281. Garamszegi, L. Z. (2009). Patterns of co-speciation and host switching in primate malaria parasites. Malaria Journal, 8, 110. Gaston, K. J. & Lawton, J. H. (1990). Effects of scale and habitat on the relationship between regional distribution and local abundance. Oikos, 58(3), 329–335. Genner, M. J., Seehausen, O., Lunt, D. H. et al. (2007). Age of cichlids: new dates for ancient lake fish radiations. Molecular Biology and Evolution, 24, 1269–1282. Genner, M. J. & Turner, G. F. (2012). Ancient hybridization and phenotypic novelty within lake Malawi’s cichlid fish radiation. Molecular Biology and Evolution, 29(1), 195–206. Geraerts, M., Muterezi Bukinga, F., Vanhove, M. P. M. et al. (2020). Six new species of Cichlidogyrus Paperna, 1960 (Platyhelminthes: Monogenea) from the gills of cichlids (Teleostei: Cichliformes) from the Lomami River Basin (DRC: Middle Congo). Parasites and Vectors, 9;13(1):187. Gillardin, C., Vanhove, M. P. M., Pariselle, A. et al. (2012). Ancyrocephalidae (Monogenea) of Lake Tanganyika: II: description of the first Cichlidogyrus spp. parasites from Tropheini fish hosts (Teleostei, Cichlidae). Parasitology Research, 110, 305–313. Good, P. (2001). Permutation Tests: a practical guide to resampling methods for testing hypotheses. 2nd edition. New York. Greenwood, P. H. (1978). A review of the pharyngeal apophysis and its significance in the classification of African cichlid fishes. Bulletin of the British Museum National History (Zoology), 33(5), 297–323. Greenwood, P. H. (1979). Towards a phyletic classification of the ‘genus’ Haplochromis (Pisces, Cichlidae) and related taxa. Part 1. Bulletin of the British Museum National History (Zoology), 39, 1–101. Greenwood, P. H. (1987). The genera of pelmatochromine fishes (Teleostei, Cichlidae). A phylogenetic review. Bulletin of the British Museum (Natural History), 53, 139–203. Grégoir, A. F., Hablützel, P. I., Vanhove, M. P. M. et al. (2015) A link between host dispersal and parasite diversity in two sympatric cichlids of Lake Tanganyika. Freshwater Biology, 60, 323–335. Guindon, S., Dufayard, J. F., Lefort, V. et al. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic

92

Biology, 59, 307–32. Gussev, A. V. (1983). The method of collecting and processing of fish parasitic monogenean material. Nauk Leningr. pp. 48 [in Russian]. Hablützel, P. I., Grégoir, A. F., Vanhove, M. P. M. et al. (2016). Weak link between dispersal and parasite community differentiation or immunogenetic divergence in two sympatric cichlid fishes. Molecular Ecology, 25(21), 5451–5466. Hafner, M. S. & Nadler, S. A. (1988). Phylogenetic trees support the coevolution of parasites and their hosts. Nature, 332(6161), 258–259. Hafner, M. S., Sudman, P. D., Villablanca, F. X. et al. (1994). Disparate rates of molecular evolution in cospeciating hosts and parasites. Science, 265, 1087–1090. Hahn, C., Fromm, B. & Bachmann, L. (2014). Comparative genomics of flatworms (Platyhelminthes) reveals shared genomic features of ecto- and endoparastic neodermata. Genome Biology and Evolution, 6(5), 1105–1117. Hahn, C., Weiss, S. J., Stojanovski, S. & Bachmann, L. (2015). Co-speciation of the ectoparasite gyrodactylus teuchis (Monogenea, Platyhelminthes) and its salmonid hosts. PLoS ONE, 10: e0127340. Hammer, Ø., Harper, D. & Ryan, P. (2001). Past: paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 9. Harris, P. D., Shinn, A. P., Cable, J. & Bakke, T. A. (2004). Nominal species of the genus Gyrodactylus von Nordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species. Systematic Parasitology, 59, 1–27. Haywood, A. M., Valdes, P. J. & Sellwood, B. W. (2000). Global scale palaeoclimate reconstruction of the middle Pliocene climate using the UKMO GCM: initial results. Global and Planetary Change, 25(3-4), 239–256. Hertel, L. A. (1993). Excretion and osmoregulation in the flatworms. Transactions of the American Microscopical Society, 112(1), 10–17. Hooge, M. D. (2001). Evolution of body-wall musculature in the Platyhelminthes (Acoelomorpha, Catenulida, Rhabditophora). Journal of Morphology, 249(3), 171–94. Humason, G. L. (1979). Animal tissue techniques. Freeman and Company, San Francisco. Humphery-Smith, I. (1989). The evolution of phylogenetic specificity among parasitic organisms. Parasitology Today, 5, 385–387. Huyse, T., Audenaert, V. & Volckaert, F. A. M. (2003). Speciation and host-parasite relationships in the parasite genus Gyrodactylus (Monogenea, Platyhelminthes) infecting gobies of the genus Pomatoschistus (Gobiidae, Teleostei). International Journal for

93

Parasitology, 33(14), 1679–1689. Huyse, T. & Volckaert, F. A. M. (2002). Identification of a host-associated species complex using molecular and morphometric analyses, with the description of Gyrodactylus rugiensoides n. sp. (Gyrodactylidae, Monogenea). International Journal for Parasitology. 32, 907–919. Huyse, T. & Volckaert, F. A. M. (2005). Comparing host and parasite phylogenies: Gyrodactylus flatworms jumping from Goby to Goby. Systematic Biology, 54, 710–718. IBM (2020). IBM Corp. Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp. Irisarri, I., Singh, P., Koblmüller, S. et al. (2018). Phylogenomics uncovers early hybridization and adaptive loci shaping the radiation of Lake Tanganyika cichlid fishes. Nature Communications, 9, 3159. Jefferies, M. J. & Lawton, J. H. (1984), Enemy free space and the structure of ecological communities. Biological Journal of the Linnean Society, 23(4), 269–286. Jiménez-García, M. I., Vidal-Martínez, V. M. & López-Jiménez, S. (2001). Monogeneans in introduced and native cichlids in México: evidence for transfer. Journal of Parasitology, 87(4), 907–909. Johnson, K. P., Adams, R. J., Page, R. D. M. & Clayton, D. H. (2003). When do parasites fail to speciate in response to host speciation? Systematic Biology, 52, 37–47. Johnson, T. C., Scholz, C. A., Talbot, M. R. et al. (1996). Late pleistocene desiccation of Lake Victoria and rapid evolution of cichlid fishes. Science, 273(5278), 1091–1093. Jorissen, M. W. P., Pariselle, A., Huyse, T. et al. (2017). Diversity and host specificity of monogenean gill parasites (Platyhelminthes) of cichlid fishes in the Bangweulu-Mweru ecoregion. Journal of Helminthology, 92(4), 417–437. Jorissen, M. W. P., Pariselle, A., Huyse, T. et al. (2018). Six new dactylogyrid species (Platyhelminthes, Monogenea) from the gills of cichlids (Teleostei, Cichliformes) from the Lower Congo Basin. Parasite, 25, 64. Jovelin, R. & Justine, J., (2001). Phylogenetic relationships within the polyopisthocotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences. International Journal for Parasitology, 31(4), 393–401. Justine, J. (1998). Non-monophyly of the monogeneans? International Journal for Parasitology, 28, 1653–1657. Kadlec, D., Šimková, A. & Gelnar, M. (2003). The microhabitat distribution of two Dactylogyrus species parasitizing the gills of the barbel, Barbus barbus. Journal of

94

Helminthology, 77, 317–325. Katongo, C., Koblmüller, S., Duftner, N. et al. (2007). Evolutionary history and biogeographic affinities of the serranochromine cichlids in Zambian rivers. Molecular Phylogenetics and Evolution. 45(1), 326–38. Katongo, C., Koblmüller, S., Duftner, N. et al. (2005). Phylogeography and speciation in the Pseudocrenilabrus philander species complex in Zambian Rivers. Hydrobiologia, 542(1), 221–233. Katongo, C., Seehausen, O. & Snoeks, J. (2017). A new species of Pseudocrenilabrus (Perciformes: Cichlidae) from Lake Mweru in the Upper Congo River System. Zootaxa. 4237(1), 181–190. Kearn, G. C. (1994). Evolutionary expansion of the Monogenea. International Journal for Parasitology, 24, 1227–1271. Kearn, G. C. (2004). Leeches, lice and lampreys. A natural history of skin and gill parasites of fishes. Dordrecht. Springer-Verlag, New York. Kearn, G. C. & Evans-Gowing, R. (1998). Attachment and detachment of the anterior adhesive pads of the monogenean (platyhelminth) parasite Entobdella soleae from the skin of the common sole (Solea solea). International Journal for Parasitology, 28(10), 1583–1593. Kennedy, C. R. (2006). Ecology of the Acanthocephala. Cambridge University Press. Kevrekidis, C., Valtl, M., Penk, S. B. R. et al. (2019). Rebekkachromis nov. gen. from the middle–upper Miocene (11 MYA) of Central Kenya: the oldest record of a haplotilapiine cichlid fish. Hydrobiologia, 832, 39–64. Khang, T. F., Soo, O. Y. M., Tan, W. B. & Lim, L. H. S. (2016). Monogenean anchor morphometry: systematic value, phylogenetic signal, and evolution. PeerJ, 4, e1668. Kingston, J. D., Fine, Jacobs, B., Hill, A. & Deino, A. (2002). Stratigraphy, age environments of the late Miocene Mpesida Beds, Tugen Hills, Kenya. Journal of Human Evolution, 42(1), 95-116. Kirchberger, P. C., Sefc, K. M., Sturmbauer, C. & Koblmüller, S. (2012). Evolutionary history of Lake Tanganyika’s predatory deepwater cichlids. International Journal of Evolutionar Biology, 2012: 716209. Klett, V. & Meyer, A. (2002) What, if anything, is a Tilapia? - mitochondrial ND2 phylogeny of tilapiines and the evolution of parental care systems in the African cichlid fishes. Molecular Biology and Evolution, 19, 865–883. Klingenberg, C. P. (2011). MorphoJ: an integrated software package for geometric morphometrics. Molecular Ecology Resources, 11, 353–357.

95

Kmentová, N., Gelnar, M., Koblmüller, S. & Vanhove, M. P. M. (2016a). Deep-water parasite diversity in Lake Tanganyika: description of two new monogenean species from benthopelagic cichlid fishes. Parasites and Vecrtors, 9, 426. Kmentová, N., Gelnar, M., Mendlova, M., et al. (2016b). Reduced host-specificity in a parasite infecting non-littoral Lake Tanganyika cichlids evidenced by intraspecific morphological and genetic diversity. Scientific Reports, 6: 39605. Kmentová, N., Koblmüller, S., Van Steenberge, M. et al. (2019). Failure to diverge in African Great Lakes: the case of Dolicirroplectanum lacustre gen. nov. comb. nov. (Monogenea, Diplectanidae) infecting latid hosts. Journal of Great Lakes Research, 46(5), 1113–1130. Kmentová, N., Koblmüller, S., Van Steenberge, M. et al. (2020). Weak population structure and recent demographic expansion of the monogenean parasite Kapentagyrus spp. infecting clupeid fishes of Lake Tanganyika, East Africa. International Journal for Parasitology, 50(6-7), 471–486. Koblmüller, S., Duftner, N., Katongo, C. et al. (2005). Ancient divergence in bathypelagic Lake Tanganyika deepwater cichlids: mitochondrial phylogeny of the tribe Bathybatini. Journal of Molecular Evolution, 60, 297–314. Koblmüller, S., Egger, B., Sturmbauer, C. & Sefc, K. M. (2007). Evolutionary history of Lake Tanganyika’s scale-eating cichlid fishes. Molecular Phylogenetics and Evolution, 44(3), 1295–1305. Koblmüller, S., Odhiambo, E. A., Sinyinza, D. et al. (2014) Big fish, little divergence: phylogeography of Lake Tanganyika’s giant cichlid, Boulengerochromis microlepis. Hydrobiologia, 748, 29–38. Koblmüller, S., Salzburger, W. & Sturmbauer, C. (2004). Evolutionary relationships in the sand-dwelling cichlid lineage of Lake Tanganyika suggest multiple colonization of rocky habitats and convergent origin of biparental mouthbrooding. Journal of Molecular Evolution 58, 79–96. Koblmüller, S., Schliewen, U. K., Duftner, N. et al. (2008a). Age and spread of the haplochromine cichlid fishes in Africa. Molecular Phylogenetics and Evolution, 49, 153– 169. Koblmüller, S., Sefc, K. M. & Sturmbauer, C. (2008b). The Lake Tanganyika cichlid species assemblage: recent advances in molecular phylogenetics. Hydrobiologia, 615, 5–20. Koch, M., Koblmüller, S., Sefc, K. M. et al. (2007). Evolutionary history of the endemic Lake Tanganyika cichlid fish Tylochromis polylepis: a recent intruder to a mature adaptive radiation. Journal of Zoological Systematics and Evolutionary Research, 45, 64–71.

96

Kocher, T. D. (2004). Adaptive evolution and explosive speciation: the cichlid fish model. Nature Reviews Genetics, 5, 288–298. Kocher, T. D., Conroy, J. A., Mckaye, K. R. et al. (1995). Evolution of NADH dehydrogenase subunit 2 in east African cichlid fish. Molecular Phylogenetics and Evolution, 4, 420–432. Kolding, J. (1993). Population dynamics and life-history styles of Nile tilapia, Oreochromis niloticus, in Ferguson’s Gulf, Lake Turkana, Kenya. Environmental Biology of Fishes, 37, 25–46. Konings, A. (2019). Tanganyika cichlids in their natural habitat. 4th edition. Hollywood Import & Export. Kornfield, I. & Smith, P. F. (2000). African cichlid fishes: model systems for evolutionary biology. Annual Review of Ecology and Systematics, 31, 163–196. Krasnov, B. R., Mouillot, D., Shenbrot, G. I., et al. (2011a). Beta-specificity: the turnover of host species in space and another way to measure host specificity. International Journal for Parasitology, 41(1), 33–41. Krasnov, B. R., Poulin ,R. & Mouillot, D. (2011b). Scale-dependence of phylogenetic signal in ecological traits of ectoparasites. Ecography, 34, 114–122. Kumar, S., Stecher, G., Li, M. et al. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549. Kumazawa, Y., Yamaguchi, M. & Nishida, M. (2000). Mitochondrial molecular clocks and the origin of euteleostean biodiversity: familial radiation of perciforms may have predated the /Tertiary boundary. In: Kato, M. (ed.), The Biology of Biodiversity. pp. 35–52. Kvach, Y. & Sasal, P. (2010). Telosentis exiguus (von Linstow, 1901) (Palaeacanthocephala: Illiosentidae), a generalist parasite of fishes in the Mediterranean basin. Systematic Parasitology, 76, 9–18 Larson, A., Echelle, A. A. & Kornfield, I. (1985). species flocks. Evolution, 39(6), 1396–1399. Laumer, C. E. & Giribet, G. (2014). Inclusive taxon sampling suggests a single, stepwise origin of ectolecithality in Platyhelminthes. Biological Journal of the Linnean Society, 111(3), 570–588. Lavoué, S., Miya, M., Inoue, J. G. et al. (2005). Molecular systematics of the gonorynchiform fishes (Teleostei) based on whole mitogenome sequences: implications for higher-level relationships within the Otocephala. Molecular Phylogenetics and Evolution, 37(1), 165– 177. le Roux, L. & Avenant-Oldewage, A. (2010). Checklist of the fish parasitic genus

97

Cichlidogyrus (Monogenea), including its cosmopolitan distribution and host species. African Journal of Aquatic Science, 35, 21–36. Legendre, P., Desdevises, Y. & Bazin, E. (2002). A statistical test for host-parasite coevolution. Systematic Biology, 51, 217–234. Lévêque, C., Oberdorff, T., Paugy, D. et al. (2008). Global diversity of fish (Pisces) in freshwater. In: Balian, E. V., Lévêque, C., Segers, H. and Martens, K. (eds.), Freshwater Animal Diversity Assessment. Springer. pp. 545-567. Lezzar, K. E., Tiercelin, J. J., Le Turdu, C. et al (2002). Control of normal fault interaction on the distribution of major Neogene sedimentary depocenters, Lake Tanganyika, East African rift. American Association of Petroleum Geologists Bulletin, 86(6), 1027–1059. Librado, P., Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452. Littlewood, D. T. J. (2006). The evolution of parasitism in flatworms. In: Maule, A. G. and Marks, N.J. (eds.), Parasitic flatworms: molecular biology, biochemistry, immunology and physiology. CABI Publishing. pp. 37–423. Littlewood, D. T. J., Rohde, K., Bray, R. A. & Herniou, E. A. (1999a). Phylogeny of the Platyhelminthes and the evolution of parasitism, Biological Journal of the Linnean Society, 68(1-2), 257–287. Littlewood, D. T. J., Rohde, K. & Clough, K. A. (1999b), The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules. Biological Journal of the Linnean Society, 66, 75–114. Littlewood, D. T. J. & Waeschenbach, A. (2015). Evolution: a turn up for the worms. Current Biology, 25(11), R457–R460. Llewellyn, J. (1984), The biology of isancistrum subulatae n.sp., a monogenean parasitic on the squid, alloteuthis subulata, at plymouth. Journal of the Marine Biological Association of the United Kingdom, 64(2), 285–302. Lowe-McConnell, R. (2003). Recent research in the African Great lakes: fisheries, biodiversity and cichlid evolution. Freshwater Forum, 20, 4–64. Macaranas, J. M., Taniguchi, N., Pante, M. J. R. et al. (1986). Electrophoretic evidence for extensive hybrid gene introgression into commercial Oreochromis niloticus (L.) stocks in the Philippines. Aquaculture Research, 17(4), 249–258. MacLeod, C. J., Paterson, A. M., Tompkins, D. M. & Duncan, R. P. (2010). Parasites lost - do invaders miss the boat or drown on arrival? Ecological Letters, 13(4), 516–527. Madanire-Moyo, G. N., Avenant-Oldewage, A. (2014). A new locality and host record for

98

Enterogyrus coronatus (Pariselle Lambert & Euzet (1991) in South Africa and a review of the morphology and distribution of Enterogyrus (Ancyrocephalidae) species. Helminthologia, 51, 13–22. Maddison, W. P. & Maddison, D. R. (2019). Mesquite: a modular system for evolutionary analysis. Version 3.61. Available from: http://mesquiteproject.org. Malmberg, G .(1957). On the occurrence of Gyrodactylus on Swedish fishes. Skr utgivna av Södra Sveriges Fisk. 1956, 19–76. [in Swedish]. Maneepitaksanti, W. & Nagasawa, K. (2012). Monogeneans of Cichlidogyrus Paperna, 1960 (Dactylogyridae), gill parasites of tilapias, from Okinawa prefecture. Japan Biogeography, 14, 111–119. Marks, N. J. & Maule, A. G. (2006). Parasitic Flatworms: molecular biology, biochemistry, immunology and physiology. 1st edition. CABI, UK. Marques, J. F., Santos, M. J., Teixeira, C. M., et al. (2011). Host-parasite relationships in flatfish (Pleuronectiformes) - The relative importance of host biology, ecology and phylogeny. Parasitology, 138, 107–121. Matějusová, I., Šimková, A., Sasal, P. & Gelnar, M. (2003). Microhabitat distribution of Pseudodactylogyrus anguillae and Pseudodactylogyrus bini among and within gill arches of the European (Anguilla anguilla L.). Parasitology Research, 89, 260–269. Matschiner, M., Böhne, A., Ronco, F. & Salzburger, W. (2020). The genomic timeline of cichlid fish diversification across continents. Nature Communications, 11, 5895. Matschiner, M., Musilová, Z., Barth, J. et al. (2017). Bayesian node dating based on probabilities of fossil sampling supports trans-Atlantic dispersal of cichlid fishes. Systematic Biology, 66(1), 3–22. Mayer, W. E., Tichy, H. & Klein, J. (1998). Phylogeny of African cichlid fishes as revealed by molecular markers. Heredity, 80, 702–714. Mccoll, R. W. (2005). Encyclopedia of World Geography, 3rd Volume. Facts on File. Meier-Kolthoff, J. P., Auch, A. F., Huson, D. H. & Göker, M. (2007). CopyCat: Cophylogenetic analysis tool. Bioinformatics, 23(7), 898–900. Meier, J. I., Stelkens, R. B., Joyce, D. A. et al. (2019). The coincidence of ecological opportunity with hybridization explains rapid adaptive radiation in Lake Mweru cichlid fishes. Nature Communications, 10, 5391. Meinilä, M., Kuusela, J., Ziȩtara, M. S. & Lumme, J. (2004). Initial steps of speciation by geographic isolation and host switch in salmonid pathogen Gyrodactylus salaris (Monogenea: Gyrodactylidae). International Journal for Parasitology, 34, 515–526.

99

Mendlová, M., Desdevises, Y, Civáňová, K., Pariselle, A. & Šimková, A. (2012) Monogeneans of West African cichlid fish: evolution and cophylogenetic interactions. PLoS ONE 7, 17. Mendlová, M., Pariselle, A., Vyskočilová, M. & Šimková, A. (2010). Molecular phylogeny of monogeneans parasitizing African freshwater Cichlidae inferred from LSU rDNA sequences. Parasitology Reseach, 107, 1405–1413. Mendlová, M. & Šimková, A. (2014). Evolution of host specificity in monogeneans parasitizing African cichlid fish. Parasites and Vectors, 7, 69. Mendoza-Palmeron, C. A., Blasco-Costa, I., Hernandez-Mena, D. & Perez-Ponce de Leon, G. (2017). Parasciadicleithrum octofasciatum n. gen., n. sp. (Monogenoidea: Dactylogyridae), parasite of Rocio octofasciata (Regan) (Cichlidae: Perciformes) from Mexico characterised by morphological and molecular evidence. Parasitology International, 66, 152–162. Messu Mandeng, F. D., Bilong Bilong, C. F., Pariselle, A., Vanhove, M.P.M., Bitja Nyom, A.R. & Agnèse, J. (2015). A phylogeny of Cichlidogyrus spp. (Monogenea, Dactylogyridea) clarifies a host-switch between fish families and reveals an adaptive component to attachment organ morphology of this parasite genus. Parasites and Vectors, 8, 582. Meyer, A., Kocher, T. D., Basasibwaki, P. & Wilson, A. C. (1990). Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences. Nature, 347, 550–553. Meyer, B. S., Indermaur, A., Ehrensperger, X. et al. (2015a). Back to Tanganyika : a case of recent trans-species-flock dispersal in East African haplochromine cichlid fishes. Royal Society Open Science, 2, 140498. Meyer, B. S., Matschiner, M. & Salzburger, W. (2017). Disentangling incomplete lineage sorting and introgression to refine species-tree estimates for Lake Tanganyika cichlid fishes. Systematic Biology, 66(4), 531–550. Meyer, B. S., Matschiner, M. & Salzburger, W. (2015b). A tribal level phylogeny of Lake Tanganyika cichlid fishes based on a genomic multi-marker approach. Molecular Phylogenetics and Evolution, 83, 56–71. Mizelle, J. (1936). New species of trematodes from the gills of Illinois fishes. The American Midland Naturalist, 17, 785–806. Mollaret, I., Jamieson, B. G., Adlard, R. D, et al. (1997). Phylogenetic analysis of the Monogenea and their relationships with Digenea and Eucestoda inferred from 28S rDNA sequences. Molecular and Biochemical Parasitology, 90, 433–438. Mollaret, I., Jamieson, B. G. & Justine, J. (2000). Phylogeny of the monopisthocotylea and

100

Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences. International Journal for Parasitology, 30, 171–185. Moran, P., Kornfield, I. & Reinthal, P. N. (1994). Molecular systematics and radiation of the haplochromine cichlids (Teleostei: Perciformes) of Lake Malawi. Copeia, 1994(2), 274– 288. Morand, S. & Guégan, J. F. (2000). Distribution and abundance of parasite nematodes: ecological specialisation, phylogenetic constraint or simply epidemiology? Oikos, 88(3), 563–573. Morand, S., Šimková, A., Matějusová, I., et al. (2002). Investigating patterns may reveal processes: evolutionary ecology of ectoparasitic monogeneans. International Journal for Parasitology, 32, 111–119. Moriarty, D. J. W., Darlington, J. P. E. C., Dunn, I. G. et al. (1973). Feeding and grazing in Lake George, Uganda. Proceedings of the Royal Society of London. Series B, 184, 299–319. Müller, J., Müller, K. (2004). TreeGraph: automated drawing of complex tree figures using an extensible tree description format. Molecular Ecology Notes, 4(4), 786–788. Mulvenna, J., Moertel, L., Jones, M. K., et al. (2010). Exposed proteins of the Schistosoma japonicum tegument. International Journal for Parasitology, 40(5), 543–554. Murray, A. M. (2001). Eocene cichlid fishes from Tanzania, East Africa. Journal of Vertebrate Paleontology, 20(4), 651–664 Muschick, M., Indermaur, A. & Salzburger, W. (2012). Convergent evolution within an adaptive radiation of cichlid fishes. Current Biology, 22, 2362–2368. Musilová, N. & Řehulková, E. & Gelnar, M. (2009). Dactylogyrids (Platyhelminthes: Monogenea) from the gills of (Teleostei: Cyprinidae) from West Africa. Zootaxa, 2241, 47–68. Muterezi Bukinga, F., Vanhove, M. P. M., Van Steenberge, M. & Pariselle, A. (2012). Ancyrocephalidae (Monogenea) of Lake Tanganyika: III: Cichlidogyrus infecting the world’s biggest cichlid and the non-endemic tribes Haplochromini, Oreochromini and Tylochromini (Teleostei, Cichlidae). Parasitology Research, 111, 2049–2061. N’Douba, V., Thys van den Audenaerde, D. F. E. & Pariselle, A. (1997). Description d’une espèce nouvelle de Monogène ectoparasite branchial de Tilapia guineensis (Bleeker, 1862) (Cichlidae) en Côte d’Ivoire. Journal of African Zoology, 111, 429–433. Nagl, S., Tichy, H., Mayer, W. E. et al. (2000). The origin and age of haplochromine fishes in Lake Victoria, East Africa. Proceedings of the Royal Society of London. Series B, 267(1447), 1049–1061.

101

Nagl, S., Tichy, H., Mayer, W. E. et al. (1998). Persistence of neutral polymorphisms in Lake Victoria cichlid fish. Proceedings of the National Academy of Sciences of the United States of America, 95(24), 14238-14243. Nguyen, L.T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. (2015). IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution, 31(1), 268–274. Nieberding, C. M. & Olivieri, I. (2007). Parasites: proxies for host genealogy and ecology? Trends in Ecology and Evolution, 22(3), 156–165. Nishida, M. (1991). Lake Tanganyika as an evolutionary reservoir of old lineages of East African cichlid fishes: inferences from allozyme data. Experientia, 47, 974–979. Nyblade, A. A. & Brazier, R. A. (2002). Precambrian lithospheric controls on the development of the East African rift system. Geology, 30(8), 755–758. Nyboer, E. A., Liang, C. & Chapman, L. J. (2019). Assessing the vulnerability of Africa’s freshwater fishes to climate change: a continent-wide trait-based analysis. Biology Conservation, 236, 505–520. Nylin, S., Agosta, S., Bensch, S. et al. (2018). Embracing colonizations: a new paradigm for species association dynamics. Trends in Ecology and Evolution, 33(1), 4–14. Ochi, H. & Yanagisawa, Y. (1998). between cichlid fishes through differential tolerance of guarding parents toward intruders. Journal of Fish Biology, 52(5), 985–996. Ofori-Amoah, B. (2019). Africa’s geography: dynamics of place, cultures and economies. 1st edition. Wiley. Page, R. D. M. (1993). Parasites, phylogeny and cospeciation. International Journal for Parasitology, 23(4), 499–506. Paperna, I. (1960). Studies on monogenetic trematodes in Israel. 2–Monogenetic trematodes of cichlids. Bamidgeh, 12, 20–33. Paperna, I. (1963). Enterogyrus cichlidarium n. gen. n. sp., a monogenetic trematode parasitic in the intestine of a fish. Bulletin of the Research Council of Israel, 11, 183–187. Paperna, I. (1964). Parasitic helminths of inland-water fishes in Israel. Israel Journal of Zoology, 13:1–20. Paperna, I. (1965). Monogenetic trematodes collected from fresh water fish in southern Ghana. Bamidgeh, 17, 107–115. Paperna, I. (1968a). Monogenetic Trematodes collected from fresh water fish in Ghana. Second report. Bamidgeh, 20, 80–100. Paperna, I. (1968b). Onchobdella n. gen. new genus of monogenetic trematodes

102

(Dactylogyridae, Bychowksi 1933) from cichlid fish from West Africa. Proceedings of the Helminthological Society of Washington, 35, 200–206. Paperna, I. (1969). Monogenetic trematodes of the fish of the Volta bassin and South Ghana. Bulletin de l’Institut Français de l’Afrique Noir, 31, 840–880. Paperna, I. (1979). Monogenea of inland aater fish in Africa. Annales du Museé Royal de l’Afrique Centrale, Sciences, Zoologiques Serie 8, 226. Paperna, I. & Thurston, J. P. (1969). Monogenetic trematodes collected from cichlid fish in Uganda; including the description of five new species of Cichlidogyrus. Revue de Zoologie et de Botanique Africaines, 79, 15–33. Pariselle A. & Euzet, L. (2004). Two new species of Cichlidogyrus Paperna, 1960 (Monogenea, ancyrocephalidae) gill parasites on Hemichromis fasciatus (Pisces, Cichlidae) in Africa, with remarks on parasite geographical distribution. Parasite, 11, 359–64. Pariselle, A., Bilong Bilong, C. F. & Euzet, L. (2003). Four new species of Cichlidogyrus Paperna, 1960 (Monogenea, Ancyrocephalidae), all gill parasites from African mouthbreeder tilapias of the genera Sarotherodon and Oreochromis (Pisces, Cichlidae), with a redescription of C. thurstonae Ergens, 1981. Systematic Parasitology, 56, 201–210. Pariselle, A., Bitja Nyom, A. R. & Bilong Bilong, C. F. (2014). Four new species of Cichlidogyrus (Monogenea, Ancyrocephalidae) from Sarotherodon mvogoi and Tylochromis sudanensis (Teleostei, Cichlidae) in Cameroon. Zootaxa, 3881, 258–266. Pariselle, A., Bitja Nyom, A. R., Bilong Bilong, C. F. (2013). Checklist of the ancyrocephalids (Monogenea) parasitizing Tilapia species in Cameroon, with the description of three new species. Zootaxa 3599, 78–86. Pariselle, A., Boeger, W. A., Snoeks, J. et al. (2011). The monogenean parasite fauna of cichlids: a potential tool for host biogeography. International Journal of Evolutionary Biology, 2011, 471–480 Pariselle, A. & Euzet, L. (1995a). Gill parasites of the genus Cichlidogyrus Paperna, 1960 (Monogenea, Ancyrocephalidae) from Tilapia guineensis (Bleeker, 1862), with descriptions of six new species. Systematic Parasitology, 30, 187–198. Pariselle, A. & Euzet, L. (1995b). Trois Monogènes nouveaux parasites branchiaux de Pelmatochromis buettikoferi (Steindachner, 1895)(Cichlidae) en Guinèe. Parasite, 2, 203– 209 Pariselle, A. & Euzet, L. (2009). Systematic revision of dactylogyridean parasites (Monogenea) from cichlid fishes in Africa, the Levant and Madagascar. Zoosystema, 31, 849–898. Pariselle, A. & Euzet, L. (1995d). Scutogyrus gen.n. (Monogenea: Ancyrocephalidae) for

103

Cichlidogyrus longicornis minus Dossou, 1982, C.l.longicornis, and C. l. ravivaginus Paperna and Thurston, 1969, with description of three new species parasitic on African cichlids. Journal of the Helminthological Society of Washington, 62, 157–173. Pariselle, A. & Euzet, L. (1997). New species of Cichlidogyrus Paperna, 1960 (Monogenea, Ancyrocephalidae) from the gills of Sarotherodon occidentalis (Daget) (Osteichthyes, Cichlidae) in Guinea and Sierra Leone (West Africa). Systematic Parasitology, 38, 221– 230. Pariselle, A. & Euzet, L. (1994). Three new species of Cichlidogyrus Paperna, 1960 (Monogenea: Ancyrocephalidae) parasitic on Tylochromis jentinki (Steindachner, 1895) (Pisces, Cichlidae) in West Africa. Systematic Parasitology, 29, 229–234. Pariselle, A. & Euzet, L. (2003). Four new species of Cichlidogyrus (Monogenea: Ancyrocephalidae), gill parasites of Tilapia cabrae (Teleostei: Cichlidae), with discussion on relative length of haptoral sclerites. Folia Parasitologica, 50, 195–201. Pariselle, A. & Euzet, L. (1996). Cichlidugyrus Paperna, 1960 (Monogenea, Ancyrocephalidae): gill parasites from West African Cichlidae of the subgenus Coptodon Regan, 1920 (Pisces), with descriptions of six new species. Systematic Parasitology, 34, 109–124. Pariselle, A. & Euzet, L. (1998). Five new species of Cichlidogyrus (Monogenea: Ancyrocephalidae) from Tilapia brevimanus , T. buttikoferi and T. cessiana from Guinea, Ivory Coast and Sierra Leone (West Africa). Folia Parasitologica, 45, 275–282. Pariselle, A., Lambert, A. & Euzet, L. (1991). A new type of haptor in mesoparasitic monogeneans of the genus Enterogyrus Paperna, 1963, with a description of Enterogyrus foratus n. sp. and E. coronatus n. sp., stomach parasites of cichlids in West Africa. Systematic Parasitology, 20, 211–220. Pariselle, A., Muterezi Bukinga, F., Van Steenberge, M. & Vanhove, M. P. M. (2015a). Ancyrocephalidae (Monogenea) of Lake Tanganyika. IV. Cichlidogyrus parasitizing species of Bathybatini (Teleostei, Cichlidae) : reduced host-specificity in the deepwater realm ? Hydrobiologia, 748, 99–119. Pariselle, A., Van Steenberge, M., Snoeks, J. et al. (2015b). Ancyrocephalidae (Monogenea) of Lake Tanganyika: does the Cichlidogyrus parasite fauna of Interochromis loocki (Teleostei, Cichlidae) reflect its host’s phylogenetic affinities? Contribution to Zoology, 84, 25–38. Park, J. K., Kim, K. H., Kang, S. et al. (2007). A common origin of complex life cycles in parasitic flatworms: evidence from the complete mitochondrial genome of Microcotyle

104

sebastis (Monogenea: Platyhelminthes). BMC Evolutionary Biology, 7, 11. Parker, G. A., Ball, M. A. & Chubb, J. C. (2015). Evolution of complex life cycles in trophically transmitted helminths. I. Host incorporation and trophic ascent. Journal of Evolutionary Biology, 28(2), 267–91. Pauquet, G., Salzburger, W. & Egger, B. (2018). The puzzling phylogeography of the haplochromine cichlid fish Astatotilapia burtoni. Ecology and Evolution, 8(11), 5637– 5648. Perkins, E. M., Donnellan, S. C., Bertozzi, T. & Whittington, I. D. (2010). Closing the mitochondrial circle on paraphyly of the Monogenea (Platyhelminthes) infers evolution in the diet of parasitic flatworms. International Journal for Parasitology, 40, 1237–1245. Plaisance, L., Littlewood, D. T. J., Olson, P. D. & Morand, S. (2005). Molecular phylogeny of gill monogeneans (Platyhelminthes, Monogenea, Dactylogyridae) and colonization of Indo-West Pacific butterflyfish hosts (Perciformes, Chaetodontidae). Zoologica Scripta, 34, 425–436. Plaisance, L., Rousset, V., Morand, S. & Littlewood, D. T. J. (2008). Colonization of Pacific islands by parasites of low dispersal ability: phylogeography of two monogenean species parasitizing butterflyfishes in the South Pacific Ocean. Journal of Biogeography, 35, 76– 87. Plisnier, P. D., Mgana, H., Kimirei, I. et al. (2009). Limnological variability and pelagic fish abundance (Stolothrissa tanganicae and Lates stappersii) in Lake Tanganyika. Hydrobiologia, 625, 117–134. Poddubnaya, L. G., Kuchta, R. & Scholz, T. (2020). Ultrastructural patterns of the excretory ducts of basal neodermatan groups (Platyhelminthes) and new protonephridial characters of basal cestodes. Parasites and Vectors, 13, 442. Poll, M. (1956). “Poissons Cichlidae,” résultats scientifiques, exploration hydrobiologique du Lac Tanganika (1946-1947). Institut Royal des Sciences Naturelles de Belgique, 3, 1–619. Poll, M. (1986) Classification des Cichlidae du lac Tanganika. tribus, genres et éspèces. Académie Royale de Belgique, Classe des Sciences. Memoires, 45, pp. 1–163. Poulin, R. (2001). Another look at the richness of helminth communities in tropical freshwater fish. Journal of Biogeography, 28(6), 737–743. Poulin, R. (1995). Phylogeny, ecology, and the richness of parasite communitities in vertebrates. Ecological Monographs, 65(3), 283–302. Poulin, R. (1992). Determinants of host-specificity in parasites of freshwater fishes. International Journal for Parasitology, 22(6), 753–758.

105

Poulin, R. (2002). The evolution of monogenean diversity. International Journal for Parasitology, 32, 245–254. Poulin, R. (2008). Evolutionary Ecology of Parasites. 2nd edition. Princeton University Press, New Jersey. Poulin, R., Krasnov, B. R. & Morand, S. (2006). Patterns of host specificity in parasites exploiting small mammals. In: Morand, S., Krasnov, B. R. and Poulin, R. (eds.), Micromammals and Macroparasites: From Evolutionary Ecology to Management. 2006th edition, Springer. pp. 233–256. Poulin, R., Krasnov, B. R., Mouillot, D. & Thieltges, D. W. (2011). The comparative ecology and biogeography of parasites. . Proceedings of the Royal Society of London. Series B, 366(1576), 2379–2390. Poulin, R. & Mouillot, D. (2003). Parasite specialization from a phylogenetic perspective: a new index of host specificity. Parasitology, 126(Pt 5), 473–480. Pouyaud, L., Desmarais, E., Deveney, M. & Pariselle, A. (2006). Phylogenetic relationships among monogenean gill parasites (Dactylogyridea, Ancyrocephalidae) infesting tilapiine hosts (Cichlidae): systematic and evolutionary implications. Molecular Phylogenetics and Evolution, 38, 242–249. Prenter, J., MacNeil, C,. Dick, J. T. A. & Dunn, A. M. (2004). Roles of parasites in animal invasions. Trends in Ecology and Evolution, 19(7), 385–390. Price, C. E. & Kirk, R. G. (1967). First description of a monogenetic trematode from Malawi. Revue de Zoologie et Botanique Africaines, 16, 137–144. Price, C. E., Peebles, H. E. & Bamford, T. (1969). The monogenean parasites of African fishes. IV. Two new species from South African hosts. Revue de Zoologie et Botanique Africaines, 79, 117–124. Rabosky, D. L. (2020). Speciation rate and the diversity of fishes in freshwaters and the oceans. Journal of Biogeography. 47, 1207–1217. Raeymaekers, J. A. M., Hablützel, P. I., Grégoir, A. F. et al. (2013). Contrasting parasite communities among allopatric colour morphs of the Lake Tanganyika cichlid Tropheus. BMC Evolutionary Biology, 13, 1–16. Rakotofiringa, S. & Euzet, L. (1983). Monogènes parasites de Cichlidae (Teleostei) endémiques de Madagascar. Bulletin de la Société Zoologique de France. 108, 107–114. Rambaut, A., 2009. FigTree. Version 1.4.4. Available from: http://tree.bio.ed.ac.uk.

Regan, C. T. (1920). III.— The classification of the fishes of the family Cichlidæ.—I. The

106

Tanganyika genera. Annals and Magazine of Natural History, 5(9), 33–53. Regan, C. T. (1922). XXXII.— The classification of the fishes of the family Cichlidæ.—II. On African and Syrian genera not restricted to the Great Lakes. Annales and Magazine of Natural History, 9(10), 249–264. Řehulková, E., Mendlová, M. & Šimková, A. (2013). Two new species of Cichlidogyrus (Monogenea: Dactylogyridae) parasitizing the gills of African cichlid fishes (Perciformes) from : morphometric and molecular characterization. Parasitology Research, 112, 1399–1410. Řehulková, E. , Seifertová, M., Přikrylová, I. & Francová, K. (2018). Monogenea. In: Scholz, T., Vanhove, M. P. M., Smit, N., et al. (eds.), A guide to the parasites of African freshwater fishes. AbcTaxa, Brussels. pp. 185–243. Ribbink, A. J., Marsh, B. A., Marsh, A. C. et al. (1983). A preliminary survey of the cichlid fishes of rocky habitats in Lake Malawi. South African Journal of Zoology, 18(3), 149– 310. Ricardo, C. K. (1939). The fishes of Lake Rukwa. Journal of the Linnean Society of London, Zoology, (40)275, 625–657 Roberts, E. M. Stevens, N. J., O’Connor, P. M. et al. (2012). Initiation of the western branch of the East African Rift coeval with the eastern branch. Nature Geoscience, 5, 289–294. Roberts, L. & Janovy, J. (2009). Foundations of parasitology. 9th edition. McGraw Hill, United States. Rodríguez-González, A. G., Míguez-Lozano, R., Llopis-Belenguer, C. & Balbuena, J. A. (2015). Phenotypic plasticity in haptoral structures of Ligophorus cephali (Monogenea: Dactylogyridae) on the flathead mullet (Mugil cephalus): a geometric morphometric approach. International Journal for Parasitology, 45(5), 295–303. Rodríguez-González, A. G., Sarabeev, V. & Balbuena, J. A. (2017). Evolutionary morphology in shape and size of haptoral anchors in 14 Ligophorus spp. (Monogenea: Dactylogyridae). PLoS ONE, 12(5), e0178367. Rognon, X. & Guyomard, R. (1997). Mitochondrial DNA differentiation among East and West African Nile tilapia populations. Journal of Fish Biology, 51(1), 204–207. Rohde, K. (1982). Ecology of marine parasites. 2nd edition. CABI. Univ. Quee. St. Lucia. Rohde, K. (1991). The evolution of protonephridia of the Platyhelminthes. Hydrobiologia, 227, 315–321. Rohde, K. (2002). Ecology and biogeography of marine parasites. Advances in Marine Biology, 43, 1–86.

107

Rohde, K. (1979). A critical evaluation of intrinsic and extrinsic factors responsible for niche restriction in parasites. The American Naturalist, 114, 648–671. Rohde, K. (1996). Robust phylogenies and adaptive radiations: a critical examination of methods used to identify key innovations. The American Naturalist, 148(3), 481–500. Rohde, K. & Hobbs, R. (1986). Species segregation: competition or reinforcement of reproductive barriers? In: Cremin, C. D. M. (eds.) Parasite lives. Papers on parasites, their hosts and their associations to honour J. F. A. Sprent, University. Saint-Lucia. pp. 189– 199. Rohde, K. & Rohde, P. P. (2008). How to measure ecological host specificity. Vie Milieu, 58, 121–124. Rohlf, F. J. (2006). TpsDig2, digitize landmarks and outlines, Version 2.10. Department of Ecology and Evolution, State University of New York at Stony Brook. Rolet, J. (1991). Structure and different kinematic development faults along the Lake Tanganyika rift valley (East African Rift system). Bulletin des Centres de Recherches Exploration-Production Elf Aquitaine, 15(2), 327–342. Roller, S., Hornung, J., Hinderer, M. & Ssemmanda, I. (2010). Middle Miocene to Pleistocene sedimentary record of rift evolution in the southern Albert Rift (Uganda). International Journal of Earth Sciences.99, 1643–1661. Ronco, F., Büscher, H. H., Indermaur A & Salzburger, W. (2020). The taxonomic diversity of the cichlid fish fauna of ancient Lake Tanganyika, East Africa. Journal of Great Lakes Reseach, 46, 1067–1078. Ronco, F., Matschiner, M., Böhne, A. et al. (2021). Drivers and dynamics of a massive adaptive radiation in cichlid fishes. Nature, 589, 78–81. Ronquist, F., Teslenko, M., Van Der Mark, P. et al. (2012). Mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542. Rüber, L., Meyer, A., Sturmbauer, C. & Verheyen, E. (2001). Population structure in two sympatric species of the Lake Tanganyika cichlid tribe Eretmodini: evidence for introgression. Molecular Ecology, 10, 1207–1225. Rüber, L., Verheyen, E. & Meyer, A. (1999). Replicated evolution of trophic specializations in an endemic cichlid fish lineage from Lake Tanganyika. Proceedings of the National Academy of Sciences of the United States, 96(18), 10230–10235. Salzburger, W. (2009). The interaction of sexually and naturally selected traits in the adaptive radiations of cichlid fishes. Molecular Ecology, 18, 169–185.

108

Salzburger, W. (2018). Understanding explosive diversification through cichlid fish genomics. Nature Reviews Genetics, 19, 705–717. Salzburger, W., Baric, S. & Sturmbauer, C. (2002a). Speciation via introgressive hybridization in East African cichlids? Molecular Ecology, 11(3), 619–625. Salzburger, W., Meyer, A., Baric, S. et al. (2002b). Phylogeny of the Lake Tanganyika cichlid species flock and its relationship to the Central and East African haplochromine cichlid fish faunas. Systematic Biology, 51, 113–135. Salzburger, W., Mack, T., Verheyen, E. & Meyer, A. (2005). Out of Tanganyika: genesis, explosive speciation, key-innovations and phylogeography of the haplochromine cichlid fishes. BMC Evolutionary Biology, 5:17. Salzburger, W. & Meyer, A. (2004). The species flocks of East African cichlid fishes: recent advances in molecular phylogenetics and population genetics. Naturwissenschaften, 91, 277–290. Salzburger, W., Van Bocxlaer, B. & Cohen, A. S. (2014). Ecology and evolution of the African Great Lakes and their faunas. Annual Review of Ecology, Evolution, and Systematics, 45, 519–545. Samonte, I. E., Satta, Y., Sato, A. et al. (2007). Gene flow between species of Lake Victoria haplochromine fishes. Molecular Biology and Evolution, 24(9), 2069–2080. Sander, S. & Rosendahl, B. R. (1989). The geometry of rifting in Lake Tanganyika, East Africa. Journal of African Earth Sciences (and the Middle East), 8(2-4), 323–354. Sasal, P., Desdevises, Y. & Morand, S. (1998). Host-specialization and species diversity in fish parasites: phylogenetic conservatism? Ecography, 21(6), 639–643. Sasal, P., Trouve, S., Muller-Graf, C., et al. (2001). Specificity and host predictability: a comparative analysis among monogenean parasites of Fish. Journal of Animal Ecology, 68, 437–444. Scarparo, G., Rugman‐Jones, P., Gebiola, M., Di Giulio, A. & Purcell, J. (2021). Social parasite distancing: RADseq reveals high inbreeding in the social parasite Microdon myrmicae but low philopatry for host ant nest. Ecological Entomology, 46, 89–99. Schedel, F. D. B., Musilová, Z. & Schliewen, U. K. (2019). East African cichlid lineages (Teleostei: Cichlidae) might be older than their ancient host lakes: new divergence estimates for the east African cichlid radiation. BMC Evolutionary Biology, 19, 94. Schelkle, B., Faria, P. J., Johnson, M. B., et al. (2012). Mixed infections and hybridisation in monogenean parasites. PLoS ONE, 7(7), e39506. Schelly, R., Stiassny, M. L. J. & Seegers, L. (2003). Neolamprologus devosi sp. n., a new

109

riverine lamprologine cichlid (Teleostei, Cichlidae) from the lower Malagarasi River, Tanzania. Zootaxa, 373: 1–11. Schliewen, U. K., Rassmann, K., Markmann, M. et al. (2001). Genetic and ecological divergence of a monophyletic cichlid species pair under fully sympatric conditions in Lake Ejagham, Cameroon. Molecular Ecology, 10(6), 1471–1488. Schliewen, U. K. & Klee, B. (2004). Reticulate sympatric speciation in Cameroonian crater lake cichlids. Frontiers in Zoology, 1, 5. Schliewen, U. K. & Stiassny, M. L. J. (2003). Etia nguti, a new genus and species of cichlid fish from the River Mamfue, Upper Cross River basin in Cameroon, West-Central Africa. Ichthyological Exploration of Freshwaters, 14, 61–71. Schmidt, H. A., Strimmer, K., Vingron, M. & Von Haeseler, A. (2002). TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics, 18(3), 502–504. Scholz, C. A., Johnson, T. C., Cohen, A. S. et al. (2007). East African megadroughts between 135 and 75 thousand years ago and bearing on early-modern human origins. Proceedings of the National Academy of Sciences of the United States of America, 104(42), 16416– 16421. Schwarzer, J., Lamboj, A., Langen, K. et al. (2014). Phylogeny and age of chromidotilapiine cichlids (Teleostei: Cichlidae). Hydrobiologia, 748, 185–199. Schwarzer, J., Misof, B. & Schliewen, U. K. (2012). Speciation within genomic networks: a case study based on Steatocranus cichlids of the lower Congo rapids. Journal of Evolutionary Biology, 25(1), 138–148. Schwarzer, J., Misof, B., Tautz, D. & Schliewen, U. K. (2009). The root of the East African cichlid radiations. BMC Evolutionary Biology, 9, 186. Seegers, L. & Tichy, H. (1999). The Oreochromis alcalicus flock (Teleostei: Cichlidae) from lakes Natron and Magadi, Tanzania and Kenya: a model for the evolution of “new” species flocks in historical times. Ichtyological Exploration of Freshwaters, 10, 147–174. Seehausen, O. (2015). Process and pattern in cichlid radiations - inferences for understanding unusually high rates of evolutionary diversification. New Phytologist, 207(2), 304–312. Seehausen, O., Koetsier, E., Schneider, M. V. et al. (2003). Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock. Proceedings of the Royal Society of London. Series B, 270, 129–137. Seehausen, O., Mayhew, P. J. & Van Alphen, J. J. M. (1999). Evolution of colour patterns in East African cichlid fish. Journal of Evolutionary Biology, 12(3), 514–534.

110

Šimková, A., Desdevises, Y., Gelnar, M. & Morand, S. (2000). Co-existence of nine gill ectoparasites (Dactylogyrus: Monogenea) parasitising the roach (Rutilus rutilus L.): history and present ecology. International Journal for Parasitology, 30, 1077–1088. Šimková, A., Desdevises, Y., Gelnar, M. & Morand, S. (2001). Morphometric correlates of host specificity in Dactylogyrus species (Monogenea) parasites of European cyprinid fish. Parasitology, 123, 169–177. Šimková, A., Kadlec, D., Gelnar, M. & Morand, S. (2002a). Abundance-prevalence relationship of gill congeneric ectoparasites: testing the core satellite hypothesis and ecological specialisation. Parasitology Research, 88, 682–686. Šimková, A., Ondráčková, M., Gelnar, M. & Morand, S. (2002b). Morphology and coexistence of congeneric ectoparasite species: reinforcement of reproductive isolation? Biological Journal of the Linnean Society, 76(1), 125–135. Šimková, A., Plaisance, L., Matějusová, I., Morand, S. & Verneau, O. (2003). Phylogenetic relationships of the Dactylogyridae Bychowsky, 1933 (Monogenea: Dactylogyridea): the need for the systematic revision of the Ancyrocephalinae Bychowsky, 1937. Systematic Parasitology, 54, 1–11. Šimková, A., Morand, S., Jobet, E., Gelnar, M. & Verneau, O. (2004). Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation. Evolution, 58, 1001–1018. Šimková, A., Řehulková, E., Rasoloariniaina, J. R. et al. (2019). Transmission of parasites from introduced tilapias: a new threat to endemic Malagasy ichthyofauna. Biological Invasions, 21, 803–819. Šimková, A., Serbielle, C., Pariselle, A. et al. (2013). Speciation in Thaparocleidus (Monogenea: Dactylogyridae) parasitizing Asian Pangasiid catfishes. BioMed Research International, 2013, 353956. Šimková, A., Verneau, O., Gelnar, M. & Morand, S. (2006). Specificity and specialization of congeneric monogeneans parasitizing cyprinid fish. Evolution, 60, 1023–1037. Sinnappah, N. D., Lim, L. H. S., Rohde, K. et al. (2001). A paedomorphic parasite associated with a neotenic amphibian host: phylogenetic evidence suggests a revised systematic position for Sphyranuridae within anuran and turtle Polystomatoineans. Molecular Phylogenetics and Evolution, 18(2), 189-201. Snoeks, J. (2000). How well known is the ichthyodiversity of the large East African Lakes? Advances in Ecological Research, 31, 17–38. Snoeks, J. (2001). Cichlid diversity, speciation and systematics: examples from the Great

111

African Lakes. Journal of Aquaculture and Aquatic Sciences 9, 150–66. Snoeks, J., Kaningini, B., Masilya Mulungula, P. et al. (2012). Fishes in Lake Kivu: diversity and fisheries. In: Descy, J., Darchambeau, F. and Schmid, M. (eds.). Lake Kivu: Limnology and Biogeochemistry of a Tropical Great Lake. 2012th edition, Springer. pp. 127–152. Sparks, J. S. & Smith, W. L. (2004). Phylogeny and biogeography of cichlid fishes (Teleostei: Perciformes: Cichlidae). Cladistics, 20, 501–517. Stankiewicz, J. & de Wit, M. J. (2006). A proposed drainage evolution model for Central Africa - Did the Congo flow east? Journal of African Earth Sciences, 44(1), 75–84. Stiassny, M. L. J. (1989). A taxonomic revision of the African genus Tylochromis (Labroidei: Cichlidae); with notes on anatomy and relationships of the groups. Ann. Zoologische wetenschappen - Koninklijk Museum voor Midden-Afrika, 258, 1–161. Stiassny, M. L. J. (1991). Phylogenetic intrarelationships of the family Cichlidae - an overview. In: Keenleyside, M. H. A. (ed.), Cichlid fishes: behaviour, ecology and evolution. Chapman and Hall., pp. 1–35. Stiassny, M. L. J. (1990). Tylochromis, relationships and the phylogenetic status of the African Cichlidae. American Museum Novitates, 2993, 14. Streelman, J. T. & Danley, P. D. (2003). The stages of vertebrate evolutionary radiation. Trends in Ecology and Evolution, 18(3), 126–131. Streelman, J. T., Zardoya, R., Meyer, A. & Karl, S. A. (1998). Multilocus phylogeny of cichlid fishes (Pisces: Perciformes): evolutionary comparison of microsatellite and single-copy nuclear loci. Molecular Biology and Evolution, 15, 798–808. Streit, A. (2008). Reproduction in Strongyloides (Nematoda): a life between sex and parthenogenesis. Parasitology, 135(3), 285–294. Strimmer ,K. & Von Haeseler, A. (1997). Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proceedings of the National Academy of Sciences of the United States of America, 94(13), 6815–6819. Sturmbauer, C. (1998). Explosive speciation in cichlid fishes of the African Great Lakes: a dynamic model of adaptive radiation. Journal of Fish Biology, 53(sA), 18–36. Sturmbauer, C., Baric, S., Salzburger, W. et al. (2001). Lake level fluctuations synchronize genetic divergences of cichlid fishes in African lakes. Molecular Biology and Evolution, 18, 144–154. Sturmbauer, C., Hainz, U., Baric, S. et al. (2003). Evolution of the tribe Tropheini from Lake Tanganyika: synchronized explosive speciation producing multiple evolutionary

112

parallelism. Hydrobiologia, 500, 51–64. Sturmbauer, C,. Husemann, M., Danley, P. (2011). Explosive speciation and adaptive radiation of East African cichlid fishes. In: Frank E. H. and Habel, J. C. (eds.). Biodiversity hotspots: Distribution and protection of conservation priority areas. Springer, London. pp. 333–362. Sturmbauer, C. & Meyer, A. (1993) Mitochondrial phylogeny of the endemic mouthbrooding lineages of cichlid fishes from lake Tanganyika in Eastern Africa. Molecular Biology and Evolution, 10, 751–768. Sturmbauer, C., Salzburger, W., Duftner, N. et al. (2010). Evolutionary history of the Lake Tanganyika cichlid tribe Lamprologini (Teleostei: Perciformes) derived from mitochondrial and nuclear DNA data. Molecular Phylogenetics and Evolution, 57, 266– 284. Takahashi, K. & Okada, N. (2002). Mosaic structure and retropositional dynamics during evolution of subfamilies of short interspersed elements in African cichlids. Molecular Biology and Evolution, 19(8), 1303–1312. Takahashi, K., Terai, Y., Nishida, M. & Okada, N. (2001). Phylogenetic relationships and ancient incomplete lineage sorting among cichlid fishes in lake Tanganyika as revealed by analysis of the insertion of retroposons. Molecular Biology and Evolution, 18(11), 2057– 2066. Takahashi, R., Watanabe, K., Nishida, M. & Hori, M. (2007). Evolution of feeding specialization in Tanganyikan scale-eating cichlids: a molecular phylogenetic approach. BMC Evolutionary Biology, 7, 195. Takahashi, T. (2003). Systematics of Tanganyikan cichlid fishes (Teleostei: Perciformes). Ichthyological Research, 50, 367–382. Takahashi, T. (2008). Description of a new cichlid fish species of the genus Benthochromis (Perciformes: Cichlidae) from Lake Tanganyika. Journal of Fish Biology, 72(3), 603–613. Takahashi, T. (2002). Systematics of the tribe Trematocarini (Perciformes: Cichlidae) from Lake Tanganyika, Africa. Ichthyological Research, 49, 253–259. Takahashi, T. & Hori, M. (2006). Description of a new Lake Tanganyikan cichlid fish of the genus Cyprichromis (Perciformes: Cichlidae) with a note on sexual dimorphism. Journal of Fish Biology, 68, 174–192. Takahashi, T. & Koblmüller, S. (2011). The adaptive radiation of cichlid fish in lake tanganyika: a morphological perspective. International Journal of Evolutionary Biology, 2011, 620754. Takahashi, T. & Koblmüller, S. (2014). A new species of Petrochromis (Perciformes:

113

Cichlidae) from Lake Tanganyika. Ichthyological Research, 61, 252–264. Takahashi, T. & Sota, T. (2016). A robust phylogeny among major lineages of the East African cichlids. Molecular Phylogenetics and Evolution, 100, 234–242. Talavera, G. & Castresana, J. (2007). Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology, 56, 564–577. Terai, Y., Takahashi, K., Nishida, M. et al. (2003). Using SINEs to probe ancient explosive speciation: “hidden” radiation of African cichlids. Molecular Biology and Evolution, 20, 924–930. Terai, Y., Takezaki, N., Mayer, W. E. et al. (2004). Phylogenetic relationships among East African haplochromine fish as revealed by short interspersed elements (SINEs). Journal of Molecular Evolution, 58(1), 64–78. Thatcher, V. E. (2006). Aquatic biodiversity in Latin America: Amazon fish parasites. 2nd edition. Coronet Books. Theis, A., Ronco. F., Indermaur, A. et al. (2014). Adaptive divergence between lake and stream populations of an East African cichlid fish. Molecular Ecology, 23(21), 5304–5322. Theisen, S., Palm, H. W., Al-Jufaili, S. H. & Kleinertz, S. (2017). Pseudempleurosoma haywardi sp. nov. (Monogenea: Ancyrocephalidae (sensu lato) Bychowsky & Nagibina, 1968): an endoparasite of croakers (Teleostei: ) from Indonesia. PLoS ONE, 12(9), e0184376. Thieme, M., Abell, R., Stiassny, M. & Skelton, P. (2005). Freshwater Ecoregions of Africa and Madagascar: a Conservation Assessment. Island Press. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680. Thurston, J. P. & Laws, R. M. (1965). Oculotrema hippopotami (Trematoda: Monogenea) in Uganda. Nature, 205, 1127. Thys van den Audenaerde, D. F. E. (1968). An annotated bibliography of Tilapia (Pisces, Cichlidae). Annales du Museé Royal de l’Afrique Centrale, Sciences, Zoologiques, 14, 406. Trewavas, E. (1983). Tilapiine fishes of the genera Sarotherodon, Oreochromis and . British Museum of Natural History, London. Trewavas, E., Green, J. & Corbet, S. A. (1972). Ecological studies on crater lakes in West

114

Cameroon Fishes of Barombi Mbo. Journal of Zoology, 167(1); 41–95. Turner, G. F., Seehausen, O., Knight, M. E. et al. (2001). How many species of cichlid fishes are there in African lakes? Molecular Ecology, 10, 293–806. Valois, A. E. & Poulin, R. (2015). Global drivers of parasitism in freshwater communities. Limnology and Oceanography, 60(5), 1707–1718. Van Steenberge, M., Pariselle, A., Huyse, T., et al. (2015). Morphology, molecules, and monogenean parasites: an example of an integrative approach to cichlid biodiversity. PLoS ONE, 10, e0124474. Vanhove, M. P. M. (2012). Species flocks and parasite evolution towards a co-phylogenetic analysis of monogenean flatworms of cichlids and gobies. Ph.D. dissertation. Leuven University, Belgium. Vanhove, M. P. M,. Briscoe, A. G., Jorissen, M. W. P. et al. (2018). The first next-generation sequencing approach to the mitochondrial phylogeny of African monogenean parasites (Platyhelminthes: Gyrodactylidae and Dactylogyridae). BMC Genomics, 19, 520. Vanhove, M. P. M., Hablützel, P. I., Pariselle, A. et al. (2016). Cichlids: a host of opportunities for evolutionary parasitology. Trends in Parasitology, 1536, 1–13. Vanhove, M. P. M., Pariselle, A., Van Steenberge, M. et al. (2015). Hidden biodiversity in an ancient lake: phylogenetic congruence between Lake Tanganyika tropheine cichlids and their monogenean flatworm parasites. Scientific Reports, 5, 13669. Vanhove, M. P. M., Snoeks, J., Volckaert, F. A. M. & Huyse, T. (2011a). First description of monogenean parasites in Lake Tanganyika: the cichlid Simochromis diagramma (Teleostei, Cichlidae) harbours a high diversity of Gyrodactylus species (Platyhelminthes, Monogenea). Parasitology, 138, 364–380. Vanhove, M. P. M., Tessens, B., Schoelinck, C. et al. (2013a). Problematic barcoding in flatworms: a case-study on monogeneans and rhabdocoels (Platyhelminthes). Zookeys, 365, 355–379. Vanhove, M. P. M., Van Steenberge, M., Dessein, S. et al. (2013b). Biogeographical implications of Zambezian Cichlidogyrus species (Platyhelminthes: Monogenea: Ancyrocephalidae) parasitizing Congolian cichlids. Zootaxa, 3608, 398–400. Vanhove, M. P. M., Volckaert, F. A. M. & Pariselle, A. (2011b). Ancyrocephalidae (Monogenea) of Lake Tanganyika: I: four new species of Cichlidogyrus from Ophthalmotilapia ventralis (Teleostei: Cichlidae), the first record of this parasite family in the basin. Zoologia (Curitiba), 28, 253–263. Vences, M., Freyhof, J., Sonnenberg, R. et al. (2001). Reconciling fossils and molecules:

115

Cenozoic divergence of cichlid fishes and the biogeography of Madagascar. Journal of Biogeography, 28, 1091–1099. Verburg, P., Antenucci, J. P. & Hecky, R. E. (2011). Differential cooling drives large-scale convective circulation in Lake Tanganyika. Limnology and Oceanography, 56(3), 910– 926. Verheyen, E., Salzburger, W., Snoeks, J. & Meyer, A. (2003). Origin of the superflock of cichlid fishes from Lake Victoria, East Africa. Science, 300(5617), 325–329. Vignon, M., Pariselle, A. & Vanhove, M. P. M. (2011). Modularity in attachment organs of African Cichlidogyrus (Platyhelminthes: Monogenea: Ancyrocephalidae) reflects phylogeny rather than host specificity or geographic distribution. Biological Journal of the Linnean Society, 102, 694–706. Vranken, N., Van Steenberge, M. & Snoeks, J. (2019). Grasping ecological opportunities: not one but five paedophagous species of Haplochromis (Teleostei: Cichlidae) in the Lake Edward system. Hydrobiologia, 832, 105–134. Wagner, C. E., Harmon, L. J. & Seehausen, O. (2012). Ecological opportunity and sexual selection together predict adaptive radiation. Nature, 487, 366–369. Wainwright, P. C., Smith, W. L., Price, S. A., et al. (2012). The evolution of pharyngognathy: a phylogenetic and functional appraisal of the pharyngeal jaw key innovation in labroid fishes and beyond. Systematic Biology, 61(6), 1001–1027. Ward, S. A. (1992). Assessing functional explanations of host-specificity. The American Naturalist, 139(4), 883–891. Weiss, J. D., Cotterill, F. P. D. & Schliewen, U. K. (2015). Lake Tanganyika - a ``melting pot’’ of ancient and young cichlid lineages (Teleostei: Cichlidae)? PLoS ONE, 10(4), e0125043. West, K., Michel, E., Todd, J. et al. (2003). The gastropods of Lake Tanganyika: diagnostic key, classification and notes on the fauna. Centre for African Wetlands. Whitfield, P. J. (1979). The biology of parasitism: an introduction to the study of associating organisms. Edward Arn, London. Whittington, I. D. (1998). Diversity “down under”: monogeneans in the Antipodes (Australia) with a prediction of monogenean biodiversity worldwide. International Journal for Parasitology, 28(10), 1481–1493. Whittington, I. D. (1997). Reproduction and host-location among the parasitic platyhelminthes. International Journal for Parasitology, 27(6), 705-714. Whittington, I. D. & Chisholm, L. A. (2008). Diseases caused by Monogenea. In: Eiras, J., Segner, H., Wahli, T., Kapoor, B. G. (eds). Fish Diseases. Science, Enfield, pp. 683–816.

116

Whittington, I. D., Chisholm, L. A. & Rohde, K. (2000a). The larvae of Monogenea (Platyhelminthes). Advances in Parasitology, 44, 139–232. Whittington, I. D., Cribb, B. W., Hamwood, T. E. & Halliday, J. A. (2000b). Host-specificity of monogenean (platyhelminth) parasites: a role for anterior adhesive areas? International Journal for Parasitology, 30, 305–320. Whittington, I. D & Cribb, B. W. (2001). Adhesive secretions in the platyhelminthes. Advances in Parasitology, 48, 101–224. Wickström, L. M., Haukisalmi, V., Varis, S. et al. (2003). Phylogeography of the circumpolar Paranoplocephala arctica species complex (Cestoda: Anoplocephalidae) parasitizing collared lemmings (Dicrostonyx spp.). Molecular Ecology, 12(12), 3359–3371. Williams, H. H., MacKenzie, K. & McCarthy, A. M. (1992). Parasites as biological indicators of the population biology, migrations, diet, and phylogenetics of fish. Reviews in Fish Biology and Fisheries 2, 144–176. Windsor, D. A. (1998). Most of the species on Earth are parasites. International Journal for Parasitology, 28, 1939–1941. Woo, P. T. K. (2006). Fish Diseases and Disorders: protozoans and metazoans infections. 2nd edition. CABI. Worthington, E. B. & Ricardo, O. K. (1936). The fish of Lake Tanganyika (other than Cichlidæ). Proceedings of the Zoological Society of London, 106(4), 1061–1112. Zamparo, D., Brooks, D. R., Hoberg, E. P. & McLennan, D. A. (2001). Phylogenetic analysis of the Rhabdocoela (Platyhelminthes) with emphasis on the Neodermata and relatives. Zoologica Scripta, 30(1), 59–77. Zardoya, R, Vollmer, D. M., Craddock, C. et al. (1996). Evolutionary conservation of microsatellite flanking regions and their use in resolving the phylogeny of cichlid fishes (Pisces: Perciformes). Proceedings of the Royal Society of London. Series B, 263(1376), 1589–1598. Zhang, D., Zou, H., Wu, S. G. et al. (2019a). Evidence for adaptive selection in the mitogenome of a mesoparasitic monogenean flatworm Enterogyrus malmbergi. Genes (Basel), 10(11), 863. Zhang, S., Zhi, T., Xu, X. et al. (2019b). Monogenean fauna of alien tilapias (Cichlidae) in south China. Parasite, 26, 4. Ziętara, M.S. & Lumme, J. (2002). Speciation by host switch and adaptive radiation in a fish parasite genus Gyrodactylus (Monogenea, Gyrodactylidae). Evolution, 56(12), 2445– 2458.

117

Study A

Underexplored diversity of gill monogeneans in cichlids from Lake

Tanganyika: eight new species of Cichlidogyrus Paperna, 1960 (Monogenea:

Dactylogyridae) from the northern basin of the lake, with remarks on the

vagina and the heel of the male copulatory organ

Rahmouni, C., Vanhove, M.P.M. and Šimková, A. (2017)

Parasites & Vectors; 10, 591

Rahmouni et al. Parasites & Vectors (2017) 10:591 DOI 10.1186/s13071-017-2460-6

RESEARCH Open Access Underexplored diversity of gill monogeneans in cichlids from Lake Tanganyika: eight new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) from the northern basin of the lake, with remarks on the vagina and the heel of the male copulatory organ Chahrazed Rahmouni1*, Maarten P. M. Vanhove1,2,3,4 and Andrea Šimková1

Abstract Background: Lake Tanganyika harbours the most diverse cichlid assemblage of the Great African Lakes. Considering its cichlid flocks consist of approximately 250 endemic species, we can hypothesize a high species-richness in their often quite host-specific monogenean ectoparasites belonging to Cichlidogyrus Paperna, 1960. Yet, only 24 species were described from Tanganyikan hosts and some host tribes have never been investigated for monogeneans. This study presents the first parasitological examination of species of the tribes Cyprichromini (Cyprichromis microlepidotus (Poll, 1956)), Eretmodini (Eretmodus marksmithi Burgess, 2012 and Tanganicodus irsacae Poll, 1950) and Ectodini (Aulonocranus dewindti (Boulenger, 1899)). Specimens of the ectodine Ophthalmotilapia nasuta (Poll & Matthes, 1962) from which four Cichlidogyrus spp. have been previously described from more southern localities were also studied. Further, we discuss the haptor configuration in Tanganyikan Cichlidogyrus spp. and highlight the morphological diversity of the vagina, and that of the heel, a sclerotized part of the male copulatory organ, absent in some species of Cichlidogyrus. Methods: Cichlidogyrus spp. were isolated from gills and fixed using GAP. Haptoral and genital hard parts were measured and drawn by means of a phase contrast microscopic examination. Results: We describe eight new species: Cichlidogyrus milangelnari n. sp. on C. microlepidotus; C. jeanloujustinei n. sp. on E. marksmithi; C. evikae n. sp. on T. irsacae; C. aspiralis n. sp., C. glacicremoratus n. sp. and C. rectangulus n. sp. on O. nasuta;andC. pseudoaspiralis n. sp. and C. discophonum n. sp. on A. dewindti. Three haptoral morphotypes were recognized among the new species. Species of Cichlidogyrus from closely related hosts exhibited the same morphotypes. Geographical variation in Cichlidogyrus spp. fauna as observed in O. nasuta and three morphotypes were distinguished. Finally, we listed 111 Cichlidogyrus species, of which 27 and three Tanganyikan species lack sclerotized vagina and heel, respectively, just like 19 and seven species outside of the lake. (Continued on next page)

* Correspondence: [email protected] 1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 2 of 21

(Continued from previous page) Conclusions: Haptoral and genital features in the Tanganyikan Cichlidogyrus fauna reflect the phylogenetic relationships of their cichlid hosts. It seems that several lineages of Cichlidogyrus spp. exist in Lake Tanganyika but further studies are necessary to confirm this hypothesis and answer questions related to Lake Tanganyika and its cichlids. Keywords: Africa, Burundi, Cichlidae, Cyprichromini, Ectodini, Eretmodini, Platyhelminthes, Monogenea, Cichlidogyrus

Background distribution of the gill monogeneans in Tanganyikan cich- The family Cichlidae Heckel, 1840 is one of the most lids may provide additional evidence for the interrelation- species-rich families of vertebrates and is characterized by ships among cichlid species [10, 17]. a high diversity in morphology, colours and behaviour [1]. As for all monogenean parasites, the description of With about 2200 described species (http://researcharchi Cichlidogyrus spp. is mainly based on the morphology ve.calacademy.org/research/ichthyology/catalog/SpeciesBy of the sclerotized structures of the attachment organ Family.asp) and their current disjunctive distribution from (i.e. haptor) and reproductive organs (i.e. vagina and Central and South America, across Africa to Madagascar, male copulatory organ, MCO) [18]. The haptoral struc- the Middle East and the Indian subcontinent, they have tures in Cichlidogyrus spp. seem to be characteristic for attracted much attention of evolutionary biologists and major phylogenetic lineages, while the MCO is important ecologists [2, 3]. The East African Great Lakes Victoria, for species-level identification [12, 17, 19]. In addition, Malawi and Tanganyika, main hotspots of cichlid biodiver- these haptoral structures in dactylogyridean monogeneans sity, alone harbour more than 1500 endemic cichlid spe- have been extensively studied in various ecological and evo- cies and have therefore been the focus of numerous lutionary contexts because of their influence on the host studies. Lake Tanganyika, the deepest and oldest lake in specificity, parasite specialization and reproductive isolation Africa, counts the genetically, morphologically and eco- among congeners through niche ecology [12, 20–22]. logically most diverse cichlid assemblages of these lakes Haptoral structures and MCO in Cichlidogyrus spp. [4]. With an estimated age of 9–12 million years (MY), it present a high morphological diversity in terms of shape holds about 75 non-cichlid and 250 endemic cichlid fish and size. The haptor of an adult specimen comprises two species. The latter belong to more than 50 genera and 12 pairs of anchors (also termed gripi) (one dorsal and one to 14 tribes [5, 6]. Cichlids have become one of the best ventral), two transversal bars (dorsal bar with two typical models for the study of biological diversification and rapid auricles and a V-shaped ventral bar) and seven dorsal and radiation [2, 3, 7]. As host-parasite systems are suitable to ventral pairs of hooks (also termed uncinuli). Four main furnish information on the evolution and distribution of morphological groups were recognized by Vignon et al. the hosts as well as to elucidate the processes of parasite [21] based on the configuration of the hook pairs. The speciation, the parasites of cichlids are the objects of vagina in Cichlidogyrus spp. can be sclerotized or not [11]. special scientific interest as well [8–10]. More than 100 The MCO consists of two main parts, i.e. copulatory tube African and Levantine cichlid species have been investi- and accessory piece (not always present; see [23]). The gated for the presence of monogenean parasites [8, 11]. copulatory tube has an ovoid basal bulb in the proximal Overall, 13 monogenean genera were proposed from part prolonged into a tube of variable size and shape, cichlid hosts worldwide. Six of them, i.e. Urogyrus Bilong with a simple or ornamented distal end. The accessory Bilong, Birgi & Euzet, 1994; Enterogyrus Paperna, 1963; piece normally extends from the basal bulb and pre- Onchobdella Paperna, 1968; Scutogyrus Pariselle & sents a simple or complicated structure [24–27]. The Euzet, 1995; Cichlidogyrus Paperna, 1960 (Dactylogyridae) sclerotized portion basal to the ovoid bulb, commonly and Gyrodactylus von Nordmann, 1832 (Gyrodactylidae), called “heel”, is relevant to species identification in were recognized in African cichlids [3, 11]. Of these, Cichlidogyrus.Thisstructure,becauseofsclerotization, Cichlidogyrus is the most species-rich with species infect- was often (but not always), considered as associated to ing almost exclusively Cichlidae (a few Cichlidogyrus repre- the accessory piece. The presence of this sclerotized sentatives occur also on Cyprinodontidae and Nandidae). portion as a part of the MCO was reported in the ori- More than 100 species of Cichlidogyrus were described ginal descriptions of Cichlidogyrus spp. from various from more than 100 cichlid hosts [11–13]. The parasite cichlid hosts (e.g. [23, 25–31]). All species of Cichlido- fauna of fishes in Lake Tanganyika is being systematically gyrus described until now from Tanganyikan cichlid investigated since recently. To date, only a limited number hosts possess a heel (e.g. [14, 28]) except for Cichlidogyrus of monogeneans was described, i.e. 24 Cichlidogyrus spp. attenboroughi Kmentová, Gelnar, Koblmüller & Vanhove, from only 20 cichlid hosts [14]. At least six cichlid tribes 2016 from Benthochromis horii Takahashi, 2008 were never investigated for monogeneans [15, 16]. The (Benthochromini) which was described recently [16]. Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 3 of 21

The aim of the present research was to study the gill When caught alive, they were sacrificed by severing the monogeneans belonging to Cichlidogyrus spp. in littoral spinal cord. Cichlid hosts (Fig. 1) were identified in situ cichlid fish communities of Lake Tanganyika in by Stephan Koblmüller (Karl-Franzens University of Burundi. Only scarce reports on these flatworms exist Graz, Austria) and photographs were taken by Radim from this stretch of the lakeshore [10, 13, 16]. Investi- Blažek (Institute of Vertebrate Biology, Czech Academy gation of five Tanganyikan hosts of three different of Sciences, Czech Republic). Cichlid species investi- tribes, i.e. Cyprichromis microlepidotus (Poll, 1956) gated and their localities of sampling are detailed in (Cyprichromini), Eretmodus marksmithi Burgess, 2012 Fig. 2. Gills were dissected by separating the gill arches and Tanganicodus irsacae Poll, 1950 (Eretmodini), and via dorsal and ventral section using standard parasito- Aulonocranus dewindti (Boulenger, 1899) and Ophthal- logical procedures, and transferred into a Petri dish con- motilapia nasuta (Poll & Matthes, 1962) (Ectodini), taining water. Monogeneans were detached from the allowed to record eight unknown Cichlidogyrus spp. gills, isolated according to Musilová et al. [29] using an which are described here. These hosts, except for O. Olympus SZX7 stereomicroscope, mounted onto a slide nasuta, were investigated for parasites for the first time. according to Vanhove et al. [23] using a drop of glycer- Indeed, three species of Ophthalmotilapia Pellegrin, ine ammonium picrate mixture (GAP) [30], and covered 1904 have already been investigated and four Cichlido- with a coverslip and sealed with nail polish. Measure- gyrus species were previously described by Vanhove ments and photographs were taken at a magnification of et al. [23] along the coasts of Lake Tanganyika in ×1000 (objective ×100 oil immersion, ocular ×10), using Congo, Zambia and Tanzania. Therefore, the present an Olympus BX51 phase-contrast microscope and paper provides additional data on the high Cichlido- Olympus Stream Image Analysis v. 1.9.3 software. All gyrus spp. richness of Tanganyikan cichlids, and on measurements (included in the species descriptions) are geographical variation in parasite fauna throughout the presented in micrometres and given as the range lake. Finally, we discussed the configuration of the hook followed by the mean and the number of specimens pairs (size and form, see [21]) in the newly described measured (n) in parentheses. Drawings of the haptoral species and the importance of the morphological diver- sclerotized parts and the copulatory organ were made sity of the vagina and heel for Cichlidogyrus systematics on flattened specimens using an Olympus BX51 micro- by indexing all species described so far as well as their type- scope equipped with a drawing tube and edited with a hosts, type-localities, and reporting the characterization of graphic tablet compatible with Adobe Illustrator CS6 v. their vagina and heel structures, based on the original de- 16.0.0 and Adobe Photoshop v. 13.0. Terminology of scriptions and/or drawings. haptoral sclerotized parts (i.e. anchors and hooks) fol- lows Gussev [31]. The numbering of the hook pairs (Roman letters I-VII) is that recommended by Mizelle Methods [32]. This method is preferred in adult specimens be- Cichlid specimens were acquired from commercial fish- cause it takes into consideration both antero-posterior ermen or caught using gill nets during snorkelling or and dorso-ventral positions of hooks [18, 33]. The length diving in September 2013 in Burundi (Lake Tanganyika). of the hook pairs i.e. “short” or “long” was assigned

Fig. 1 Cichlid hosts examined for the present study. a Cyprichromis microlepidotus (Poll, 1956). b Eretmodus marksmithi Burgess, 2012. c Tanganicodus irsacae Poll, 1950. d Ophthalmotilapia nasuta (Poll & Matthes, 1962). e Aulonocranus dewindti (Boulenger, 1899). Photos by R. Blažek (Burundi, 2013) Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 4 of 21

Fig. 2 Map of Lake Tanganyika (blue) indicating the localities of sampling along the coast in Burundi following Pariselle & Euzet [11]. The metrics used for Results the hard structures are shown in Fig. 3. Investigation of the five cichlid host species revealed The type-material was deposited in the Invertebrate thepresenceofeightnewspeciesofCichlidogyrus: C. collection of the Royal Museum for Central Africa milangelnari n. sp. on Cyprichromis microlepidotus (RMCA), Tervuren, Belgium; the Muséum National d’His- (Cyprichromini); C. jeanloujustinei n. sp. on Eretmo- toire Naturelle (MNHN), Paris, France; and the Iziko dus marksmithi (Eretmodini); C. evikae n. sp. on Tan- South African Museum (SAMC), Cape Town, Republic of ganicodus irsacae (Eretmodini); C. aspiralis n. sp., C. South Africa. Prevalence and intensity of infection were glacicremoratus n. sp. and C. rectangulus n. sp. on calculated according to Bush et al. [34]. Cichlidogyrus spe- Ophthalmotilapia nasuta (Ectodini); and C. pseudoaspiralis cies isolated from Ophthalmotilapia spp. in Burundi were n. sp. and C. discophonum n. sp. on Aulonocranus compared to those previously described on representatives dewindti (Ectodini). of Ophthalmotilapia spp. in Congo, Tanzania and Zambia by examination of museum specimens (Table 1). To high- Family Dactylogyridae Bychowski, 1933 light the importance of the vagina and heel in Cichlido- Genus Cichlidogyrus Paperna, 1960 gyrus species and give an overview of the morphological diversity of these sclerotized structures (shape and size when available), we looked for the reproductive organ fea- Cichlidogyrus milangelnari n. sp. tures in the original descriptions and/or on drawings. These are provided in an alphabetic list of the African Type-host: Cyprichromis microlepidotus (Poll, 1956) (Fig. 1a); Cichlidogyrus species (from Tanganyika and elsewhere, tribe Cyprichromini (Perciformes: Cichlidae). see Additional file 1: Table S1), their type-hosts and type- Type-locality: Nyaruhongoka (3°41′S, 29°20′E), Lake localities, authors, and date of citation based mainly on Tanganyika, Burundi. the original descriptions and on the systematic revision of Type-material: Holotype: MRAC_vermes_37940. Paratypes: dactylogyridean cichlid parasites made by Pariselle & MRAC_vermes_37940; MNHN HEL583; SAMC-A088695. Euzet [11]. Host nomenclature follows FishBase [35]. Site in host: Gills. Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 5 of 21

Fig. 3 Measurements used in the descriptions of the new species of Cichlidogyrus. Abbreviations: A, anchor (DA, dorsal anchor; VA, ventral anchor; a, total length; b, blade length; c, shaft length; d, guard length; e, point length); DB, dorsal bar (h, auricle length; w; maximum straight width; x, total length; y, distance between auricles); VB, ventral bar (x, length of one ventral bar branch; w, maximum width); H, hook length; MCO, male copulatory organ straight length; Ct, copulatory tube curved length; He, heel straight length; Ap, accessory piece straight length; Vg, vagina (V, vagina total length; v, vagina width)

Prevalence and intensity of infection: 100% (3/3); 7–37 daily support for the research on monogeneans in Lake monogeneans per infected host. Tanganyika. ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the Description International Code of Zoological Nomenclature (ICZN) [36], [Based on 13 specimens fixed in GAP; Fig. 4]. Body details of the new species have been submitted to ZooBank. 412–826 (597; n = 10) long, 69–156 (98; n = 10) wide at The Life Science Identifier (LSID) of the article is urn:lsid: mid-body. Dorsal anchors with short shaft and more zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A4754 pronounced guard (c.2 times length of shaft) and curved 242. The LSID for the new name Cichlidogyrus milangelnari blade with arched point: a = 33–36 (35; n =12);b=19– is urn:lsid:zoobank.org:act:789E0B84-18E1-46A4-8029-FC7A 30 (23; n =12);c=2–6 (4; n = 11); d = 9–13 (10; n = 11); 63140721. e=5–7 (6; n = 12). Dorsal bar relatively small, curved, Etymology: The specific epithet of the new species, “milangelnari”, thick in middle part, with short auricles: h = 6–9 (9; n = honors the Czech parasitologist Professor Milan Gelnar, head 12); w = 7–9 (8; n = 12); x = 26–31 (29; n = 12); y = 11–14 of the Laboratory of Parasitology (Department of Botany and (12; n = 12). Ventral anchors similar to dorsal ones: a = Zoology, Faculty of Science, Masaryk University, Czech 32–35 (33; n = 12); b = 22–33 (29; n = 12); c = 1–6 (4; n Republic) as the recognition for his kind guidance and = 12), d = 5–10 (8; n =12); e=6–9 (8; n = 12). Ventral

Table 1 List of Cichlidogyrus spp. described from species of Ophthalmotilapia by Vanhove et al. [23] Cichlidogyrus species Host species Locality Material deposition Cichlidogyrus centesimus Ophthalmotilapia ventralis Wonzye and Kasenga points (Zambia); MRAC 37680 (paratype, 1 slide) Kikoti (D.R. Congo) Ophthalmotilapia nasuta; O. boops Mtosi (Tanzania) Cichlidogyrus makasai O. ventralis Wonzye and Kasenga points (Zambia); Paratype (2 slides) MRAC 37676 and 37,677 Kikoti (D.R. Congo) Cichlidogyrus sturmbaueri O. ventralis Wonzye and Kasenga points (Zambia) Paratype (2 slides) MRAC 37681 and 37,682 O. nasuta Musamba (Tanzania) Cichlidogyrus vandekerkhovei O. ventralis Wonzye and Kasenga points (Zambia); Paratype (2 slides) MRAC 37675 and 37,679 Kikoti (D.R. Congo) O. nasuta; O. boops Mtosi (Tanzania) Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 6 of 21

Fig. 4 Sclerotized structures of Cichlidogyrus milangelnari n. sp. ex Cyprichromis microlepidotus. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; Ct, copulatory tube; Ap, accessory piece bar V-shaped, with constant width: w = 4–9 (5; n = 12); Pariselle & Vanhove, 2015 [17]; C. gillardinae Muterezi x=26–31 (29; n = 12). Haptor with 7 pairs of short Bukinga, Vanhove, Van Steenberge & Pariselle, 2012 hooks, hooks V with larval size (sensu [11, 37]); each [41]; C. gistelincki Gillardin, Vanhove, Pariselle, Huyse & hook with erect thumb and shank comprised of 2 subunits: Volckaert, 2011 [42]; C. halli Price & Kirk, 1967 [43]; C. pair I = 8–12 (10; n = 12) long, pair II = 10–12 (11; n =12) haplochromii Paperna & Thurston, 1969 [44]; C. irenae long, pair III = 9–12 (11; n = 12) long, pair IV = 11–13 (12; Gillardin, Vanhove, Pariselle, Huyse & Volckaert, 2012 n = 12) long, pair V = 7–11 (9; n = 12) long, pair VI = 10–13 [42]; C. longipenis Paperna & Thurston, 1969 [44]; C. (12; n = 12) long, pair VII = 9–12 (11; n = 12) long. makasai Vanhove, Volckaert & Pariselle, 2011 [23]; C. Male copulatory organ composed of long copulatory mulimbwai Muterezi Bukinga, Vanhove, Van Steenberge tube with thick wall, associated to small bulb, curved & Pariselle, 2012 [41]; C. muterezii Pariselle & Vanhove, at proximal third with fan-like ending: MCO = 39–45 2015 [17]; C. nageus Řehulková, Mendlová & Šimková, (41; n = 13); Ct = 44–52 (48; n = 13). Heel absent. 2013 [18]; C. raeymaekersi Pariselle & Vanhove, 2015 [17]; Accessory piece thick, composed of 2 superimposed C. rognoni Pariselle, Bilong Bilong & Euzet, 2003 [45]; C. parts with forked ending, Ap = 34–38 (36; n =13).Vagina schreyenbrichardorum Pariselle & Vanhove, 2015 [15]; C. non-sclerotized. sanjeani Pariselle & Euzet, 1997 [40]; C. sigmocirrus Pariselle, Bitja Nyom & Bilong Bilong, 2014 [46]; C. steenbergei Gillardin, Vanhove, Pariselle, Huyse & Differential diagnosis Volckaert, 2012 [42]; C. tilapiae Paperna, 1960 [24], Cichlidogyrus milangelnari n. sp. belongs to the group of C. vandekerkhovei Vanhove, Volckaert & Pariselle, species with short hook pairs I-IV, VI and VII (sensu 2011 [23]; and C. vealli Pariselle & Vanhove, 2015 Vignon et al. [21]), a copulatory tube without a swollen [15]. Cichlidogyrus attenboroughi was the first record proximal portion and non-sclerotized vagina (see [37]), of Cichlidogyrus spp. from Lake Tanganyika lacking a heel just like C. attenboroughi Kmentová, Gelnar, Koblmüller [16]. However, the new species is easily distinguish- & Vanhove, 2016 [16]; C. banyankimbonai Pariselle & able from the latter by the (i) length of the dorsal bar Vanhove, 2015 [17]; C. berminensis Pariselle, Bitja Nyom auricles (6–9 μminC. milangelnari n. sp. vs 14–23 μmin & Bilong Bilong, 2013 [38]; C. bifurcatus Paperna, 1960 C. attenboroughi), (ii) the MCO (curved at the proximal [24]; C. brunnensis Kmentová, Gelnar, Koblmüller & third with fan ending vs L-shaped, strongly curved halfway Vanhove, 2016 [16]; C. buescheri Pariselle & Vanhove, with constricted ending in C. attenboroughi), and (iii) 2015 [15]; C. consobrini Jorissen, Pariselle & Vanhove, the accessory piece (thick, composed of two superim- 2017 [39]; C. fontanai Pariselle & Euzet, 1997 [40]; C. posed parts with forked ending in C. milangelnari n. frankwillemsi Pariselle & Vanhove, 2015 [17]; C. frans- sp. vs C-shaped, broader than copulatory tube in C. wittei Pariselle & Vanhove, 2015 [17]; C. georgesmertensi attenboroughi). Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 7 of 21

Cichlidogyrus jeanloujustinei n. sp. with short auricles: h = 7–9 (8; n = 6); w = 3–5 (4; n = 6); x=21–25 (23; n = 6); y = 7–11 (9; n = 6). Ventral anchors Type-host: Eretmodus marksmithi Burgess, 2012 (Fig. 1b); with shaft shorter than guard, blade longer than in dor- tribe Eretmodini (Perciformes: Cichlidae). sal anchors, with arched point: a = 27–30 (28; n = 6); b = Type-locality: Mukuruka (4°14′S, 29°33′E), Lake Tanganyika, 21–25 (23; n = 6); c = 3–6 (4; n = 6); d = 8–11 (10; n = 6); Burundi. e=8–10 (9; n = 6). Ventral bar V-shaped, with constant Type-material: Holotype: MRAC_vermes_37939. Paratypes width: w = 4–6 (5; n = 6); x = 27–35 (31; n = 6). Haptor MRAC_vermes_37947; MNHN HEL582; SAMC-A088694. with 7 pairs of short hooks, hooks V with larval size Site in host: Gills. (see above); each hook with erect thumb and shank Prevalence and intensity of infection: 30% (11/36); 1–3 comprised of 2 subunits: pair I = 11–13 (12; n =6) monogeneans per infected host. long, pair II = 15–19 (17; n = 6) long, pair III = 15–22 ZooBank registration: To comply with the regulations set (19; n =6) long, pair IV=19–24 (21; n =6) long, pair out in article 8.5 of the amended 2012 version of the V=10–12 (11; n = 6) long, pair VI = 18–23 (21; n =6) International Code of Zoological Nomenclature (ICZN) [36], long, pair VII = 15–18 (17; n = 6) long. Male copula- details of the new species have been submitted to ZooBank. tory organ composed of long, straight copulatory The Life Science Identifier (LSID) of the article is urn:lsid: tube, associated to ovoid basal bulb with thick wall: zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A4754 MCO = 52–60 (56; n = 10); Ct = 51–59 (56; n =10). 242. The LSID for the new name Cichlidogyrus jeanloujustinei Heel poorly developed, He = 1–2(1;n = 9). Accessory is urn:lsid:zoobank.org:act:51509F53-0C47-48F5-9E26- piece proximally with 2 thick portions attached to 4252B83565AE. basal bulb, slightly curved in middle distal part, with Etymology: The specific epithet “jeanloujustinei” honors the blunt ending, Ap = 41–45 (43; n =10). Vagina non- French parasitologist Jean-Lou Justine, Professor at the sclerotized. Muséum National d’Histoire Naturelle, Paris, France, who is extensively studying the systematics and biodiversity Differential diagnosis of monogeneans, digeneans, and nematodes. According to the relative length of the hook pairs, C. jeanloujustinei n. sp. belongs to the same morphological Description group as C. milangelnari n. sp. (see above). The charac- [Based on 10 specimens fixed in GAP; Fig. 5]. Body teristic structures of the MCO (reduced heel and two at- 590–1397 (831; n = 5) long, 158–255 (194; n = 5) wide at tached thick portions in the proximal part of the mid-body. Dorsal anchors with short shaft and more accessory piece) make C. jeanloujustinei n. sp. unique pronounced guard (c.3 times length of shaft) and curved within this group. The new species exhibits haptoral blade with arched point: a = 23–29 (25; n = 6); b = 16–20 structures similar to C. milangelnari n. sp. but differs in (17; n = 6); c = 2–5 (4; n = 6), d = 8–13 (10; n = 6); e = 7– having shorter dorsal anchors (23–29 vs 33–36 μm) and 10 (8; n = 6). Dorsal bar relatively small, well arched, a longer MCO (52–60 vs 39–45 μm).

Fig. 5 Sclerotized structures of Cichlidogyrus jeanloujustinei n. sp. ex Eretmodus marksmithi. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 8 of 21

Cichlidogyrus evikae n. sp. 10 (8; n = 10); w = 3–5(4;n = 10); x = 17–26 (21; n =10);y =5–9(7;n = 10). Ventral anchors with shaft shorter than Type-host: Tanganicodus irsacae Poll, 1950; tribe Eretmodini guard, curved blade with arched point: a = 22–24 (23; n = (Perciformes: Cichlidae) (Fig. 1c). 10); b = 19–21 (20; n = 10); c = 3–5(4;n =10);d=6–9(7; Type-locality: Mukuruka (4°14′S, 29°33′E), Lake Tanganyika, n = 10); e = 7–10 (8; n = 10). Ventral bar V-shaped: w = 3–6 Burundi. (5; n =10);x=25–31 (28; n = 10). Haptor with 7 pairs of Type-material: Holotype: MRAC_vermes_37946. Paratypes: short hooks, hooks V with larval size (see above), thumb MRAC_vermes_37958; MNHN HEL586; SAMC-A088701. broad and junction with shank well pronounced with prox- Site in host: Gills. imal protrusion: pair I = 11–13 (12; n =11)long,pairII= Prevalence and intensity of infection: 71% (5/7); 1–3 12–17 (15; n = 11) long, pair III = 15–19 (17; n = 11) long, monogeneans per infected host. pair IV = 19–23 (20; n =6)long,pairV=10–12 (11; n =11) ZooBank registration: To comply with the regulations set long, pair VI = 13–21 (18; n =11) long, pair VII=14–17 out in article 8.5 of the amended 2012 version of the (16; n = 11) long. Male copulatory organ composed International Code of Zoological Nomenclature (ICZN) [36], of long copulatory tube with thick wall, slightly details of the new species have been submitted to ZooBank. curved where associated to irregularly shaped bulb, The Life Science Identifier (LSID) of the article is urn:lsid: linked to accessory piece with thin filament: MCO = zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A475 53–58 (56; n =12);Ct=52–57 (54; n =10).Heelreduced 4242. The LSID for the new name Cichlidogyrus evikae is to inconspicuous, He = 0–2(1;n = 12). Accessory piece urn:lsid:zoobank.org:act:85036A92-0337-4428-9E88-6CC27 with 2 distinct parts variable in thickness, superimposed E6205C2. with irregular surface, endings blunt, one extremity Etymology: The name is given in honour of the Czech shorter than the other, Ap = 46–52 (49; n = 12). Vagina parasitologist Dr. Eva Řehulková (Department of Botany non-sclerotized. and Zoology, Faculty of Science, Masaryk University, Czech Republic) who studies monogenean flatworms for her Differential diagnosis contributions to our research. Cichlidogyrus evikae n. sp. belongs to the same morpho- logical group as C. milangelnari n. sp. and C. jeanloujustinei Description n. sp. (see above). It is most similar to C. jeanloujustinei n. [Based on 12 specimens fixed in GAP; Fig. 6]. Body 400– sp. in having (i) a MCO with an ovoid basal bulb prolonged 1496 (883; n = 9) long, 109–305 (198; n = 9) wide at mid- into a copulatory tube with thick wall, (ii) a poorly devel- body. Dorsal anchors with short shaft and more oped to inconspicuous heel, and (iii) an accessory piece pronounced guard (c.2 times length of shaft) and curved composed of two superimposed parts. Cichlidogyrus evikae blade with arched point: a = 20–23 (22; n =10);b=12–20 n. sp. can be distinguished from C. jeanloujustinei because (16; n =10);c=3–5(4;n = 10); d = 7–9(8;n = 10); e = 7–9 the shape of the hook pairs (except for pairs I and V) is dif- (8; n = 10). Dorsal bar relatively small, well arched with ferent: thumbs are broad in C. evikae n. sp. which gives an equal thickness over entire width and short auricles: h = 6– articulated appearance.

Fig. 6 Sclerotized structures of Cichlidogyrus evikae n. sp. ex Tanganicodus irsacae. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 9 of 21

Cichlidogyrus aspiralis n. sp. in middle with slightly curved point: a = 39–43 (41; n = 3); b = 32–33 (32; n = 3); c = 2–4 (3; n = 3); d = 10–14 Type-host: Ophthalmotilapia nasuta (Poll & Matthes, 1962) (12; n = 3); e = 7–10 (9; n = 3). Dorsal bar long, straight (Fig. 1d); tribe Ectodini (Perciformes: Cichlidae). with short appendages of anterior face of dorsal trans- Type-locality: Magara (3°44′S, 29°19′E), Lake Tanganyika, verse bar: h = 6–10 (8; n = 3); w = 6–8 (7; n = 3); x = 45– Burundi. 47 (46; n = 3); y = 19–21 (20; n = 3). Ventral anchors with Type-material: Holotype: MRAC_vermes_37943; Paratype: shorter shaft than guard and arched point: a = 33–34 MRAC_vermes_37954; SAMC-A088698. (34; n = 3); b = 32–33 (33; n = 3); c = 2–5 (3; n = 3); d = Site in host: Gills. 7–9 (8; n = 3); e = 9–10 (9; n = 3). Ventral bar V-shaped, Prevalence and intensity of infection: 75% (3/4); 1–2 with constant width: w = 4–5 (5; n = 3); x = 37–40 (39; n monogeneans per infected host. = 3). Hook pair I with well-developed shank, long in ZooBank registration: To comply with the regulations set comparison with remaining pairs which are similarly out in article 8.5 of the amended 2012 version of the short (sensu [11, 37]), pair V retains its larval size; each International Code of Zoological Nomenclature (ICZN) [36], hook with erect thumb and shank comprised of 2 sub- details of the new species have been submitted to ZooBank. units: pair I = 26–27 (27; n = 3) long, pair II = 19–21 (20; The Life Science Identifier (LSID) of the article is urn:lsid: n = 3) long, pair III = 20–23 (21; n = 3) long, pair IV = zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A4754 20–21 (20; n = 3) long, pair V = 11–12 (12; n = 3) long, 242. The LSID for the new name Cichlidogyrus aspiralis is pair VI = 20–22 (21; n = 3) long, pair VII = 20–21 (20; n urn:lsid:zoobank.org:act:1918864B-1B58-4DCA-B97C- = 3) long. Male copulatory organ beginning in an ovoid 57A80C79A576. bulb, with short straight copulatory tube: MCO = 36–38 Etymology: The specific epithet “aspiralis” refers to the (37; n = 4); Ct = 20–21 (21; n = 4). Heel long, straight, absence of a spiral thickening in the copulatory tube in He = 14–18 (16; n = 4). Accessory piece thin, proximally comparison to the species C. centesimus described by connected to basal bulb, rounded and slightly enlarged Vanhove et al. [23]. distally, Ap = 14–19 (16; n = 4). Vagina short, sclerotized: V=15–16 (15; n = 2); v = 5–7 (6; n = 2). Description [Based on 4 specimens fixed in GAP; Fig. 7]. Body 336– Differential diagnosis 407 (373; n = 3) long, 76–120 (102; n = 3) wide at mid- Cichlidogyrus aspiralis n. sp. belongs to the group of body. Dorsal anchors with short shaft and pronounced species exhibiting long hook pair I (pair V with larval guard (c.4 times length of shaft) and blade slightly bent size) and short pairs II-IV, VI and VII (see [21]), a

Fig. 7 Sclerotized structures of Cichlidogyrus aspiralis n. sp. ex Ophthalmotilapia asuta. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece; Vg, vagina Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 10 of 21

copulatory tube without a swollen proximal portion, and thin often extending beyond penis and ending in a well a sclerotized vagina (see [37]). This group includes C. developed, enlarged and bulbous extremity, attached albareti Pariselle & Euzet, 1998 [47]; C. dageti Dossou & by a filament to the distal extremity of the basal bulb, Birgi, 1984 [48]; C. digitatus Dossou, 1982 [49]; C. dra- 26–38 μmlonginC. casuarinus). colemma Řehulková, Mendlová & Šimková, 2013 [18]; C. euzeti Dossou & Birgi, 1984 [48]; C. falcifer Dossou & Cichlidogyrus glacicremoratus n. sp. Birgi, 1984 [48]; C. longicirrus Paperna, 1965 [50]; and C. sanseoi Pariselle & Euzet, 2004 [27]. The Tanganyikan Type-host: Ophthalmotilapia nasuta (Poll & Matthes, 1962) species C. muzumanii isolated from Tylochromis polyle- (Fig. 1d); tribe Ectodini Perciformes: Cichlidae). pis (Boulenger, 1900) in the Congo is the only species Type-locality: Magara (3°44′S, 29°19′E), Lake Tanganyika, hitherto known to have a long hook pair I and short Burundi. pairs II-IV, VI and VII [41], and therefore C. aspiralis n. Type-material: Holotype: MRAC_vermes_37941; Paratypes: sp. is the second representative with this haptoral con- MRAC_vermes_37942; MRAC_vermes_37948; MRAC_ figuration in the Lake. In addition, the dorsal bar auri- vermes_37950; MRAC_vermes_37952; MRAC_vermes_ cles in C. aspiralis n. sp. have small hollow outgrowths 37953; MNHN HEL584; SAMC-A088696. on the anterior face, a feature observed in congeners in- Site in host: Gills. fecting representatives of Tylochromis Regan, 1920, Prevalence and intensity of infection: 75% (3/4); 4–28 such as the Tanganyikan C. mulimbwai and C. muzu- monogeneans per infected host. manii, both parasites of T. polylepis [41] (see above), ZooBank registration: To comply with the regulations set and the non-Tanganyikan C. chrysopiformis, C. djietoi outinarticle8.5oftheamended2012versionofthe and C. sigmocirrus Pariselle, Bitja Nyom & Bilong International Code of Zoological Nomenclature (ICZN) [36], Bilong, 2014 from T. sudanensis Daget, 1945 [46], C. details of the new species have been submitted to ZooBank. kothiasi Pariselle & Euzet, 1994 from T. jentinki The Life Science Identifier (LSID) of the article is urn:lsid: (Steindachner, 1862) [51], and also C. dageti,C. euzeti zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A475 and C. falcifer found on Hemichromis fasciatus Peters, 4242. The LSID for the new name Cichlidogyrus glaci 1857 [48]. Cichlidogyrus aspiralis n. sp. resembles C. cremoratus is urn:lsid:zoobank.org:act:54CA15E9-086B- centesimus Vanhove, Volckaert & Pariselle, 2011 iso- 4113-9C94-A56D47A14F51. lated from O. boops, O. nasuta and O. ventralis [23] Etymology: The specific epithet “glacicremoratus” is derived based on a similarly shaped MCO with a relatively slen- from the Latin “glacies” and “cremor” and refers to the der and long heel. However, C. aspiralis n. sp. is mainly shape of the proximal part of the MCO which reminds of distinguishable from C. centesimus by (i) the absence of an ice-cream. a spirally coiled thickening in the distal part of the copulatory tube (present in C. centesimus), and (ii) the Description presence of an accessory piece (absent in C. centesi- [Based on 14 specimens fixed in GAP; Fig. 8]. Body mus). Cichlidogyrus aspiralis n. sp. is also similar to C. 356–546 (459; n = 13) long, 88–145 (108; n = 13) wide at casuarinus Pariselle, Muterezi Bukinga & Vanhove, mid-body. Dorsal anchors relatively small with short 2015, described from Bathybates minor Boulenger, shaft and more pronounced guard (c.2 times length of 1906, in having a similarly shaped dorsal bar (see shaft), arched blade with slightly curved point: a = 19–23 above), a relatively long straight heel, and a sclerotized (21; n = 13); b = 14–20 (18; n = 13); c = 1–3 (2; n = 13), d vagina. However, the new species is easily distinguish- =4–6 (5; n = 13); e = 7–10 (8; n = 13). Dorsal bar slightly ablefromthelatterby(i)theshorterdorsal(39–43 vs curved, with straight long auricles: h = 14–18 (16; n = 52–64 μm) and ventral anchors (33–34 vs 47–59 μm, 13); w = 3–5 (4; n = 13); x = 23–27 (25; n = 13); y = 5–7 (ii) the shorter dorsal (45–47 vs 64–85 μm) and ventral (6; n = 13). Ventral anchors with shaft shorter than guard bars (37–40 vs 54–67), (iii) the hook pair I (long and and curved blade with arched point: a = 20–24 (21; n = well-developed in C. aspiralis n. sp. vs long but not 12); b = 17–19 (18; n =12); c=1–3 (2; n =12), d=4–6 thick in C. casuarinus), (iv) the shorter heel (14–18 μm (5; n = 12); e = 7–9 (8; n = 12). Ventral bar V-shaped, vs 40–59 μm), (v) the differently sized and shaped with constant width: w = 2–4 (3; n = 13); x = 24–28 (26; copulatory tube (short straight copulatory tube, 20–21 μm n = 13). Haptor with 7 pairs of short hooks, each hook in C. aspiralis n. sp. vs straight and pointed, with distal with erect thumb and shank comprised of 2 subunits: external wall exhibiting a typical spirally coiled thickening, pair I = 11–13 (12; n = 13) long, pair II = 11–13 (12; n = 34–44 μminC. casuarinus), and (vi) the shorter and 13) long, pair III = 12–14 (13; n = 13) long, pair IV = 13– differently shaped accessory piece (thin, proximally 15 (14; n = 13) long, pair V = 10–12 (11; n = 13) long, connected to the basal bulb, rounded and slightly enlarged pair VI = 13–15 (14; n = 13) long, pair VII = 12–14 (13; distally, 14–19 μm long in C. aspiralis n. sp. vs simple and n = 13) long. Male copulatory organ composed of Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 11 of 21

Fig. 8 Sclerotized structures of Cichlidogyrus glacicremoratus n. sp. ex Ophthalmotilapia nasuta. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece irregularly shaped basal bulb and long, wavy copulatory Type-locality: Magara (3°44′S, 29°19′E), Lake Tanganyika, tube with thick wall, constricted and curved approxi- Burundi. mately at proximal third, with wide terminal opening: Type-material: Holotype: MRAC_vermes_37942. Paratypes: MCO = 31–41 (37; n =14);Ct=42–47 (45; n = 14). Small, MRAC_vermes_37949; MRAC_vermes_37951; MNHN irregular sclerotized structure flange-like, probably part HEL585; SAMC-A088697. of the accessory piece, surrounds basal bulb and is Site in host: Gills. considered to be the heel, He = 1–2(1;n =14). Prevalence and intensity of infection: 75% (3/4); 1–5 Accessory piece with proximal constrictions, distal monogeneans per infected host. part similar to copulatory tube in thickness, ending in ZooBank registration: To comply with the regulations a composed portion, one extremity shorter than the set out in article 8.5 of the amended2012 version of the other, Ap = 28–35 (32; n = 14). Vagina non-sclerotized. International Code of Zoological Nomenclature (ICZN) [36], details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid: zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D-B1D4A47 Differential diagnosis 54242. The LSID for the new name Cichlidogyrus rectangulus Cichlidogyrus glacicremoratus n. sp. belongs to the is urn:lsid:zoobank.org:act:7CFFD130-2733-46CF-984C- same group as C. milangelnari n. sp., C. jeanloujustinei 48482EAB2734. n. sp., and C. evikae n. sp. These species share the small Etymology: The specific name “rectangulus” is derived from size of all hook pairs. Cichlidogyrus glacicremoratus n. sp. the Latin “rectangulum” which refers to the geometric shape is similar to C. vandekerkhovei isolated from O. boops, O. of the heel. ventralis and O. nasuta [23] regarding the morphology of the dorsal and ventral anchors. However, the new species Description is easily distinguishable from the latter by (i) the shorter [Based on 12 specimens fixed in GAP; Fig. 9]. Body 307– dorsal bar auricles (14–18 vs 24–34 μm), (ii) the heel 568 (513; n = 7) long, 109–165 (142; n =8)wideatmid- (irregular flange-like vs well-developed), (iii) the copu- body. Dorsal anchors with shaft slightly shorter than guard, latory tube (constricted proximally, wavy with wide blade curved in distal third, with arched point: a = 26–29 terminal opening vs narrowing distally), and (iv) the (28; n =6);b=22–26 (24; n =6);c=6–8(7;n = 6), d = 8– accessory piece (curved vs straight). 12 (10; n =6);e=6–8(7;n = 6). Dorsal bar strongly arched, thick in middle part, with straight narrow auricles: h = 12– Cichlidogyrus rectangulus n. sp. 18 (14; n =6);w=5–7(6;n =6);x=25–29 (27; n =6),y= 6–8(7;n = 6). Ventral anchors with more pronounced Type-host: Ophthalmotilapia nasuta (Poll & Matthes, 1962) guard than shaft (c.2 times length of shaft), curved blade (Fig. 1d); tribe Ectodini (Perciformes: Cichlidae). with arched point: a = 25–27 (26; n =6);b=21–25 (22; Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 12 of 21

n =6); c=6–8(7;n =6),d=13–15 (14; n =6);e=8–12 representative in the lake. Cichlidogyrus rectangulus n. (9; n = 16). Ventral bar V-shaped, with constant width: sp. shares the host species O. nasuta with C. sturm- w=4–8(6;n =7); x=26–38 (34; n = 7). Haptor with baueri. In addition, both species possess similarly-sized 5 pairs of long hooks, hooks I and V shorter (sensu transversal bars, a similarly-shaped accessory piece, and [11, 37]); each hook with erect thumb and shank a heel in the MCO. However, C. rectangulus n. sp. differs comprised of 2 subunits: pair I = 17–19 (18; n =6) from C. sturmbaueri by the (i) longer dorsal anchors long, pair II = 32–38 (35; n = 6) long, pair III = 37–41 (26–29 vs 19–21 μm), (ii) longer hook pairs (almost (39; n =6) long, pair IV=38–41 (40; n =6) long, pair twice as long in C. rectangulus n. sp. compared to C. V=13–15 (14; n = 5) long, pair VI = 32–41 (36; n =6) sturmbaueri), (iii) size and shape of the heel (long rect- long, pair VII = 32–41 (37; n = 6) long. Male copula- angular, 18–22 μm vs short heel, 4–7 μm), (iv) longer tory organ bulky, with copulatory tube beginning in copulatory tube (42–53 vs 34–39 μm), and (v) longer ovoid bulb and relatively thick wall, S-shaped and accessory piece (32–36 vs 24–28 μm). narrower in distal portion: MCO = 57–65 (62; n =9); Ct = 42–53 (47; n = 9). Heel long, thick, rectangular, Cichlidogyrus discophonum n. sp. He = 18–22 (20; n = 12). Accessory piece linked to basal bulb by broad connection, thick and curved in Type-host: Aulonocranus dewindti (Boulenger, 1899) (Fig. 1e); middle, with bifurcate ending: Ap = 32–36 (34; n =9). tribe Ectodini (Perciformes: Cichlidae). Vagina non-sclerotized. Type-locality: Nyaruhongoka (3°41′S, 29°20′E), Lake Tanganyika Burundi. Differential diagnosis Type-material: Holotype: MRAC_vermes_37945. Paratypes: Based on the haptoral sclerites, C. rectangulus n. sp. be- MRAC_vermes_37945; MRAC_vermes_37956; SAMC- longs to the group of species with shorter hook pair I A088700. (pair V with larval size) and longer pairs II-IV, VI and Site in host: Gills. VII (see [21]), a copulatory tube without a swollen prox- Prevalence and intensity of infection: 33% (1/3); 1–8 imal portion, and a non-sclerotized vagina (see [37]). monogeneans per infected host. This group includes a single species, C. sturmbaueri ZooBank registration: To comply with the regulations Vanhove, Volckaert & Pariselle, 2011, a parasite previ- set out in article 8.5 of the amended 2012 version of the ously found on O. nasuta and O. ventralis. The latter International Code of Zoological Nomenclature (ICZN) was the first species of Cichlidogyrus hitherto described [36], details of the new species have been submitted to from endemic Tanganyikan cichlids displaying short ZooBank. The Life Science Identifier (LSID) of the article is hook pair I and long hook pairs II-IV, VI and VII (see urn:lsid:zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D- [23]), and therefore C. rectangulus n. sp. is the second B1D4A4754242. The LSID for the new name Cichlidogyrus

Fig. 9 Sclerotized structures of Cichlidogyrus rectangulus n. sp. ex Ophthalmotilapia nasuta. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 13 of 21

discophonum is urn:lsid:zoobank.org:act:CFC631DE-63E3- Heel absent. Accessory piece short, with 2 thick distinct 4A0C-BC20-AD2FC3AB3CDA. parts, twisted distally, ending in hook, Ap = 15–22 Etymology: The specific name “discophonum” is derived (18; n = 8). Vagina non-sclerotized. from the Latin “discophonum”, meaning compact disk reader, which refers to the characteristic shape of the Differential diagnosis copulatory organ. Cichlidogyrus discophonum n. sp. belongs to the same morphological group as C. milangelnari n. sp., C. jean- Description loujustinei n. sp., C. evikae n. sp. and C. glacicremoratus [Based on 8 specimens fixed in GAP; Fig. 10]. Body n. sp. This species is similar to C. milangelnari n. sp. in 537–658 (612; n = 5) long, 88–146 (111; n = 6) wide at the morphology of the haptoral structures (ventral bar mid-body. Dorsal anchors with short shaft and more and hook pairs) and in having a MCO without a heel. pronounced guard (c.2 times length of shaft) and curved However, it is easily distinguishable from C. milangelnari blade with slightly arched point: a = 21–23 (22; n =6);b= n. sp. by (i) the size of the dorsal and ventral anchors 18–21 (19; n =6);c=1–3(2;n =6);d=4–6(5;n =6);e= [21–22 μm (same size for both anchors) vs 33–36 and 7–9(8;n = 6). Dorsal bar slightly arched, thick in middle 32–35 μm, respectively], (ii) the copulatory tube (large part, with blunt endings and long auricles: h = 18–20 (19; ovoid basal bulb and C-shaped copulatory tube, thick n =7);w=3–5(4;n =7);x=22–27 (25; n =7);y=4–7(5; proximally and tapering distally vs small bulb, curved at n = 7). Ventral anchors with shorter shaft than guard and the distal third), and (iii) the accessory piece (short with slightly arched point: a = 20–22 (21; n =6);b=18–20 (19; two thick distinct parts, distally twisted ending in hook, n =6);c=1–3(2;n =6),d=4–6(5;n =6);e=5–8(7;n = 15–22 μm long vs thick and two superimposed parts 6). Ventral bar V-shaped: w = 2–4(3;n =7); x=26–28 with forked ending, 34–38 μm long). Cichlidogyrus dis- (27; n = 7). Haptor with 7 pairs of short hooks, hooks V cophonum n. sp. resembles C. makasai, a gill parasite of with larval size (see above); each hook with erect thumb the ectodine cichlids O. nasuta, O. boops (Boulenger, and shank comprised of 2 subunits: pair I = 10–12 (11; n 1901) and O. ventralis (Boulenger, 1898) [23] by the =12)long,pairII=11–13 (12; n =12)long,pairIII=12– morphology of haptoral and reproductive structures: (i) 14 (13; n = 6) long, pair IV = 13–15 (14; n = 6) long, pair small, slender dorsal and ventral anchors with shorter V=9–11 (10; n = 5) long, pair VI = 13–16 (15; n =7)long, shaft than guard, and (ii) curved copulatory tube taper- pair VII = 11–15 (13; n = 6) long. Male copulatory organ ing distally. However, it can be easily distinguished from composed of long C-shaped copulatory tube, thick C. makasai by (i) the absence of a heel (vs pronounced proximally, with large ovoid basal bulb, tapering dis- heel in C. makasai), (ii) the length of the copulatory tube tally: MCO = 26–34 (29; n =8);Ct=41–47 (44; n =8). (41–47 vs 69–79 μm), and (iii) the shape of the accessory

Fig. 10 Sclerotized structures of Cichlidogyrus discophonum n. sp. ex Aulonocranus dewindti. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 14 of 21

piece (two thick distinct parts, twisted distally with hook with erect thumb and shank comprised of 2 sub- hook-like ending vs simple and slightly bent at distal third units: pair I = 22–24 (23; n = 3) long, pair II = 20–22 (21; with spanner-like ending). Cichlidogyrus discophonum n. n = 3) long, pair III = 21–23 (22; n = 3) long, pair IV = sp. is easily distinguishable from C. vandekerkhovei (also 21–24 (22; n = 3) long, pair V = 10–12 (11; n = 3) long, found on O. boops, O. nasuta and O. ventralis [23]) in hav- pair VI = 15–16 (16; n = 3) long, and pair VII = 17–18 ing a shorter accessory piece with different shape (two thick (18; n = 3) long. Male copulatory organ beginning in distinct parts, distally twisted ending in hook, 15–22 μm vs ovoid bulb, with relatively long, curved and thin copula- straight with forked ending, one extremity shorter than the tory tube: MCO = 52–56 (54; n = 8); Ct = 40–43 (42; n = other and sometimes crossed, 24–34 μm). In addition, C. 8). Heel long, straight, He = 14–17 (16; n = 8). Accessory discophonum n. sp. lacks a heel unlike C. vandekerkhovei. piece thin, straight, proximally with thin elbow-shaped connection to copulatory tube, Ap = 26–31 (29; n = 8). Cichlidogyrus pseudoaspiralis n. sp. Vagina non-sclerotized.

Type-host: Aulonocranus dewindti (Boulenger, 1899) (Fig. 1e); Differential diagnosis tribe Ectodini (Perciformes: Cichlidae). The new species C. pseudoaspiralis n. sp. belongs to the Type-locality: Nyaruhongoka (3°41′S, 29°20′E), Lake group of Cichlidogyrus spp. characterized by a long hook Tanganyika, Burundi. pair I (pair V with larval size) and short pairs II-IV, VI Type-material: Holotype: MRAC_vermes_37944. Paratypes: and VII (see [21]), a copulatory tube without a swollen MRAC_vermes_37955; MNHN HEL587; SAMC-A088699. proximal portion and a non-sclerotized vagina (see [37]). Site in host: Gills. This group includes C. arfii Pariselle & Euzet, 1995 [25]; Prevalence and intensity of infection: 33% (1/3); 1–8 C. berradae Pariselle & Euzet, 2003 [38]; C. dionchus monogeneans per infected host. Paperna, 1968 [52]; C. halinus Paperna, 1969 [53]; C. ZooBank registration: To comply with the regulations set muzumanii Muterezi Bukinga, Vanhove, Van Steenberge out in article 8.5 of the amended 2012 version of the & Pariselle, 2012 [41]; C. nuniezi Pariselle & Euzet, 1998 International Code of Zoological Nomenclature (ICZN) [47]; C. papernastrema Price, Peebles & Bamford, 1969 [36], details of the new species have been submitted to [54]; C. philander Douëllou, 1993 [55]; C. quaestio ZooBank. The Life Science Identifier (LSID) of the article is Douëllou, 1993 [55]; C. reversati Pariselle & Euzet, 2003 urn:lsid:zoobank.org:pub:3B9F16F6-8E3F-44F5-8D5D- [38]; and C. yanni Pariselle & Euzet, 1996 [56]. Cichlido- B1D4A4754242. The LSID for the new name Cichlidogyrus gyrus pseudoaspiralis n. sp. is similar to the new species pseudoaspiralis is urn: urn:lsid:zoobank.org:act:F69F41FB- C. aspiralis n. sp. described above in the morphology of C806-40E4-99F0-7FE8BA1FBCC7. the haptoral structures (hook pairs, dorsal and ventral Etymology: The specific epithet is the combination of anchors) and the relatively straight heel. However, it is the Latin prefix “pseudo” and “aspiralis”, referring to easily distinguished from C. aspiralis n. sp. by (i) the the similarity of the new species to C. aspiralis n. sp. shorter dorsal (31–32 vs 45–47 μm) and ventral bars described above. (30–32 vs 37–40 μm), (ii) the longer copulatory tube (40–43 vs 20–21 μm), (iii) the longer accessory piece Description (26–31 vs 14–19 μm), and (iv) the vagina (absent in C. [Based on 8 specimens fixed in GAP; Fig. 11]. Body pseudoaspiralis n. sp.). Further, as in C. aspiralis n. sp., 545–714 (639; n = 3) long, 113–129 (121; n = 3) wide at the dorsal bar in C. pseudoaspiralis n. sp. is similar to mid-body. Dorsal anchors with short shaft and elongated that exhibited by the monogenean species of the ty- guard (c.5 times length of shaft) and short, slightly bent lochromine cichlid hosts (see above). Thus, the new blade and curved point: a = 37–42 (40; n = 3); b = 27–28 species is mainly distinguishable from C. muzumanii (27; n = 3); c = 2–4 (3; n = 3); d = 13–16 (15; n = 3); e = by (i) the differently sized and shaped dorsal bar (31– 6–7 (7; n = 3). Dorsal bar straight, thick, long with short 32 vs 45–62 μm), (ii) the copulatory tube (relatively appendages of anterior face of dorsal transverse bar: h = long, curved and thin copulatory tube, 40–43 μminC. 6–7 (7; n = 3); w = 4–6 (5; n = 3); x = 31–32 (32; n = 3); y pseudoaspiralis n. sp. vs penis starting in a considerable =14–15 (14; n = 3). Ventral anchors with shaft shorter bulb, with broad and thick walled spirally coiled tube, than guard, blade longer than in dorsal anchors, with 57–68 μminC. muzumanii), (iii) the heel (14–17 vs 4– arched point: a = 37–40 (39; n = 3); b = 34–37 (36; n = 3); 7 μminC. muzumanii), and (iv) the accessory piece (thin c=2–4 (3; n = 3); d = 9–11 (10; n = 3); e = 10–11 (10; n elbow-shaped connection to the copulatory tube, 26– = 3). Ventral bar V-shaped: w = 3–4 (4; n = 3); x = 30–32 31 μminC. pseudoaspiralis n. sp. vs not attached to the (31; n = 3). Hook pair I with well-developed shank, long copulatory tube, 17–20 μminC. muzumanii). In addition, in comparison with remaining pairs which are similarly like C. aspiralis n. sp., C. pseudoaspiralis n. sp. is easily dis- short (sensu [11, 37]), pair V retains its larval size; each tinguishable from C. centesimus by the absence of a spiral Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 15 of 21

Fig. 11 Sclerotized structures of Cichlidogyrus pseudoaspiralis n. sp. ex Aulonocranus dewindti. Abbreviations: DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I-VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece in the copulatory tube and the presence of an accessory congener C. centesimus possesses long and large hook piece (see above). Further, the new species is distinguish- pair I, and long pairs II-IV, VII and VII, a hook config- able from C. casuarinus by (i) the shorter dorsal (37–42 vs uration observed in the type-species C. arthracanthus 52–64 μm) and ventral anchors (37–40 vs 47–59 μm), (ii) Paperna, 1960 described on Coptodon zillii (Gervais, the shorter dorsal (31–32 vs 64–85 μm) and ventral bars 1848) (see [23, 24], figures and diagnosis). According to (30–32 vs 54–67 μm), (iii) the hook pair I (long and well Vignon et al. [21], C. arthracanthus displays character- developed vs long but not thick), (iv) the shorter heel (14– istic configuration of the hook pairs and therefore be- 17 vs 40–59 μm), (v) the longer and differently shaped longs to none of the major groups. Moreover, in Lake copulatory tube (relatively long, curved and thin copulatory Tanganyika, C. casuarinus found on B. minor and C. tube, 40–43 μminC. pseudoaspiralis n. sp. vs straight and nshomboi Muterezi Bukinga, Vanhove, Van Steenberge pointed, with distal external wall exhibiting a typical spir- & Pariselle, 2012 described from Boulengerochromis ally coiled thickening, 34–44 μminC. casuarinus), and (vi) microlepis (Boulenger, 1899) possess a characteristic the sclerotized vagina (present in C. casuarinus). hook configuration with a thin hook pair I and long pairs II-IV, VI and VII [28, 41]. Cichlidogyrus rectangu- Remarks on the diversity of the sclerotized structures in lus n. sp. (Fig. 9) exhibits a short hook pair I and long species of Cichlidogyrus pairs II-IV, VI and VII, with C. sturmbaueri hitherto as The new species of Cichlidogyrus described herein be- sole known representative of this morphological group long to three morphological groups according to the in Lake Tanganyika ([23], see above). Finally, following relative length of their haptoral hook pairs following Pariselle & Euzet [11], some other species of Cichlido- Pariselle & Euzet [11], and Vignon et al. [21]. As gyrus, with their hook pairs, “escape” from the classifi- already mentioned in the diagnoses, C. milangelnari n. cation based on the hook configuration: we find the sp. (Fig. 4), C. jeanloujustinei n. sp. (Fig. 5), C. evikae n. non-Tanganyikan species C. nandidae Birgi & Lambert, sp. (Fig. 6), C. glacicremoratus n. sp. (Fig. 8), and C. dis- 1986 found on the non-cichlid host Polycentropsis abbre- cophonum n. sp. (Fig. 10) belong to the group of species viata Boulenger, 1901 possess long hook pairs I-IV, VI and with short hook pairs I-IV, IV and VII. However, C. evi- VII (pair I not large) [57], C. kothiasi showing well devel- kae n. sp. displays a characteristic shape of the hooks oped and long pair I with resembling pairs II-IV, VI and (broad thumb with a proximal protrusion). The closely VII in size [51], and finally C. chrysopiformis Pariselle, Bitja related C. aspiralis n. sp. (Fig. 7) and C. pseudoaspiralis Nyom & Bilong Bilong, 2013 from T. bemini Thys van der n. sp. (Fig. 11) belong to the group of species with long Audenaerde, 1972, with hook pair I of medium size, but and well-developed hook pair I (pair V with larval size) not large, and short pairs II-IV, VI and VII [37]. and short pairs II-IV, VI and VII. Conversely, according Based on the original descriptions and the systematic to the definition of Pariselle & Euzet [11], its Tanganyikan review of African monogenean species published by Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 16 of 21

Pariselle & Euzet [11], an overview of 111 species within Euzet, 1995 [26]; C. gillesi Pariselle, Bitja Nyom & Bilong Cichlidogyrus (including the new species described in Bilong, 2013 [38]; C. guirali Pariselle & Euzet, 1997 [40]; C. this study) is provided (see Additional file 1: Table S1) hemi Pariselle & Euzet, 1998 [47]; C. kouassii N’Douba, focusing on the structural diversity of two reproductive Thys van den Audenaerde & Pariselle, 1997 [61]; C. legen- organ features, i.e. the vagina and the heel as part of the drei Pariselle & Euzet, 2003 [38]; C. lemoallei Pariselle & MCO, and reviewing their presence/absence in Cichlido- Euzet, 2003 [38]; C. microscutus Pariselle & Euzet, 1996 gyrus spp. First, our summary data (Additional file 1: [56]; C. ouedraogoi Pariselle & Euzet, 1996 [56]; C. paganoi Table S1) show that a total of 56 Cichlidogyrus spp. de- Pariselle & Euzet, 1997 [40]; C. testificatus Dossou, 1982 scribed on non-Tanganyikan cichlid hosts exhibit a [49]; C. thurstonae Ergens, 1981 [60]; C. tiberianus sclerotized vagina. This feature has been mentioned in Paperna, 1960 [24]; and C. vexus Pariselle & Euzet, 1995 the original descriptions and drawings. Conversely, in 19 [26] (see Additional file 1: Table S1). species of Cichlidogyrus, the vagina is non-sclerotized Finally, as shown in Additional file 1: Table S1, a heel and therefore, not visible. However, a few species of was reported, drawn and measured in most of the descrip- Cichlidogyrus show a sclerotization in the vagina but only tions (70 species). Next, few papers reported the presence in the opening. In the last two cases, the authors did not of the heel without measurements (19 species) or only in provide any drawing or morphological characterization of the drawings of the MCO (12 species). In such case, the the vagina. From Lake Tanganyika, we listed only five structure of the heel was deduced and a short character- Cichlidogyrus spp. possessing a sclerotized vagina while isation based on the original drawings is suggested. More- the vagina of the remaining species (27 species) is non- over, seven species of non-Tanganyikan Cichlidogyrus are sclerotized. Within the first haptoral group (i.e. short hook totally lacking a heel: C. arfii, C. haplochromii, C. karibae, pairs I-IV, IV and VII), we listed 18 non-Tanganyikan spe- C. longicirrus,C.longipenis, C. sanseoi,andC. tilapiae cies exhibiting a sclerotized vagina. These are C. acerbus (see Additional file 1: Table S1); in Lake Tanganyika only Dossou, 1982 [49]; C. amieti Birgi & Euzet, 1983 [58]; C. C. attenboroughi shows this character ([16], see above and amphoratus Pariselle & Euzet, 1996 [56]; C. berrebii Pari- Additional file 1: Table S1). The original drawing of the selle & Euzet, 1994 [51]; C. cirratus Paperna, 1964 [59]; C. non-Tanganyikan species C. papernastrema shows a cubitus Dossou, 1982 [49]; C. djietoi [46]; C. giostrai MCO lacking a heel, while the holotype slide reveals a vis- Pariselle, Bilong Bilong & Euzet, 2003 [45]; C. karibae ible heel on the bottom of the basal bulb ([54, 39]). Fur- Douëllou, 1993 [55]; C. levequei Pariselle & Euzet, 1996 thermore, different heel sizes are sometimes found in [56]; C. louipaysani Pariselle & Euzet, 1995 [26]; C. mvogoi species exhibiting a similar heel shape, i.e. C. berradae iso- Pariselle, Bitja Nyom & Bilong Bilong, 2014 [46]; C. njinei lated from Tilapia cabrae Boulenger, 1899, C. digitatus Pariselle, Bilong Bilong & Euzet, 2003 [45]; C. ornatus and C. yanni from C. zillii which present a relatively thin Pariselle & Euzet, 1996 [56]; C. pouyaudi Pariselle & and slender heel, C. ergensi isolated from C. zillii and C. Euzet, 1994 [51]; C. sclerosus Paperna & Thurston, 1969 ouedraogoi from T. coffea Thys van den Audenaerde, 1970 [44]; C. slembroucki Pariselle & Euzet, 1998 [47]; and C. present a bean-shaped heel [37, 49, 56]. zambezensis Douëllou, 1993 [55]. From Lake Tanganyika, a single species, C. mbirizei Muterezi Bukinga, Vanhove, Discussion Van Steenberge & Pariselle, 2012, possesses a sclerotized Monogeneans are an ideal group of organisms for study- vagina [46]. The three Tanganyikan species, C. casuarinus, ing evolutionary mechanisms because of their remark- C. centesimus and C. nshomboi, belonging to none of the able species richness, morphological diversity and wide morphological groups (see above) were originally de- distribution [62]. They developed a broad range of spe- scribed lacking a sclerotized vagina. Later, this feature has cialized attachment organs, probably linked with host been reported by Pariselle et al. Unlike C. rectangulus n. specificity [63]. Considering the high diversity of cichlids sp. and C. sturmbaueri showing the last hook configur- in the Lake Tanganyika where the cichlid flocks consist ation (i.e. short hook pair I and long pairs II-IV, VI and of hundreds genetically and morphologically highly di- VII, see above), all Cichlidogyrus spp. described so far be- verse endemic species [64], we can hypothesize a high longing to this morphological group (all non-Tanganyikan) diversity of cichlid-specific monogenean parasites, e.g. possess a sclerotized vagina. These are C. aegypticus Cichlidogyrus spp. As already mentioned, the number of Ergens, 1981 [60]; C. agnesi Pariselle & Euzet, 1995 [26]; C. species of Cichlidogyrus studied and described so far re- anthemocolpos Dossou, 1982 [49]; C. bilongi Pariselle & mains small compared to the extraordinary diversity of Euzet, 1995 [26]; C. bonhommei Pariselle & Euzet, 1998 their potential cichlid hosts in Lake Tanganyika. [47]; C. bouvii Pariselle & Euzet, 1997 [40]; C. dossoui Our investigation of five cichlid species from the Douëllou, 1993 [55]; C. douellouae Pariselle, Bilong Bilong Burundi coast revealed eight new Cichlidogyrus spp. & Euzet, 2003 [45]; C. ergensi Dossou, 1982 [49]; C. flexi- which are described herein. Three of these cichlid spe- colpos Pariselle & Euzet, 1995 [26]; C. gallus Pariselle & cies belong to tribes that were not previously Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 17 of 21

investigated for the presence of monogeneans, i.e. the with short straight auricles (see diagnosis), and a MCO with cyprichromine C. microlepidotus and the eretmodines E. a straight heel. The morphotype of C. discophonum n. sp., marksmithi and T. irsacae; these are new host records C. glacicremoratus n. sp., C. makasai and C. vandekerkhovei for representatives of Cichlidogyrus. Three species of displays short hook pairs and a dorsal bar with long auri- Cichlidogyrus were described from these cichlids: C. cles. The morphotype represented by C. rectangulus n. sp. milangelnari n. sp. from C. microlepidotus, C. jeanlou- and C. sturmbaueri presents a curved dorsal bar, long hook justinei n. sp. from E. marksmithi and C. evikae n. sp. pairs II-IV, VI and VII and a MCO with a short copulatory from T. irsacae. Several haptoral (small dorsal and ven- tube associated to an h-shaped accessory piece. tral anchors, dorsal bar relatively short and ventral bar The morphological diversity within the newly de- similar in shape and size), as well as some general copu- scribed Cichlidogyrus spp. isolated from Ophthalmo- latory organ characteristics (ovoid basal bulb prolonged tilapia in Burundi and its congeners from southernmost into a copulatory tube with thick wall and accessory localities (Table 1) is probably influenced by the dis- piece composed of two superimposed parts) clearly sug- tribution of the cichlid host in the lake and by an allo- gest an affinity between C. jeanloujustinei n. sp. and C. patric evolution. Indeed, it is well known now that Lake evikae n. sp., and therefore reflect the phylogenetic rela- Tanganyika harbours several Ophthalmotilapia spp. tionship between their hosts E. marksmithi and T. irsa- which vary morphologically and genetically along Lake cae, both belonging to the tribe Eretmodini. Similar Tanganyika [67, 68]. They are well distributed along the observations, i.e. the morphological similarity of the lake [69]; Konings [70] reported five different popula- sclerotized parts of closely related Tanganyikan Cichlido- tions of O. nasuta in (i) Uvira (D.R. Congo) where the gyrus spp. have been reported for tropheine hosts and holotype was caught, (ii) the Burundese shore (similar to host choice was clearly associated to phylogenetic re- the Uvira population), (iii) the Ubwari Peninsula (Eastern latedness of the cichlid hosts [15, 17, 42, 65]. D.R. Congo) and further south to Kalemie (D.R. Congo), Furthermore, the distribution of the morphologically (iv) south of Kalemie along the western shore as far as similar new species C. pseudoaspiralis n. sp. and C. Chimba in Zambia with a small isolated population at aspiralis n. sp. on A. dewindti and O. nasuta, respectively Cape Nangu across Cameron Bay, and (v) Zambian and (O. nasuta was previously investigated for the presence of Tanzanian waters which harbour the widest range of all monogenean species in more southern localities in Lake Ophthalmotilapia spp. [69, 71]. Thus, it would be inte- Tanganyika) mirrors the relatedness between the two resting to investigate whether the Cichlidogyrus fauna in hosts, both belonging to the tribe Ectodini [66]. Indeed, Burundese O. ventralis follows a similar geographical vari- the two new Cichlidogyrus spp. exhibit the same morpho- ation in community composition as found for O. nasuta. type and similarities in the shape and/or size of the In addition, the endemic O. heterodonta (Poll & Matthes, sclerotized structures are mainly visible in the ventral and 1962) inhabiting various localities in the northern and dorsal anchors, the hook pairs and the heel in the MCO central parts of Lake Tanganyika is the only species of this (see diagnoses and drawings). Intraspecific variability was genus that has never been investigated for its parasite reported in the heel length of C. centesimus, a species exhi- fauna. A study of gill ectoparasites on this cichlid host biting the same morphotype as the two new species de- may provide additional data on the lake’s parasite species scribed here, i.e. C. aspiralis n. sp. and C. pseudoaspiralis diversity. Moreover, it has been reported that Ophthalmo- n. sp. [23]. However, the shorter dorsal and ventral bars in tilapia spp. show low genetic diversity but present a high addition to the longer MCO (longer copulatory tube and morphological diversity and colour plasticity, and even accessory piece) and non-sclerotized vagina in C. pseu- morphologically intermediate populations among geo- doaspiralis n. sp., make it distinct from C. aspiralis n. sp. graphically separated species were found [65]. Thus, the Our study of O. nasuta in Burundi revealed the pres- geographical variation of Tanganyikan O. nasuta probably ence of three new monogenean species i.e. C. aspiralis n. played a role in its Cichlidogyrus spp. speciation and dis- sp., C. glacicremoratus n. sp. and C. rectangulus n. sp.: tribution. Few studies have been performed on Cichlido- these were well differentiated from the four species pre- gyrus spp. infecting Tanganyikan cichlids incorporating viously described by Vanhove et al. [23] on O. nasuta the diversity among host populations. Cichlidogyrus spe- and its congeners (see Table 1). Morphologically, C. cies richness and assemblage composition in several sym- aspiralis n. sp., C. pseudoaspiralis n. sp., C. discophonum patric Simochromis diagramma (Günther, 1894) and n. sp. and C. rectangulus n. sp., share various sclerotized Tropheus moorii Boulenger, 1898 populations in southern features with C. centesimus, C. makasai, C. vandekerkhovei Lake Tanganyika (Zambia) was studied by Grégoir et al. and C. sturmbaueri. Three distinct morphotypes were dis- [72]. These authors showed seven morphologically distinct tinguished. The first morphotype represented by C. aspira- Cichlidogyrus spp. and significant variation of the parasite lis n. sp., C. pseudoaspiralis n. sp. and C. centesimus,is assemblages among sampling sites for T. moorii in con- characterized mainly by a long hook pair I, a dorsal bar trast to S. diagramma which displayed a less species-rich Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 18 of 21

and more homogeneous parasite fauna. Grégoir et al. [72] sclerotized vagina unlike the remaining new Cichlidogyrus proposed that this difference is related to differences in species described herein. In fact, most Tanganyikan dispersal capacity and hence population structure of the Cichlidogyrus spp. exhibit a non-sclerotized vagina host species. (see above and Additional file 1: Table S1). On the The present study illustrates the morphological diver- other hand, a comparison of Cichlidogyrus spp. based sity of the sclerotized parts of monogeneans in cichlids on the vagina and the length of hook pairs revealed from Lake Tanganyika through the new species descrip- that only two Tanganyikan representatives of the tions and the checklist of Tanganyikan and non- group of species with short hooks I and long pairs II- Tanganyikan Cichlidogyrus spp. Four different haptoral IV, VI and VII, lack a sclerotized vagina. Our results morphotypes of Cichlidogyrus species have previously shed light on the necessity to elucidate the evolution- been reported [11, 21, 23, 38]. Haptoral characteristics ary scenarios and the significance of the sclerotization are usually used to differentiate between major lineages in the vagina in Cichlidogyrus spp. It would be inter- within Cichlidogyrus, whereas the morphology of the esting to analyse whether there is a correlation be- copulatory organ is more appropriate to distinguish be- tween the reproductive organs (presence/absence of the tween closely related species [17, 19, 23]. The correlation sclerotized vagina) and the haptoral sclerites (morphology between the different hook pairs and anchors in Cichli- of the hook pairs). dogyrus spp. was studied by Pariselle & Euzet [37] and Examination of all original drawings and descriptions of three main groups were defined, i.e. (i) long hook pair I, Cichlidogyrus spp. allowed us to highlight the high di- short hook pair VI and long anchors, (ii) short hook pair versity in the heel structure (Additional file 1: Table S1). I, short hook pair VI and medium-sized anchors, and fi- The descriptions of the type-species, i.e. C. arthracanthus nally, (iii) short hook pair I, long hook pair VI and small on C. zillii reported a sclerotized structure associated with anchors. Later, Pariselle & Euzet [11] standardized the the copulatory tube, different from the accessory piece length of the hooks by dividing their total length by the and the auxiliary plate [24, 47]. In fact, the sclerotized por- total length of the hook pair V (larval size). This method tion considered as a heel is part of the accessory piece was adopted to classify the length of the hook pairs i.e. [18]. This feature is absent in two of the newly described “short” or “long”. Vignon et al. [21] proposed an evolu- species, C. milangelnari n. sp. and C. discophonum n. sp., tionary scenario for the configuration of the hook pairs. representing the second record of Cichlidogyrus spp. Morphological data in addition to phylogenetic analysis parasitizing Tanganyikan cichlids that lack heel, the suggested that short hook pairs represent a putative first being C. attenboroughi from the benthochromine primitive feature state in Cichlidogyrus and species later B. horii [16]. A clear example of morphologically related developed large hook pair I and longer pairs II-IV, VI Cichlidogyrus spp. in endemic Tanganyikan cichlids is and VII [12, 21]. Further, the study of Vignon et al. [21] the long straight heel present in C. casuarinus, C. allowed to classify for instance C. kothiasi within the centesimus, C. aspiralis n. sp., C. pseudoaspiralis n. sp. group of species exhibiting short hook pairs and C. and C. nshomboi. The characteristic shape of the heel nandidae within the group possessing large pair I in addition to the spirally-coiled wall of the MCO in and short pairs II-IV, VI and VII. On the other hand, Cichlidogyrus spp. infecting Bathybatini (C. casuarinus), some Tanganyikan and non-Tanganyikan species (see Ectodini (C. centesimus, absent in C. aspiralis n. sp. and C. above), with their “new” hook configurations, were pseudoaspiralis n. sp., see above) and Boulengerochromini not yet described, and therefore, not included. (C. nshomboi) are found exclusively in these species Therefore, we suggest that there are more than four [16, 23, 28, 41]. previously reported haptoral groups. Moreover, due to the incomplete taxonomic coverage, it is still not Conclusions possible to fully elucidate the evolution of the differ- It is too early for conclusions about the role of host- ent haptoral configurations in Cichlidogyrus spp. It specificity in Lake Tanganyika due to limited data on would be interesting to re-investigate the structural ectoparasite monogeneans in this system. Further studies diversity of the hook pairs in Cichlidogyrus spp. and to investigate cichlid fishes in the lake for parasites be- identify the exact “borders” between the haptoral longing to Cichlidogyrus spp. are necessary. The high groups. morphological diversity of haptoral structures and repro- In addition to the haptoral sclerites, species of ductive organs of the new species described herein and Cichlidogyrus described so far can be clustered based other species identified so far confirms the existence of on the vagina being sclerotized or non-sclerotized. In various lineages of Cichlidogyrus in Lake Tanganyika. Cichlidogyrus spp. from Lake Tanganyika the vagina is However, further morphological studies and molecular sclerotized or not (see above). Cichlidogyrus pseudoaspiralis data are needed to elucidate their origin and evolution- n. sp. isolated from Burundese O. nasuta exhibits a ary history. Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 19 of 21

Additional file Deberiotstraat 32, B-3000 Leuven, Belgium. 4Centre for Environmental Sciences, Research Group Zoology: Biodiversity & Toxicology, Hasselt University, Agoralaan Gebouw D, B-3590 Diepenbeek, Belgium. Additional file 1: Table S1. Overview of the vagina and the heel structures of African Cichlidogyrus species. (DOCX 100 kb) Received: 16 November 2016 Accepted: 9 October 2017

Abbreviations MCO: Male copulatory organ References Acknowledgments 1. Maan ME, Sefc KM. Colour variation in cichlid fish: developmental mechanisms, The authors are most grateful to C. Sturmbauer (University of Graz, Austria), selective pressures and evolutionary consequences. Semin Cell Dev Biol. M. Reichard, R. Blažek (Czech Academy of Sciences, Czech Republic), A. 2013;24:516–28. Meyer (University of Konstanz, Germany), G. Banyankimbona (University of 2. Takahashi T, Koblmüller S. The adaptive radiation of cichlid fish in Lake Burundi), the Schreyen-Brichard family (Burundi/Belgium) and the technical Tanganyika: a morphological perspective. Int J Evol Biol. 2011;2011:620754. staff of Fishes of Burundi for fish sampling and identification. We thank our 3. Pariselle A, Boeger WA, Snoeks J, Bilong Bilong CF, Morand S, Vanhove colleagues and students E. Řehulková, I. Přikrylová, Š.Mašová, V. Michálková MPM. The monogenean parasite fauna of cichlids: a potential tool for host (Masaryk University, Czech Republic) for their help with fish dissection, para- biogeography. Int J Evol Biol. 2011;2011:471480. site isolation and fixation, and all the group working in the Laboratory of 4. Snoeks J. How well known is the ichthyodiversity of the large east African Parasitology (Masaryk University). Our special thanks go to R. Blažek for his lakes? Adv Ecol Res. 2000;31:17–38. help with fish dissection and providing us with the photographs of cichlid 5. Meyer BS, Matschiner M, Salzburger W. A tribal level phylogeny of Lake species, to J.-L. Justine for nomenclatural advice and to S. Koblmüller for fish Tanganyika cichlid fishes based on a genomic multi-marker approach. Mol identification, co-organisation of the fieldwork and scientific input on Tan- Phylogenet Evol. 2015;83:56–71. ganyika cichlids. We acknowledge both anonymous referees for contributing 6. Takahashi T, Sota T. A robust phylogeny among major lineages of the east substantially to improving the manuscript. African cichlids. Mol Phylogenet Evol. 2016;100:234–42. 7. Barluenga M, Meyer A. Phylogeography, colonization and population history Funding of the Midas cichlid species complex (Amphilophus spp.) in the Nicaraguan This research was funded by the Czech Science Foundation project no. crater lakes. BMC Evol Biol. 2010;10:326. P505/12/G112 - European Centre of Ichtyoparasitology (ECIP). 8. Vanhove MPM, Hablützel PI, Pariselle A, Šimková A, Huyse T, Raeymaekers JAM. Cichlids: a host of opportunities for evolutionary parasitology. Trends Availability of data and materials Parasitol. 2016;32:820–32. The data supporting the conclusions of this article are included within the 9. Pariselle A, Morand S, Deveney MR, Pouyaud L. Parasite species richness of article. The type-material of the new species described in this study is closely related hosts : historical scenario and “genetic” hypothesis. In: deposited in the invertebrate collection of the Royal Museum for Central Combes C, Jourdane J, editors. Taxonomie, écologie et évolution des Africa (RMCA), Tervuren, Belgium; the Muséum National d’Histoire Naturelle métazoaires parasites (livre hommage à ). Perpignan: Perpignan (MNHN), Paris, France; and the Iziko South African Museum (SAMC), Cape University Press; 2003. p. 147–63. Town, Republic of South Africa (see taxonomic summaries for details on 10. Vanhove MPM, Pariselle A, Van Steenberge M, Raeymaekers JAM, Hablützel repositories and accession numbers). PI, Gillardin C, et al. Hidden biodiversity in an ancient lake: phylogenetic congruence between lake Tanganyika tropheine cichlids and their Authors’ contributions monogenean flatworm parasites. Sci Rep. 2015;5:13669. AŠ and MPMV designed and supervised this study. MPMV co-organised the 11. Pariselle A, Euzet L. Systematic revision of dactylogyridean parasites field trip, parasite collection, preparation of specimens and provided scientific (Monogenea) from cichlid fishes in Africa, the Levant and Madagascar. background in the field of cichlids from Lake Tanganyika and their gill Zoosystema. 2009;31:849–98. monogeneans. CR performed the microscopical observations, identified the 12. Mendlová M, Desdevises Y, Civáňová K, Pariselle A, Šimková A. Monogeneans new species, drew the hard parts and constructed the checklist. MPMV and of west African cichlid fish: evolution and cophylogenetic interactions. PLoS CR wrote the paper. AŠ, CR and MPMV discussed the results. AŠ and MPMV One. 2012;7:e37268. revised the manuscript. All authors read and approved the final manuscript. 13. Kmentová N, Gelnar M, Koblmüller S, Vanhove MPM. First insights into the diversity of gill monogeneans of “Gnathochromis” and Limnochromis Ethics approval (Teleostei, Cichlidae) in Burundi: do the parasites mirror host ecology and All applicable institutional, national and international guidelines for the care phylogenetic history? PeerJ. 2016;4:e1629. and use of were followed. The research was approved by the Ethics 14. Kmentová N, Gelnar M, Mendlová M, Van Steenberge M, Koblmuller S, Committee of Masaryk University. The approval number which allows us to Vanhove MPM. Reduced host-specificity in a parasite infecting non-littoral work with vertebrate animals is CZ01308. Lake Tanganyika cichlids evidenced by intraspecific morphological and genetic diversity. Sci Rep. 2016;6:39605. Consent for publication 15. Pariselle A, Van Steenberge M, Snoeks J, Volckaert FAM, Huyse T, Vanhove Not applicable. MPM. Ancyrocephalidae (Monogenea) of Lake Tanganyika: does the Cichlidogyrus parasite fauna of Interochromis loocki (Teleostei, Cichlidae) ’ – Competing interests reflect its host s phylogenetic affinities? Contrib Zool. 2015;84:25 38. The authors declare that they have no competing interests. 16. Kmentová N, Gelnar M, Koblmüller S, Vanhove MPM. Deep-water parasite diversity in Lake Tanganyika: description of two new monogenean species from benthopelagic cichlid fishes. Parasit Vectors. 2016;9:426. Publisher’sNote 17. Van Steenberge M, Pariselle A, Huyse T, Volckaert FAM, Snoeks J, Vanhove Springer Nature remains neutral with regard to jurisdictional claims in MPM. Morphology, molecules, and monogenean parasites: an example of published maps and institutional affiliations. an integrative approach to cichlid biodiversity. PLoS One. 2015;10:e0124474. 18. Řehulková E, Mendlová M, Šimková A. Two new species of Cichlidogyrus Author details (Monogenea: Dactylogyridae) parasitizing the gills of African cichlid fishes 1Department of Botany and Zoology, Faculty of Science, Masaryk University, (Perciformes) from Senegal: morphometric and molecular characterization. Kotlářská 2, CZ-611 37 Brno, Czech Republic. 2Capacities for Biodiversity and Parasitol Res. 2013;112:1399–410. Sustainable Development (CEBioS), Operational Directorate Natural 19. Pouyaud L, Desmarais E, Deveney M, Pariselle A. Phylogenetic relationships Environment, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, among monogenean gill parasites (Dactylogyridea, Ancyrocephalidae) B-1000 Brussels, Belgium. 3Laboratory of Biodiversity and Evolutionary infesting tilapiine hosts (Cichlidae): systematic and evolutionary implications. Genomics, Department of Biology, University of Leuven, Charles Mol Phylogenet Evol. 2006;38:242–9. Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 20 of 21

20. Šimková A, Morand S. Co-evolutionary patterns in congeneric monogeneans: a 42. Gillardin C, Vanhove MPM, Pariselle A, Huyse T, Volckaert FAM. review of Dactylogyrus species and their cyprinid hosts. J Fish Biol. 2008;73:2210–27. Ancyrocephalidae (Monogenea) of Lake Tanganyika: II: description of the 21. Vignon M, Pariselle A, Vanhove MPM. Modularity in attachment organs of first Cichlidogyrus spp. parasites from Tropheini fish hosts (Teleostei, African Cichlidogyrus (Platyhelminthes: Monogenea: Ancyrocephalidae) Cichlidae). Parasitol Res. 2012;110:305–13. reflects phylogeny rather than host specificity or geographic distribution. 43. Price CE, Kirk RG. First description of a monogenetic trematode from Malawi. Biol J Linn Soc. 2011;102:694–706. Rev Zool Bot Afr. 1967;16:137–44. 22. Messu Mandeng FD, Bilong Bilong CF, Pariselle A, Vanhove MPM, Bitja Nyom 44. Paperna I, Thurston JP. Monogenetic Trematodes collected from cichlid fish AR, Agnèse JF. A phylogeny of Cichlidogyrus spp. (Monogenea, Dactylogyridea) in Uganda; including the description of five new species of Cichlidogyrus. clarifies a host-switch between fish families and reveals an adaptive component Rev Zool Bot Afr. 1969;79:15–33. to attachment organ morphology of this parasite genus. Parasit Vectors. 45. Pariselle A, Bilong Bilong CF, Euzet L. Four new species of Cichlidogyrus 2015;8:582. Paperna, 1960 (Monogenea, Ancyrocephalidae), all gill parasites from African 23. Vanhove MPM, Volckaert FAM, Pariselle A. Ancyrocephalidae (Monogenea) mouthbreeder tilapias of the genera Sarotherodon and Oreochromis (Pisces, of Lake Tanganyika: I: four new species of Cichlidogyrus from Ophthalmotilapia Cichlidae), with a redescription of C. thurstonae Ergens, 1981. Syst Parasitol. ventralis (Teleostei: Cichlidae), the first record of this parasite family in the basin. 2003;56:201–10. Zool. (Curitiba, Impresso). 2011;28:253–63. 46. Pariselle A, Bitja Nyom AR, Bilong Bilong CF. Four new species of Cichlidogyrus 24. Paperna I. Studies on monogenetic trematodes in Israel. 2 monogenetic (Monogenea, Ancyrocephalidae) from Sarotherodon mvogoi and Tylochromis trematodes of cichlids. Bamidgeh, Bull Fish Cult Isr. 1960;12:20–33. sudanensis (Teleostei, Cichlidae) in Cameroon. Zootaxa. 2014;3881:258–66. 25. Pariselle A, Euzet L. Trois Monogènes nouveaux parasites branchiaux de 47. Pariselle A, Euzet L. Five new species of Cichlidogyrus (Monogenea: Pelmatochromis buettikoferi (Steindachner, 1895) (Cichlidae) en Guinée. Ancyrocephalidae) from Tilapia brevimanus, T. buttikoferi and T. cessiana Parasite. 1995;2:203–9. from Guinea, Ivory Coast and Sierra Leone (West Africa). Folia Parasitol. 26. Pariselle A, Euzet L. Gill parasites of the genus Cichlidogyrus Paperna, 1960 1998;45:275–82. (Monogenea, Ancyrocephalidae) from Tilapia guineensis (Bleeker, 1862), with 48. Dossou C, Birgi E. Monogènes parasites d’Hemichromis fasciatus Peters, 1857 descriptions of six new species. Syst Parasitol. 1995;30:187–98. (Teleostei, Cichlidae). Ann Sci Nat Zool. 1984;6:101–9. 27. Pariselle A, Euzet L. Two new species of Cichlidogyrus Paperna, 1960 49. Dossou C. Parasites de poissons d’eau douce du Bénin III. Espèces nouvelles (Monogenea, Ancyrocephalidae) gill parasites on Hemichromis fasciatus du genre Cichlidogyrus (Monogenea) parasites de Cichlidae. Bull Inst Fond (Pisces, Cichlidae) in Africa, with remarks on parasite geographical Afr Noire. 1982;44:295–322. distribution. Parasite. 2004;11:359–64. 50. Paperna I. Monogenetic trematodes collected from freshwater fish in 28. Pariselle A, Muterezi Bukinga F, Van Steenberge M, Vanhove MPM. southern Ghana. Bamidgeh, Bull Fish Cult Isr. 1965;17:107–15. Ancyrocephalidae (Monogenea) of Lake Tanganyika. IV. Cichlidogyrus 51. Pariselle A, Euzet L. Three new species of Cichlidogyrus Paperna, 1960 parasitizing species of Bathybatini (Teleostei, Cichlidae): reduced host- (Monogenea: Ancyrocephalidae) parasitic on Tylochromis jentinki (Steindachner, specificity in the deepwater realm? Hydrobiologia. 2015;748:99–119. 1895) (Pisces, Cichlidae) in West Africa. Syst Parasitol. 1994;29:229–34. 29. Musilová N, Řehulková E, Gelnar M. Dactylogyrids (Platyhelminthes: Monogenea) 52. Paperna I. Monogenetic trematodes collected from fresh water fish in from the gills of Labeo (Teleostei: Cyprinidae) from West Africa. Zootaxa. Ghana. Second report. Bamidgeh, Bull Fish Cult Isr. 1968;20:80–100. 2009;2241:47–68. 53. Paperna I. Monogenetic trematodes of the fish of the Volta bassin and 30. Malmberg G. [On the occurrence of Gyrodactylus on Swedish fishes.] Skr. South Ghana. Bull Inst Fond Afr Noire. 1969;31:840–80. utgivna av Södra Sveriges Fisk. 1957;1956:19–76. (In Swedish). 54. Price CE, Peebles HE, Bamford T. The monogenean parasites of African 31. Gussev AV. [The method of collecting and processing of fish parasitic fishes. IV. Two new species from south African hosts. Rev Zool Bot Afr. monogenean material.] Nauk. Leningr. 1983;48 (In Russian). 1969;79:117–24. 32. Mizelle J. New species of trematodes from the gills of Illinois fishes. Am Midl 55. Douëllou L. Monogeneans of the genus Cichlidogyrus Paperna, 1960 Nat. 1936;17:785–806. (Dactylogyridae: Ancyrocephalinae) from cichlid fishes of Lake Kariba 33. Kritsky DC, Thatcher VE, Boeger WA. Neotropical Monogenea. 8. Revision of (Zimbabwe) with descriptions of five new species. Syst Parasitol. Urocleidoides (Dactylogyridae, Ancyrocephalinae). Proc Helminthol Soc 1993;25:159–86. Wash. 1986;53:1–37. 56. Pariselle A, Euzet L. Cichlidogyrus Paperna, 1960 (Monogenea, 34. Bush AO, Lafferty KD, Lotz JM, Shostak AW. Parasitology meets ecology on Ancyrocephalidae): gill parasites from west African Cichlidae of the its own terms: Margolis et al. revisited. J Parasitol. 1997;83:575–83. subgenus Coptodon Regan, 1920 (Pisces), with descriptions of six new 35. Froese R, Pauly D, editors. FishBase. World Wide Web electronic publication. species. Syst Parasitol. 1996;34:109–24. http://www.fishbase.org/search.php. Version 06/2017. Accessed Sept 2017. 57. Birgi E, Lambert A. Présence chez un Nandidae (Téléostéen), Polycentropsis 36. International Commission on Zoological Nomenclature. Amendment of abbreviata Boulenger, 1901, du genre Cichlidogyrus (Monogenea, articles 8, 9, 10, 21 and 78 of the international code of zoological nomenclature Monopisthocotylea, Ancyrocephalidae). Description de Cichlidogyrus to expand and refine methods of publication. Bull Zool Nomenclature. nandidae n. sp. Ann Parasitol Hum Comp. 1986;61:521–8. 2012;69:161–9. 58. Birgi E, Euzet L. Monogènes parasites des poissons des eaux douces du 37. Pariselle A, Euzet L. Four new species of Cichlidogyrus (Monogenea: Cameroun. Présence des genres Cichlidogyrus et Dactylogyrus chez Ancyrocephalidae), gill parasites of Tilapia cabrae (Teleostei: Cichlidae), with Aphyosemion (Cyprinodontidae). Bull Soc Zool Fr. 1983;108:101–6. discussion on relative length of haptoral sclerites. Folia Parasitol. 2003;50: 59. Paperna I. Parasitic helminths of inland-water fishes in Israel. Isr J Zool. 195–201. 1964;13:1–26. 38. Pariselle A, Bitja Nyom AR, Bilong Bilong CF. Checklist of the ancyrocephalids 60. Ergens R. Nine species of the genus Cichlidogyrus Paperna, 1960 (Monogenea: (Monogenea) parasitizing Tilapia species in Cameroon, with the description of Ancyrocephalinae) from Egyptian fishes. Folia Parasitol. 1981;28:205–14. three new species. Zootaxa. 2013;3599:78–86. 61. N’Douba V. Thys van den Audenaerde DFE, Pariselle A. Description d’une 39. Jorissen MWP, Pariselle A, Huyse T, Vreven EJ, Snoeks J, Volckaert FAM, espèce nouvelle de Monogène ectoparasite branchial de Tilapia guineensis Manda AC, Kasembele GK, Artois T, Vanhove MPM. Diversity and host (Bleeker, 1862) (Cichlidae) en Côte d’Ivoire. J Afr Zool. 1997;111:429–33. specificity of monogenean gill parasites (Platyhelminthes) of cichlid fishes 62. Poulin R. The evolution of monogenean diversity. Int J Parasitol. 2002;32:245–54. in the Bangweulu-Mweru ecoregion. J Helminthol. 2017:1–21. 63. Šimková A, Verneau O, Gelnar M, Morand S. Specificity and specialization of 40. Pariselle A, Euzet L. New species of Cichlidogyrus Paperna, 1960 (Monogenea, congeneric monogeneans parasitizing cyprinid fish. Evolution. 2006;60:1023–37. Ancyrocephalidae) from the gills of Sarotherodon occidentalis (Daget) 64. Meyer BS, Indermaur A, Ehrensperger X, Egger B, Banyankimbona G, Snoeks J, (Osteichthyes, Cichlidae) in Guinea and Sierra Leone (West Africa). Syst Salzburger W. Back to Tanganyika: a case of recent trans-species-flock dispersal Parasitol. 1997;38:221–30. in east African haplochromine cichlid fishes. R Soc Open Sci. 2015;2:140498. 41. Muterezi Bukinga F, Vanhove MPM, Van Steenberge M, Pariselle A. 65. Vanhove MPM. Species flocks and parasite evolution towards a co-phylogenetic Ancyrocephalidae (Monogenea) of Lake Tanganyika: III: Cichlidogyrus analysis of monogenean flatworms of cichlids and gobies. PhD thesis. Belgium: infecting the world’s biggest cichlid and the non-endemic tribes University of Leuven; 2012. Haplochromini, Oreochromini and Tylochromini (Teleostei, Cichlidae). 66. Koblmüller S, Salzburger W, Sturmbauer C. Evolutionary relationships in the Parasitol Res. 2012;111:2049–61. sand-dwelling cichlid lineage of Lake Tanganyika suggest multiple colonization Rahmouni et al. Parasites & Vectors (2017) 10:591 Page 21 of 21

of rocky habitats and convergent origin of biparental mouthbrooding. J Mol Evol. 2004;58:79–96. 67. Van Steenberge M, Vanhove MPM, Muzumani Risasi D, Mulimbwa N'sibula T, Muterezi Bukinga F, Pariselle A, et al. A recent inventory of the fishes of the north-western and centrtal western coast of Lake Tanganyika (Democratic Republic Congo). Acta Ichtyol Pisc. 2011;41:201–14. 68. Sturmbauer C, Baric S, Salzburger W, Rüber L, Verheyen E. Lake level fluctuations synchronize genetic divergences of cichlid fishes in African lakes. Mol Biol Evol. 2001;18:144–54. 69. Nevado B, Fazalova V, Backeljau T, Hanssens M, Verheyen E. Repeated unidirectional introgression of nuclear and mitochondrial DNA between four congeneric Tanganyikan cichlids. Mol Biol Evol. 2011;28:2253–67. 70. Konings A. Tanganyika cichlids in their natural habitat. 3rd ed. El Paso: Cichlid Press; 2015. 71. Hanssens M, Snoeks J, Verheyen E. A morphometric revision of the genus Ophthalmotilapia (Teleostei, Cichlidae) from Lake Tanganyika (East Africa). Zool J Linnean Soc. 1999;125:487–512. 72. Grégoir AF, Hablützel PI, Vanhove MPM, Pariselle A, Bamps J, Volckaert FAM, Raeymaekers JAM. A link between host dispersal and parasite diversity in two sympatric cichlids of Lake Tanganyika. Freshw Biol. 2015;60:323–35.

Submit your next manuscript to BioMed Central and we will help you at every step:

• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research

Submit your manuscript at www.biomedcentral.com/submit Study B

Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea:

Dactylogyridae) parasitizing the gills of Congolese cichlids from northern

Lake Tanganyika

Rahmouni, C., Vanhove, M.P.M. and Šimková, A. (2018)

PeerJ 6:e5604

Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) parasitizing the gills of Congolese cichlids from northern Lake Tanganyika

Chahrazed Rahmouni1, Maarten P.M. Vanhove1,2,3,4 and Andrea Šimková1 1 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 2 Zoology Unit, Finnish Museum of Natural History, University of Helsinki, Helsinki, Finland 3 Laboratory of Biodiversity and Evolutionary Genomics, Department of Biology, University of Leuven, Leuven, Belgium 4 Centre for Environmental Sciences, Research Group Zoology: Biodiversity and Toxicology, Universiteit Hasselt, Diepenbeek, Belgium

ABSTRACT Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) isolated from the gills of six cichlid host species belonging to four tribes and sampled from the Congolese coastline of Lake Tanganyika (LT) are described: Cichlidogyrus adkoningsi sp. nov. from Cyphotilapia frontosa (tribe Cyphotilapiini); C. koblmuelleri sp. nov. from Cardiopharynx schoutedeni (Ectodini); C. habluetzeli sp.nov.from C. schoutedeni and C. frontosa; C. antoineparisellei sp.nov.fromInterochromis loocki (Tropheini); C. masilyai sp. nov. from Petrochromis orthognathus (Tropheini); C. salzburgeri sp.nov.fromP. trewavasae,andC. sergemorandi sp.nov.from Tylochromis polylepis (Tylochromini). This study represents the first parasitological examination of cyphotilapiine cichlid hosts. Representatives of the Tanganyikan Submitted 2 March 2018 ectodine, tropheine, and tylochromine cichlids previously sampled from various Accepted 16 August 2018 localities in the lake yielded nine, twelve, and two described species of Cichlidogyrus, Published 23 October 2018 respectively. The study further includes a morphological characterization of the male Corresponding author copulatory organ of six undescribed species of Cichlidogyrus found on the gills of Chahrazed Rahmouni, the tropheines I. loocki and P. orthognathus, and on those of Callochromis melanostigma [email protected] and Xenotilapia flavipinnis (both Ectodini). Geographical variation in the monogenean Academic editor fauna of I. loocki was observed. The most closely related cichlid species investigated Jean-Lou Justine in this study harboured Cichlidogyrus spp. exhibiting some similarities in their Additional Information and sclerotized structures. Thus, our paper provides additional evidence of the high species Declarations can be found on ’ fi page 31 richness of Cichlidogyrus and the link with their hosts sphylogeneticaf nities in LT. DOI 10.7717/peerj.5604 Copyright Subjects Biodiversity, Parasitology, Taxonomy, Freshwater Biology 2018 Rahmouni et al. Keywords Congo, Cichlidae, Interochromis, Petrochromis, Callochromis, Platyhelminthes, Distributed under Tylochromis, C. koblmuelleri sp. nov., C. habluetzeli sp. nov., C. masilyai sp. nov., C. antoineparisellei Creative Commons CC-BY 4.0 sp. nov., Cardiopharynx, C. sergemorandi sp. nov., Cyphotilapia, C. adkoningsi sp. nov., Xenotilapia, C. salzburgeri sp. nov.

How to cite this article Rahmouni et al. (2018), Seven new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) parasitizing the gills of Congolese cichlids from northern Lake Tanganyika. PeerJ 6:e5604; DOI 10.7717/peerj.5604 INTRODUCTION With an estimated 3,000 species distributed from Central and South America, across Africa to Madagascar, and to the Middle East and the Indian subcontinent (Chakrabarty, 2004), cichlid fishes represent one of the most species-rich families of vertebrates, accounting for about 10% of total teleost diversity (Takahashi & Koblmüller, 2011; Wanek & Sturmbauer, 2015). The Great African Rift Lakes Malawi, Victoria, and Tanganyika harbour cichlid flocks exhibiting high morphological, ecological, and genetic diversity (Takahashi & Sota, 2016). The exact number of species inhabiting these three lakes is still unknown, but approximately 2,000 species have been described (Koblmüller, Sefc & Sturmbauer, 2008). Lake Tanganyika (LT), located in the Great Rift Valley in central East Africa, is the deepest and oldest lake in Africa (Cohen et al., 1997) and the second deepest and oldest lake in the world (Salzburger et al., 2005). It holds the most diverse cichlid assemblages, comprised of several lineages of mostly endemic species classified into more than 50 genera and 12–14 tribes (Snoeks, 2000; Koblmüller, Sefc & Sturmbauer, 2008; Takahashi & Sota, 2016). Over 250 cichlid species are known to inhabit this lake (Takahashi & Koblmüller, 2011). Cichlids represent a textbook model in evolutionary biology (Kocher, 2004). Their mechanisms of speciation by rapid radiation make them crucial to the study of biological diversification, dynamics, and functions (Barluenga & Meyer, 2010; Takahashi & Koblmüller, 2011). Cichlid monogeneans are a promising tool for elucidating the speciation of both fish and parasites (Vanhove et al., 2015, 2016). Among the 14 monogenean parasite genera known to infect cichlids, six (Urogyrus Bilong Bilong, Birgi & Euzet, 1994; Enterogyrus Paperna, 1963; Onchobdella Paperna, 1968; Scutogyrus Pariselle & Euzet, 1995; Cichlidogyrus (Dactylogyridae Bychowski, 1933), and Gyrodactylus von Nordmann, 1832 (Gyrodactylidae Van Beneden & Hesse, 1863)) were reported from African cichlids (Pariselle & Euzet, 2009; Pariselle et al., 2011; Mendoza-Palmero et al., 2017). More than 100 African and Levantine cichlid species have been investigated for the presence of monogenean parasites (Pariselle & Euzet, 2009; Vanhove et al., 2016). Cichlidogyrus Paperna, 1960 is the most species-rich genus and is mostly restricted to African and Levantine hosts (a few species were isolated from non-cichlid hosts, see for instance Birgi & Lambert (1986))(Pariselle & Euzet, 2009). To date, 111 valid species of Cichlidogyrus have been recognized in African cichlids (see the overview of Tanganyikan and non-Tanganyikan species of Cichlidogyrus published recently by Rahmouni et al. (2017)). Some Tanganyikan cichlid tribes remain to be investigated for their gill flatworms. No parasitological data are available on the Cyphotilapiini Salzburger et al., 2002 with its three endemic representatives Cyphotilapia frontosa (Boulenger, 1906), Cyphotilapia gibberosa (Takahashi & Nakaya, 2003), and Trematochromis benthicola (Matthes, 1962) (Muschick, Indermaur & Salzburger, 2012; Meyer, Matschiner & Salzburger, 2015; Takahashi & Sota, 2016). Among the 34 valid cichlid species that belong to the endemic Tanganyikan tribe Ectodini Poll, 1986, which includes 10 genera, only four species were studied for the presence of parasites and nine Cichlidogyrus spp. were described.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 2/37 Vanhove, Volckaert & Pariselle (2011) described four Cichlidogyrus spp. (C. centesimus, C. makasai, and C. vandekerkhovei Vanhove, Volckaert & Pariselle, 2011 on Congolese and Zambian Ophthalmotilapia ventralis (Boulenger, 1898) and Tanzanian O. boops (Boulenger, 1901) and O. nasuta (Poll & Matthes, 1962), and C. sturmbaueri Vanhove, Volckaert & Pariselle, 2011 on Zambian O. ventralis and Tanzanian O. nasuta). Later, Rahmouni et al. (2017) investigated Aulonocranus dewindti (Boulenger, 1899) and O. nasuta from the Burundese part of LT and described two Cichlidogyrus spp. on A. dewindti (C. discophonum and C. pseudoaspiralis Rahmouni, Vanhove & Šimková, 2017), and three species on O. nasuta (C. aspiralis, C. glacicremoratus and C. rectangulus Rahmouni, Vanhove & Šimková, 2017). Cardiopharynx Poll, 1942 is monotypic and represented by C. schoutedeni Poll, 1942 (Konings, 2015). Still in the Ectodini, Callochromis Regan, 1920 consists of three nominal species: (Boulenger, 1898), C. melanostigma (Boulenger, 1906), and C. pleurospilus (Boulenger, 1906) (Konings, 2015), whereas Xenotilapia Boulenger, 1898 includes 13–17 species (Kidd et al., 2012). Xenotilapia flavipinnis Poll, 1985 has a lake-wide distribution (Konings, 2015). In contrast to some other members of the Ectodini, there are no data available on the parasite fauna hosted by representatives of Callochromis and Xenotilapia. The Tropheini is one of the most species-rich cichlid tribes endemic to LT with nine genera including approximately 24 species (Takahashi & Koblmüller, 2014). At least eight species of Petrochromis Boulenger, 1898, a representative of the Tropheini, have been described (Sturmbauer et al., 2003; Takahashi & Sota, 2016). Petrochromis orthognathus Matthes, 1959 is restricted to the northern two-thirds of the lake. Petrochromis trewavasae Poll, 1948 is found in the southern part of the lake, usually in sympatry with Petrochromis ephippium Brichard, 1989, a morphologically similar species considered conspecifictoP. trewavasae (Konings, 2015). Interochromis Yamaoka, Hori & Kuwamura, 1998 is a monotypic genus erected because of the morphological and ecological similarities between I. loocki and species of Petrochromis, and the differences between I. loocki and species of the tropheine Simochromis Boulenger, 1898 (see overview in Pariselle et al., 2015b). Several studies have been carried out on the parasitic flatworms of these cichlids. Gillardin et al. (2012) described three Cichlidogyrus spp. (C. steenbergei and C. irenae Gillardin et al., 2012 from Zambian and Congolese Limnotilapia dardennii (Boulenger, 1899) and ‘Gnathochromis’ pfefferi (Boulenger, 1898), respectively, and C. gistelincki Gillardin et al., 2012 from Congolese, Tanzanian, and Zambian ‘Ctenochromis’ horei (Günther, 1894)). Then, Pariselle et al. (2015b) examined Zambian I. loocki and described three Cichlidogyrus spp. (C. buescheri, C. schreyenbrichardorum, and C. vealli Pariselle & Vanhove, 2015). In the same study, they compared the haptoral structures of representatives of Cichlidogyrus infecting I. loocki with those observed in some undescribed Cichlidogyrus spp. isolated from representatives of Petrochromis. The same team described six species of Cichlidogyrus infecting Congolese, Tanzanian and Zambian tropheine cichlids (C. banyankimbonai, C. muterezii, and C. raeymaekersi Pariselle & Vanhove, 2015 on Simochromis diagramma (Günther, 1894); C. georgesmertensi Pariselle & Vanhove, 2015 on

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 3/37 Pseudosimochromis babaulti (Pellegrin, 1927); C. franswittei Pariselle & Vanhove, 2015 on P. marginatus (Poll, 1956) and P. curvifrons (Poll, 1942); and finally C. frankwillemsi Pariselle & Vanhove, 2015 on P. curvifrons (Van Steenberge et al., 2015)). Lake Tanganyika harbours representatives of a few non-endemic tribes resulting from colonisation from the lacustrine environment.ThisisthecaseofTylochromini Poll, 1986 with Tylochromis polylepis (Boulenger, 1900) as its sole representative in the lake. Members of Tylochromis Regan, 1920 inhabit rivers, lakes, and coastal lagoons throughout central and western Africa. Muterezi Bukinga et al. (2012) studied the gill monogeneans of T. polylepis and described two species of Cichlidogyrus from Congolese host specimens, that is, C. mulimbwai and C. muzumanii Muterezi Bukinga et al., 2012. In addition, they presented drawings of the hard parts (haptor and reproductive organs) of an undescribed species referred to as Cichlidogyrus sp. ‘T. polylepis 3’. The aim of this paper is to study gill monogenean diversity in cichlids belonging to four tribes from the Congolese lakeshore of northern LT. We describe seven new species of Cichlidogyrus and provide the morphological characterization of six undescribed congeners.

MATERIAL AND METHODS Cichlid specimens were obtained in September 2013 and August 2016 from LT along the shoreline of the Democratic Republic of the Congo (DRC) (Fig. 1). In total, 26 fish specimens belonging to eight cichlid species of four tribes were purchased from fish markets or captured using gill nets during snorkelling or diving. Fish were placed in a cool box containing ice, transported to the laboratory, and dissected immediately. Cichlid hosts were identified in situ on the basis of morphological characters by Walter Salzburger (Zoological Institute, University of Basel, Switzerland), Donatien Muzumani Risasi (Centre de Recherche en Hydrobiologie, Uvira, DRC), and Maarten Van Steenberge (the Royal Museum for Central Africa (MRAC), Tervuren and the Royal Belgian Institute of Natural Sciences, Brussels, Belgium). We performed molecular analysis on the samples, and obtained sequence data from the partial cytochrome b (cyt-b) mitochondrial gene to confirm the identity of the investigated cichlids. Fin clips from the cichlid specimens were preserved in 96% ethanol. Cichlid DNA was extracted using the DNeasy Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The partial cyt-b gene was amplified following Mendlová et al. (2012). The PCR products were loaded onto a 1% agarose gel and subsequently purified, and sequencing was performed following Rahmouni et al. (2017). Nucleotide sequences were edited using Sequencher software v. 5.0 (Gene Codes, Ann Arbor, MI, USA). The identification of cichlid species based on the sequence similarity approach was carried out using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi: blastn, default settings), available through the website of the National Centre for Biotechnology Information (NCBI Resource Coordinators, 2017). The newly generated sequences were deposited in GenBank under the accession numbers MH297985–MH298008.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 4/37 Figure 1 Map of sampling localities in LT. Full-size  DOI: 10.7717/peerj.5604/fig-1

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 5/37 Gill arches were separated via dorsal and ventral section using standard parasitological procedures and transferred into a Petri dish containing water. Monogeneans were detached from the gills and isolated according to Musilová, Řehulková & Gelnar (2009) using an MST130 stereoscopic microscope and mounted on slides with glycerine ammonium picrate mixture (GAP) (Malmberg, 1957). Parasite identification was conducted using original descriptions, the systematic revision of dactylogyridean parasites of African cichlids by Pariselle & Euzet (2009), and the recent overview focusing on the genitals of African Cichlidogyrus spp. by Rahmouni et al. (2017). Measurements and photographs were taken using an Olympus BX51 phase-contrast microscope and Olympus Stream Image Analysis v. 1.9.3 software. All measurements are included in the species descriptions. They are in micrometres, and are given as the mean followed by the range and the number of measurements (n) in parentheses (measurements of some undescribed species are given as the length of the structure in question followed by the number of measurements in parentheses). Drawings of the haptoral sclerotized parts and copulatory organs were made on flattened specimens using an Olympus BX51 microscope equipped with a drawing tube and edited with a graphic tablet compatible with Adobe Illustrator CS6 v. 16.0.0 and Adobe Photoshop v. 13.0. The terminology of haptoral sclerotized parts (anchors and hooks; also termed gripi and uncinuli, respectively) follows Gussev (1983). The numbering of hook pairs (Roman letters I–VII) is that recommended by Mizelle (1936). This method is preferred in adult specimens because it takes into consideration both antero-posterior and dorso–ventral positions of hooks (Kritsky, Thatcher & Boeger, 1986; Řehulková, Mendlová & Šimková, 2013). The lengths of hook pairs(shortorlong)wasassignedfollowingPariselle & Euzet (2009).Theclassification of haptoral groups follows Vignon, Pariselle & Vanhove (2011).Themetricsusedfor the hard structures are shown in Fig. 2. The type material was deposited in the Invertebrate collection of the MRAC, Tervuren, Belgium; the Finnish Museum of Natural History (MZH), Helsinki, Finland; and the Muséum National d’Histoire Naturelle (MNHN), Paris, France. Host nomenclature follows FishBase (Froese & Pauly, 2017). The list of museum specimens used for comparison with the new species is presented in Table 1. Sampling was carried out under mission statements 022/ MINEURS/CRH-U/2013 and 031/MINRST/CRH-U/2016 from the Centre de Recherche en Hydrobiologie-Uvira. In the absence of relevant animal welfare regulations in the D.R. Congo, the same strict codes of practice enforced within the EuropeanUnionwereapplied.ThisstudywasapprovedbytheAnimalCareandUse Committee of the Faculty of Science, Masaryk University, Brno (Czech Republic), approval nuber CZ01308. The electronic version of this article in portable document format will constitute a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed through any standard web browser by appending the

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 6/37 Figure 2 Measurements used in the descriptions of the new species of Cichlidogyrus. (A) anchor. (DA) dorsal anchor. (VA) ventral anchor. (a) total length. (b) blade length. (c) shaft length. (d) guard length. (e) point length. (DB) dorsal bar: (h) auricle length. (w) maximum straight width. (x) total length. (y) distance between auricles. (VB) ventral bar: (x) length of one ventral bar branch. (w) maximum width. (H) hook length. (MCO) male copulatory organ straight length. (Ct) copulatory tube curved length. (He) heel straight length. (Ap) accessory piece straight length. (Vg) vagina: (V) vagina total length. (v) vagina width. Full-size  DOI: 10.7717/peerj.5604/fig-2

Table 1 Comparative museum material examined in the present study. Cichlidogyrus spp. Host species Locality Accession number Cichlidogyrus mulimbwai Muterezi Bukinga T. polylepis (Boulenger, 1900) Mulembwe & Moba (DRC) MRAC 37701 et al., 2012 Cichlidogyrus muzumanii Muterezi Bukinga T. polylepis (Boulenger, 1900) Mulembwe & Moba (DRC) MRAC 37699 et al., 2012 Cichlidogyrus buescheri Pariselle & Vanhove, I. loocki (Poll, 1949) Kalambo Lodge (Zambia) MRAC 37744 2015 Cichlidogyrus schreyenbrichardorum Pariselle I. loocki (Poll, 1949) Kalambo Lodge (Zambia) MRAC, MRAC 37741 & Vanhove, 2015 Cichlidogyrus vealli Pariselle & Vanhove, I. loocki (Poll, 1949) Kalambo Lodge (Zambia) MRAC, MRAC 37743 2015 Cichlidogyrus discophonum Rahmouni, A. dewindti (Boulenger, 1899) Nyaruhongoka (Burundi) MRAC, MRAC 37956 Vanhove & Šimková, 2017 Cichlidogyrus pseudoaspiralis Rahmouni, A. dewindti (Boulenger, 1899) Nyaruhongoka (Burundi) MRAC 37955 Vanhove & Šimková, 2017 Cichlidogyrus aspiralis Rahmouni, Vanhove O. nasuta (Poll & Matthes, 1962) Magara (Burundi) MRAC 37954 & Šimková, 2017

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 7/37 LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank. org:pub:7076794A-B9EB-4FFC-AC49-66C304EC5BFB. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS. RESULTS Molecular identification of cichlid hosts The mitochondrial cytochrome b (cyt-b) gene fragment of 25 cichlid specimens was successfully amplified. The length of each consensus sequence was 419 bp. The Blast search processed on the NCBI website confirmed the species identification of the cichlid species investigated for the presence of gill parasitic flatworms. SPECIES DESCRIPTIONS

Dactylogyridae Bychowski, 1933 Cichlidogyrus Paperna, 1960 Cichlidogyrus adkoningsi sp. nov. Fig. 3 urn:lsid:zoobank.org:act:526C2A74-E3D6-4357-B1E8-03B08B95CE38.

Description. Based on three specimens fixed in GAP. Body 516 (482–550; n =3) long, 93 (80–107; n = 3) wide at mid-body. Dorsal anchors with short shaft and more pronounced guard and curved blade with arched point: a =31(30–32; n =3);b =25 (24–26; n =3);c =1–2(n =3);d =9(7–10; n =3);e =10(9–12; n = 3). Dorsal bar curved with constant width and relatively long auricles: h =38(35–44; n =3);w =3–4 (n =3);x =28(26–30; n =3);y =2(1–4; n = 3). Ventral anchors with shorter shaft than guard, curved blade with arched point: a =28(26–29; n =3);b =24(23–25; n =3); c =1–2(n =3),d =7(6–8; n =3);e =11(10–13; n = 3). V-shaped ventral bar: w =4(3–5; n =3);x =32(29–36; n = 3). Haptor with seven short hook pairs, hooks V retain their larval size (sensu Pariselle & Euzet, 2003, 2009), each hook with erect thumb and shank comprised of two subunits: pair I = 11 (10–12; n = 3) long, pair II = 14–15 (n =3) long, pair III = 15–16 (n =3)long,pairIV=16–17 (n = 3) long, pair V = 11–12 (n =3) long, pair VI = 17–18 (n =3)long,andpairVII=17(16–18, n = 3) long. Male copulatory organ with relatively short copulatory duct, slightly curved halfway and tapered distally: MCO = 45 (42–47; n = 3); Ct = 31 (29–33; n = 3). Heel irregularly shaped, He = 10 (9–11; n = 3). Accessory piece linked to the basal bulb, C-shaped, thick in the middle part, ending in hook, Ap = 26 (24–28; n = 13). Vagina non-sclerotized.

Diagnosis. Cichlidogyrus adkoningsi sp. nov. belongs to the group of species which exhibit short hook pairs I–IV, VI, and VII (sensu Vignon, Pariselle & Vanhove, 2011), a copulatory duct without a swollen proximal portion, and a non-sclerotized vagina (see Pariselle & Euzet, 2003), just like C. attenboroughi Kmentová, Gelnar, Koblmüller et al., 2016; C. banyankimbonai; C. berminensis Pariselle, Bitja Nyom & Bilong Bilong, 2013; C. bifurcatus Paperna, 1960; C. brunnensis Kmentová, Gelnar, Koblmüller et al., 2016; C. buescheri; C. consobrini Jorissen, Pariselle & Vanhove, 2018;

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 8/37 Figure 3 Sclerotized structures of Cichlidogyrus adkoningsi sp. nov. ex Cyphotilapia frontosa. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copu- latory organ; Ct, copulatory tube; Ap, accessory piece. Full-size  DOI: 10.7717/peerj.5604/fig-3

C. discophonum; C. evikae Rahmouni, Vanhove & Šimková, 2017; C. fontanai Pariselle & Euzet, 1997; C. frankwillemsi; C. franswittei; C. georgesmertensi; C. gillardinae; C. gistelincki; C. glacicremoratus; C. haplochromii Paperna & Thurston, 1969; C. irenae; C. jeanloujustinei Rahmouni, Vanhove & Šimková, 2017; C. longipenis Paperna & Thurston, 1969; C. makasai; C. milangelnari Rahmouni, Vanhove & Šimková, 2017; C. mulimbwai; C. muterezii; C. nageus Řehulková, Mendlová & Šimková, 2013; C. raeymaekersi; C. rognoni Pariselle, Bilong Bilong & Euzet, 2003; C. schreyenbrichardorum; C. sanjeani Pariselle & Euzet, 1997; C. sigmocirrus Pariselle, Bitja Nyom & Bilong Bilong, 2014; C. steenbergei; C. tilapiae Paperna, 1960; C. vandekerkhovei; and C. vealli. Dorsal and ventral bars as well as the accessory piece in C. adkoningsi sp. nov. and C. sturmbaueri described from O. ventralis and O. nasuta are of similar size. Additionally, the copulatory duct in both species is similarly shaped (see Vanhove, Volckaert & Pariselle, 2011). However, the new species is distinguishable from C. sturmbaueri by (i) the longer dorsal anchors (30–32 mminC. adkoningsi sp. nov. vs 19–21 mmin C. sturmbaueri), (ii) the longer dorsal bar auricles (35–44 mminC. adkoningsi sp. nov. vs 12–15 mminC. sturmbaueri), (iii) the different hook pairs (C. adkoningsi sp. nov. exhibits short hook pairs I–IV, VI, and VII while C. sturmbaueri displays short hook pair I and long pairs II–IV, VI, and VII (see Pariselle & Euzet, 2009; Rahmouni, Vanhove & Šimková, 2017 for the importance of hook length in the systematics of Cichlidogyrus), and (iv) the accessory piece (C-shaped, thick in the middle part ending in hook in C. adkoningsi sp. nov. vs H-shaped in C. sturmbaueri). The long auricles of C. adkoningsi sp. nov. are reminiscent of C. vandekerkhovei from O. ventralis, O. boops and O. nasuta, C. glacicremoratus from O. nasuta, and C. discophonum from A. dewindti, which are

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 9/37 all parasites of ectodine cichlids (Vanhove, Volckaert & Pariselle, 2011; Rahmouni, Vanhove & Šimková, 2017). Further, C. adkoningsi sp. nov. shares with C. makasai similarly sized dorsal and ventral bars, hook pairs, and accessory pieces. However, in C. adkoningsi sp. nov. (i), the dorsal anchors are longer (30–32 mminC. adkoningsi sp. nov. vs 19–23 mminC. makasai), (ii) the dorsal bar auricles are much longer (35–44 in C. adkoningsi sp. nov. vs 17–23 mminC. makasai), (iii) the heel is longer (9–11 in C. adkoningsi sp. nov. vs 2–4 mminC. makasai), (iv) the copulatory duct is much shorter and differently shaped (relatively short copulatory duct, slightly curved halfway, with a length of 33–29 mminC. adkoningsi sp. nov. vs thin curved duct which tapers distally, 69–79 mm long in C. makasai), and (v) the accessory piece is differently shaped (thick, C-shaped, ending in a hook in C. adkoningsi sp. nov. vs simple, slightly bent at distal third, resembling a spanner in C. makasai).

Type-host: Cyphotilapia frontosa (Boulenger, 1906) (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Cyphotilapiini Salzburger et al., 2002).

Host accession numbers: MH297995–96

Site of infection: Gills.

Type-locality: Makabola village (332′S, 299′E; purchase from fisherman), DRC, LT.

Prevalence & intensity of infection: one fish specimen infected/two fish specimens examined, three parasite specimens on the infected fish.

Holotype: MRAC M.T.38432.

Paratype: MRAC M.T.38433.

Etymology: the specific epithet of the new species, ‘adkoningsi’, honours the Dutch biologist Dr. Adrianus Johannes Franciscus Marinus Maria Konings, known as Ad Konings, who has published extensively on cichlids. His books on the cichlids of LT have been crucial to our research on the parasite fauna of these fishes.

Cichlidogyrus koblmuelleri sp. nov. Fig. 4 urn:lsid:zoobank.org:act:473DB764-6798-43EE-8DD8-3B10D1AC1BBF.

Description. Based on seven specimens fixed in GAP. Body 462 (402–564; n = 4) long, 85 (68–121; n = 4) wide at mid-body. Dorsal anchors long with short shaft and more pronounced guard, curved blade and arched point: a = 27 (26–28; n = 4); b = 22 (21–23; n = 4); c =1–2(n = 4); d =9(8–10; n = 4); e =9–10 (n = 4). Dorsal bar slightly curved with blunt endings and long auricles: h = 26 (25–28; n = 4); w =4–5(n = 4); x =26 (25–27; n = 4); y =4(2–5; n = 4). Ventral anchors similar to dorsal ones: a =24–25 (n = 4); b =21–22 (n = 4); c =1–2(n = 4), d =6(5–7; n = 4); e =9–10 (n = 4). V-shaped ventral bar: w =2–3(n = 4); x = 29 (28–30; n = 10). Haptor with seven short hook pairs, hooks V retain their larval size (see above), each hook with erect thumb and shank comprised of two subunits: pair I = 11 (10–12; n = 4) long, pair II = 13–14 (n =4)

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 10/37 Figure 4 Sclerotized structures of Cichlidogyrus koblmuelleri sp. nov. ex Cardiopharynx schoutedeni. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copu- latory organ; Ct, copulatory tube; Ap, accessory piece. Full-size  DOI: 10.7717/peerj.5604/fig-4

long, pair III = 14–15 (n = 4) long, pair IV = 15–16 (n = 4) long, pair V = 10–11 (n = 4) long, pair VI = 16 (15–17; n = 4) long, and pair VII = 13–14 (n = 4) long. Male copulatory organ with a C-shaped copulatory duct: MCO = 46 (36–49; n = 7); Ct = 63 (59–65; n = 7). No heel. Accessory piece curved with two superimposed parts, one of them thicker and longer than the other, ending in moderately curved hook, Ap = 38 (37–40; n = 7). Vagina non-sclerotized.

Diagnosis. Cichlidogyrus koblmuelleri sp. nov. belongs to the same group as C. adkoningsi sp. nov. as it shares the small size of all hook pairs. The new species most closely resembles C. discophonum described from A. dewindti (see Rahmouni, Vanhove & Šimková, 2017) regarding the morphology of the dorsal and ventral anchors and the absence of a heel. However, it differs from the latter by (i) the longer copulatory duct (59–65 mminC. koblmuelleri sp. nov. vs 41–47 mminC. discophonum), and (ii) the longer and differently shaped accessory piece (curved with two superimposed parts, one of them thicker and longer than the other ending in a hook, 37–40 mminC. koblmuelleri sp. nov. vs short with two thick distinct parts, twisted distally, ending in hook, 15–22 mm in C. discophonum). Like C. adkoningsi sp. nov. and the other Cichlidogyrus spp. of the ectodine cichlids listed in the previous diagnosis, C. koblmuelleri sp. nov. exhibits long dorsal bar auricles. However, they are longer in the new species than those of C. glacicremoratus (25–28 mminC. koblmuelleri sp. nov. vs 14–18 mmin C. glacicremoratus). Moreover, C. koblmuelleri sp. nov. is mainly distinguishable from C. glacicremoratus by (i) the longer and differently shaped copulatory duct (C-shaped copulatory duct, 59–65 mminC. koblmuelleri sp. nov. vs wavy copulatory duct, with thick

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 11/37 Figure 5 Sclerotized structures of Cichlidogyrus habluetzeli sp. nov. ex Cardiopharynx schoutedeni and Cyphotilapia frontosa. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece; Vg, vagina. Full-size  DOI: 10.7717/peerj.5604/fig-5

wall, constricted and curved approximately at proximal third, with wide terminal opening, 42–47 mminC. glacicremoratus), and (ii) the heel (no heel in C. koblmuelleri sp. nov. vs small, irregular sclerotized flange-like structure in C. glacicremoratus).

Type-host: Cardiopharynx schoutedeni Poll, 1942 (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Ectodini Poll, 1986).

Host accession numbers: MH297989–94.

Site of infection: Gills.

Type-locality: Mulongwe fish market (322′S, 296′E), Uvira, DRC, LT.

Prevalence & intensity of infection: four fish specimens infected/six fish specimens examined, one to three parasite specimens per infected host.

Holotype: MRAC M.T.38434.

Paratypes: MRAC M.T.38435, MRAC M.T.38439.

Etymology: the specific epithet of the new species, ‘koblmuelleri’ honours the biologist Dr. Stephan Koblmüller (Austria), an all-round specialist in LT’s ichthyofauna, in recognition of his crucial contribution to parasitological work.

Cichlidogyrus habluetzeli sp. nov. Fig. 5 urn:lsid:zoobank.org:act:EC79CE69-2D88-4D96-A255-AC0E31B76EAE.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 12/37 Description. Based on 28 specimens fixed in GAP. Body 507 (286–790; n = 19) long, 99 (62–152; n = 19) wide at mid-body. Dorsal anchors long with short shaft and more pronounced guard, curved blade with slightly arched point: a = 60 (53–66; n = 20); b =43 (38–46; n = 20); c =6(3–9; n = 21); d = 22 (16–26; n = 20); e =11(9–14; n = 20). Dorsal bar relatively curved, thick in the middle part with blunt endings and straight auricles: h = 14 (10–18; n = 19); w =11(9–14; n = 18); x = 59 (54–63; n = 18); y =25 (22–29; n = 19). Ventral anchors shorter than dorsal ones with shorter shaft than guard, curved blade with arched point: a = 47 (44–52; n = 12); b = 44 (40–48; n = 20); c =6 (5–8; n = 18), d =12(8–15; n = 18); e = 11 (10–13; n = 21). Long and thick V-shaped ventral bar: w =7(6–10; n = 20); x = 53 (47–60; n = 20). Haptor with seven long hook pairs, pair I large in comparison with remaining pairs, hooks V retain their larval size (sensu Pariselle & Euzet, 2003, 2009), and each hook with erect thumb and shank comprised of two subunits: pair I = 38 (33–42; n = 20) long, pair II = 27 (23–31; n = 18) long, pair III = 27 (22–31; n = 19) long, pair IV = 24 (21–28; n = 19) long, pair V = 12 (11–13; n = 19) long, pair VI = 28 (24–31; n = 20) long, and pair VII = 28 (25–32; n = 20) long. Male copulatory organ with a relatively short straight copulatory duct: MCO = 44 (39–53; n = 23); Ct = 21 (19–23; n = 24). Well-developed straight heel, He = 23 (18–29; n = 24). Thin accessory piece distally with leaf-shaped ending, Ap = 15 (13–21; n = 21). Sclerotized vagina: V = 23 (19–32; n = 6); v = 11 (10–14; n = 7).

Diagnosis. Cichlidogyrus habluetzeli sp. nov. belongs to the group of species with long hook pairs I–IV, VI and VII, large hook pair I, a copulatory duct without a swollen proximal portion, and a sclerotized vagina (see above). This group is restricted to a single species, the Tanganyikan C. centesimus (see Vanhove, Volckaert & Pariselle, 2011). Therefore, C. habluetzeli sp. nov. is only the second record with this configuration of sclerotized structures throughout the entire genus. The dorsal bar auricles in C. habluetzeli sp. nov. are small hollow outgrowths on the anterior face, a feature observed in congeners infecting representatives of Tylochromini and Ectodini. With this morphology, we find Tanganyikan C. mulimbwai and C. muzumanii, both from T. polylepis (Muterezi Bukinga et al., 2012), C. pseudoaspiralis from A. dewindti, and C. aspiralis from O. nasuta (Rahmouni, Vanhove & Šimková, 2017) to be just like non-Tanganyikan C. chrysopiformis, C. djietoi, and C. sigmocirrus Pariselle, Bitja Nyom & Bilong Bilong, 2014, all parasites of T. sudanensis Daget, 1954 (Pariselle, Bitja Nyom & Bilong Bilong, 2014), C. kothiasi Pariselle & Euzet, 1994 from T. jentinki (Steindachner, 1862) (Pariselle & Euzet, 1994), and C. dageti, C. euzeti, and C. falcifer Dossou & Birgi, 1984 from Hemichromis fasciatus (Dossou & Birgi, 1984; Pariselle & Euzet, 2009). In addition to its haptoral features, C. habluetzeli sp. nov. exhibits a similar morphotype of the reproductive organs as C. aspiralis, C. pseudoaspiralis (Rahmouni, Vanhove & Šimková, 2017), C. casuarinus Pariselle, Muterezi Bukinga & Vanhove, 2015 isolated from a range of cichlid representatives of the tribe Bathybatini Poll, 1986 (Pariselle et al., 2015a), C. centesimus (Vanhove, Volckaert & Pariselle, 2011), and C. nshomboi Muterezi Bukinga et al., 2012 from Boulengerochromis microlepis (Boulenger, 1899) Boulengerochromini Takahashi, 2003 (all Tanganyikan species) (Muterezi Bukinga et al., 2012). C. habluetzeli sp. nov. can

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 13/37 be compared to C. aspiralis regarding the similarly shaped and sized copulatory duct and accessory piece, and the presence of a sclerotized vagina. However, the new species differs from C. aspiralis by (i) the longer dorsal and ventral anchors (respectively, 60–66; 46–52 mminC. habluetzeli sp. nov. vs 39–43; 33–34 mminC. aspiralis), (ii) the longer dorsal and ventral bars (respectively, 54–63; 52–60 mminC. habluetzeli sp. nov. vs 45–47, 37–40 mminC. aspiralis), (iii) and the longer dorsal bar auricles (14–18 mmin C. habluetzeli sp. nov. vs 6–10 mminC. aspiralis). On the other hand, the dorsal and ventral anchors in C. habluetzeli sp. nov. are similar to those of C. casuarinus and C. nshomboi, while in C. centesimus and C. pseudoaspiralis, they are shorter (respectively, 53–66; 44–52 mminC. habluetzeli sp. nov. vs 41–55; 34–44 mminC. centesimus and 37–42; 37–40 mminC. pseudoaspiralis). Also, the dorsal bar in the new species is similar to that of C. nshomboi, shorter than that of C. casuarinus (54–63 mminC. habluetzeli sp. nov. vs 64–85 mminC. casuarinus), and longer than those of C. centesimus and C. pseudoaspiralis (54–63 mminC. habluetzeli sp. nov. vs 37–52; 31–32 mmin C. centesimus, and C. pseudoaspiralis, respectively). Like C. aspiralis and C. pseudoaspiralis, C. habluetzeli sp. nov. lacks a spirally coiled thickening in the distal part of its copulatory duct, a feature present in C. casuarinus, C. centesimus and C. nshomboi. Further, the copulatory duct in C. habluetzeli sp. nov. is similar in size to that in C. centesimus and C. nshomboi, while it is longer in C. casuarinus and C. pseudoaspiralis (19–23 mmin C. habluetzeli sp. nov. vs 33–44; 40–43 mminC. casuarinus and C. pseudoaspiralis, respectively). Unlike C. pseudoaspiralis, C. habluetzeli sp. nov. exhibits a sclerotized vagina similarly to C. casuarinus, C. centesimus, and C. nshomboi (see Vanhove, Volckaert & Pariselle, 2011; Muterezi Bukinga et al., 2012; Pariselle et al., 2015a; Rahmouni, Vanhove & Šimková, 2017).

Type-host: Cardiopharynx schoutedeni Poll, 1942 (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Ectodini Poll, 1986).

Site of infection: Gills.

Type-locality: Mulongwe fish market (322′S, 296′E), Uvira, DRC, LT.

Prevalence & intensity of infection in the type host: three fish specimens infected/six fish specimens examined, one to six parasite specimens per infected host.

Additional host: found on the gills of C. frontosa, one fish specimen infected/two fish specimens examined, nine parasite specimens on the infected host.

Holotype: MRAC M.T.38437.

Paratypes: MRAC M.T.38436, MRAC M.T.38438; MNHN HEL748–49, HEL752; MZH KN10058–59.

Etymology: the specific epithet of the new species, ‘habluetzeli’ honours the biologist Dr. Pascal István Hablützel (Switzerland/Belgium) in honour of his pioneering work in the eco-immunology of Tanganyikan cichlids.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 14/37 Figure 6 Sclerotized structures of Cichlidogyrus antoineparisellei sp. nov. ex Interochromis loocki. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece; Vg, vagina. Full-size  DOI: 10.7717/peerj.5604/fig-6

Cichlidogyrus antoineparisellei sp. nov. Fig. 6 urn:lsid:zoobank.org:act:B324AD44-4520-4129-8DBB-8985A981783D.

Description. Based on 12 specimens fixed in GAP. Body 436 (330–509; n = 5) long, 82 (76–86; n = 5) wide at mid-body. Dorsal anchors with poorly marked shaft and more pronounced guard (approximately four times the length of shaft), bent blade with arched point: a = 25 (24–26; n = 7); b =22–23 (n = 7); c =1–2(n = 7); d =6(4–7; n = 7); e =9(8–10; n = 7). Dorsal bar curved, thick in the middle part with blunt endings and well-developed auricles: h =11–12 (n = 7); w =5–6(n = 7); x = 27 (26–29; n = 7); y =9(8–11; n = 7). Ventral anchors similar in shape and size to dorsal ones: a = 26 (25–27; n = 7); b = 24 (23–25; n = 7); c =2(1–3; n = 7); d =6(3–7; n = 7); e =10 (9–12; n = 7). V-shaped ventral bar: w =4–5(n = 7); x = 26 (24–27; n = 7). Haptor with seven short hook pairs, hooks V retain their larval size (see above), each hook with erect thumb and shank comprised of two subunits: pair I = 11–12 (n = 6) long, pair II = 13 (12–14; n = 7) long, pair III = 15 (13–16; n = 7) long, pair IV = 17 (15–18; n =7) long, pair V = 11 (9–12; n = 6) long, pair VI = 16 (14–17; n = 7) long, and pair VII = 13–14 (n = 7) long. Male copulatory organ composed of long, curved copulatory duct that tapers distally: MCO = 42 (39–46; n = 10); Ct = 59 (54–62; n = 11). Heel relatively short, He = 6 (5–7; n = 11). Accessory piece is linked to basal bulb by thin filament, C-shaped in the middle part, composed of two distinct parts, one extremity ending in hook while the other connected to the latter with swollen base, Ap = 29 (22–33; n = 10). Long tubular vagina with two characteristic extremities, a broadened part and one extremity covered by a triangular-like structure: V = 40 (35–47; n =11);v =9(7–13; n = 11).

Diagnosis. According to the sclerites of the haptoral and reproductive organs, C. antoineparisellei sp. nov. belongs to the group which includes species with short hook

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 15/37 pairs I–IV, VI, and VII, and a copulatory duct without a swollen proximal portion, and exhibits a sclerotized vagina (see above). This group includes C. acerbus Dossou, 1982; C. amieti Birgi & Euzet, 1983; C. amphoratus Pariselle & Euzet, 1996; C. berrebii Pariselle & Euzet, 1994; C. cirratus Paperna, 1964; C. cubitus Dossou, 1982; C. djietoi; C. giostrai Pariselle, Bilong Bilong & Euzet, 2003; C. karibae Douëllou, 1993; C. kothiasi Pariselle & Euzet, 1994; C. lagoonaris Paperna, 1969; C. levequei Pariselle & Euzet, 1996; C. louipaysani Pariselle & Euzet, 1995; C. mvogoi Pariselle, Bitja Nyom & Bilong Bilong, 2014; C. njinei Pariselle, Bilong Bilong & Euzet, 2003; C. ornatus Pariselle & Euzet, 1996; C. pouyaudi Pariselle & Euzet, 1994; C. sclerosus Paperna & Thurston, 1969; C. slembroucki Pariselle & Euzet, 1998; and C. zambezensis Douëllou, 1993. C. mbirizei Muterezi Bukinga et al., 2012, a parasite described from a representative of the tribe Oreochromini Dunz & Schliewen, 2013, Oreochromis tanganicae (Günther, 1894), is the only species hitherto known to have short hook pairs I–IV, VI, and VII and a sclerotized vagina in LT (see Muterezi Bukinga et al., 2012), and therefore C. antoineparisellei sp. nov. is the second known representative with the combination of these sclerotized structures in the lake. However, C. antoineparisellei sp. nov. is the first representative of Cichlidogyrus recognized on Tanganyikan tropheines which has a sclerotized vagina. Further, because of the systematic and phylogenetic affinities among the Tropheini and the Haplochromini Poll, 1986 (see Salzburger et al., 2005), C. antoineparisellei sp. nov. is considered to be the second representative species exhibiting this feature within the parasites infecting representatives of the Haplochromini lineage, like its congener C. zambezensis reported from the non-Tanganyikan haplochromines Serranochromis macrocephalus (Boulenger, 1899), S. robustus jallae (Günther, 1864) (Vanhove et al., 2013), S. mellandi (Boulenger, 1905), S. stappersi Trewavas, 1964, S. thumbergi (Castelnau, 1861), and S. angusticeps (Boulenger, 1905) (C. zambezensis was also found on O. mortimeri Trewavas, 1966; see Douëllou, 1993; Jorissen et al., 2018). Morphologically, C. antoineparisellei sp. nov. exhibits similar haptoral features to those of Tanganyikan C. brunnensis described from Boulenger, 1901 Trematocarini Poll, 1986 (Kmentová et al., 2016a), and a variety of West African parasite species such as C. amphoratus from Tilapia louka Thys van den Audenaerde, 1969 (Pariselle & Euzet, 1996), C. sclerosus Paperna & Thurston, 1969 from O. mossambicus (Peters, 1852), O. niloticus (Linnaeus, 1758), Haplochromis sp., O. leucosticus (Trewavas, 1933), C. zillii (Gervais, 1848), O. spilurus (Günther, 1894), O. aureus (Steindachner, 1864) (Paperna & Thurston, 1969; Pariselle & Euzet, 2009), O. mortimeri, S. microcephalus (Douëllou, 1993), and O. mweruensis Trewavas, 1983 (Jorissen et al., 2018), and C. giostrai from Sarotherodon caudomarginatus (Boulenger, 1916) (Pariselle, Bilong Bilong & Euzet, 2003). These include the characteristic broad base and almost non-incised roots of the anchors. C. antoineparisellei sp. nov. shares its host species I. loocki with C. buescheri in addition to the morphology of its hook pairs and dorsal and ventral anchors (see Pariselle et al., 2015b). However, it is mainly distinguishable from the latter by (i) the shorter dorsal bar (26–29 mmin C. antoineparisellei sp. nov. vs 31–47 mminC. buescheri), and (ii) the sclerotized vagina (no sclerotized vagina in C. buescheri).

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 16/37 Figure 7 Sclerotized structures of Cichlidogyrus masilyai sp. nov. ex Petrochromis orthognathus. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece. Full-size  DOI: 10.7717/peerj.5604/fig-7

Type-host: Interochromis loocki (Poll, 1949) (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Tropheini Poll, 1986).

Host accession numbers: MH298001–02.

Site of infection: Gills.

Type-locality: Pemba (340′S, 2910′E; caught when snorkelling), DRC, LT.

Prevalence & intensity of infection: two fish specimens infected/two fish specimens examined, one to seven parasite specimens per infected host.

Holotype: MRAC M.T.38440.

Paratypes: MRAC M.T.38441–43; MNHN HEL750–51.

Etymology: the species epithet ‘antoineparisellei’ honours the French parasitologist Dr. Antoine Pariselle, researcher at the Institut de Recherche pour le Développement, who extensively studied the monogeneans of African fish, trained countless people in fish parasitology, and described more than fifty species of Cichlidogyrus, of which three parasitize I. loocki.

Cichlidogyrus masilyai sp. nov. Fig. 7 urn:lsid:zoobank.org:act:4D5AF9E3-9CCE-4D3F-95B4-CCDA7B1A5C91.

Description. Based on 12 specimens fixed in GAP. Body 622 (523–721; n = 2) long, 107 (56–158; n = 2) wide at mid-body. Dorsal anchors with short shaft and pronounced

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 17/37 guard (approximately two times the length of shaft), bent blade with arched point: a = 28 (27–29; n = 3); b = 21 (20–23; n = 3); c =5(4–6; n = 3); d =10(9–11; n = 3); e =7(5–8; n = 4). Dorsal bar well-arched, C-shaped with relatively short auricles: h =10 (8–11; n = 3); w =6(5–7; n = 3); x = 28 (22–24; n = 3); y = 14 (13–15; n = 3). Ventral anchors with shorter shaft than guard and arched point: a = 28 (27–29; n = 3); b = 24 (23–25; n = 3); c =4(2–5; n = 3); d =9(6–10; n = 3); e =9(8–10; n = 3). W-shaped ventral bar: w =5(4–6; n = 3); x = 32 (31–33; n = 3). Haptor with seven short hook pairs, hooks V retain their larval size (see above), each hook with erect thumb and shank comprised of two subunits: pair I = 12–13 (n = 3) long, pair II = 16 (15–17; n =3) long, pair III = 20 (19–21; n = 3) long, pair IV = 22 (21–23; n = 3) long, pair V = 12 (10–13; n = 3) long, pair VI = 22–23 (n = 3) long, and pair VII = 19 (17–21; n = 3) long. Male copulatory organ beginning as an irregularly shaped bulb with thin copulatory duct, slightly curved proximally, straight halfway, folded back distally: MCO = 33 (31–34; n = 3); Ct = 35 (32–36; n = 3). Poorly developed heel at the side of the basal bulb, He = 1–2(n = 3). Accessory piece seems to be unattached to basal bulb, is thin, slightly curved in the middle part with pincer-like ending, Ap = 26 instead of 29 (24–29; n = 3). Vagina non-sclerotized.

Diagnosis. Cichlidogyrus masilyai sp. nov. belongs to the same group as C. adkoningsi sp. nov. and C. koblmuelleri sp. nov. (see above). The new species shows a similar morphotype to several representatives of Cichlidogyrus described from non-Tanganyikan haplochromine cichlids such as C. bifurcatus from Astatotilapia flaviijosephi (Lortet, 1883) (Paperna, 1960), C. haplochromii from H. guiarti (Pellegrin, 1904) (Paperna & Thurston, 1969) and C. zambezensis, in addition to C. gillardinae from the Tanganyikan haplochromine A. burtoni (Günther, 1894) (Muterezi Bukinga et al., 2012). Further, the exceptional shape of the dorsal bar in the new species, that is, well-arched, C-shaped, is observed for the first time in Cichlidogyrus spp. infecting a tropheine cichlid. The new species exhibits the same hook pairs, similarly sized dorsal and ventral anchors, a non-sclerotized vagina, and an accessory piece ending in a pincer-like structure as in C. buescheri, a parasite of I. loocki. However, the shape of the dorsal and ventral anchors is different, that is, the shaft is more clearly marked in C. masilyai sp. nov. compared to that observed in C. buescheri. Further, it differs from C. buescheri by (i) the shorter and differently shaped dorsal bar (C-shaped, 22–24 mminC. masilyai sp. nov. vs moderately arched, 31–45 mminC. buescheri), (ii) the copulatory duct (thin, slightly curved proximally, straight halfway, folded distally, 31–34 mminC. masilyai sp. nov. vs curved duct with narrow extremity, 49–58 mminC. buescheri), (iv) the heel (poorly developed, lateral to the bulb, 1–2 mminC. masilyai sp. nov. vs large heel of irregular shape at the basis of the bulb, 6–11 mminC. buescheri), and (v) the accessory piece (seems to be unattached to basal bulb and thin, 24–29 mminC. masilyai sp. nov. vs wide, directly attached to the basal bulb, 31–52 mminC. buescheri).

Type-host: Petrochromis orthognathus Matthes, 1959 (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Tropheini Poll, 1986).

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 18/37 Figure 8 Sclerotized structures of Cichlidogyrus salzburgeri sp. nov. ex Petrochromis trawavasae. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece. Full-size  DOI: 10.7717/peerj.5604/fig-8

Host accession numbers: MH298003–06.

Site of infection: Gills.

Type-locality: Pemba (340′S, 2910′E; caught when snorkelling), DRC, LT.

Prevalence & intensity of infection: two fish specimens infected/four fish specimens examined, one to two parasite specimens per infected host.

Holotype: MRAC M.T.38447.

Paratype: MRAC M.T.38446.

Etymology: the species epithet ‘masilyai’ is given in honour of the Congolese biologist Prof. Dr. Pascal Masilya Mulungula of the Institut Supérieur Pédagogique de Bukavu and general director of the Centre de Recherche en Hydrobiologie-Uvira (DRC), in appreciation of the hospitality and help received during the fieldtrip for the present study.

Cichlidogyrus salzburgeri sp. nov. Fig. 8 urn:lsid:zoobank.org:act:612A6D2B-09DD-4902-8640-A4877277E7F3.

Description. Based on four specimens fixed in GAP. Dorsal anchors with short shaft and more pronounced guard (approximately three times the length of shaft), bent blade with arched point: a = 27 (26–29; n = 3); b = 18 (17–19; n = 3); c =4–5(n = 3); d =12–13 (n = 3); e =8(7–9; n = 4). Dorsal bar slightly curved with well-developed auricles: h = 22 (21–24; n = 3); w =6–7(n = 3); x = 32 (35–38; n = 3); y =11–12 (n = 4). Ventral anchors with shorter shaft than guard and arched point: a = 32 (31–33; n = 3);

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 19/37 b =26–27 (n = 3); c =5–6(n = 3); d =11–12 (n = 3); e =12–13 (n = 3). V-shaped ventral bar: w =7–8(n = 3); x = 39 (38–40; n = 3). Haptor with short hook pair I, hooks V retain their larval size (see above), pairs II–IV, VI, and VII long, each hook with erect thumb and shank comprised of two subunits: pair I = 12–13 (n = 3) long, pair II = 23–24 (n = 3) long, pair III = 28–29 (n = 3) long, pair IV = 31–32 (n = 3) long, pair V = 11–12 (n = 3) long, pair VI = 25–26 (n = 3) long, and pair VII = 25–26 (n = 3) long. Male copulatory organ with straight, wide copulatory duct and large distal opening: MCO = 42 (41–44; n = 4); Ct = 40 (39–42; n = 4). Short heel, He = 3 (2–4; n = 4). Accessory piece linked to basal bulb, thin proximally, straight, pincer-like ending with one extremity shorter than the other, Ap = 29 (28–30; n = 4). Vagina non-sclerotized.

Diagnosis. Cichlidogyrus salzburgeri sp. nov. belongs to the group of Cichlidogyrus which are characterized by a shorter hook pair I (pair V with larval size) and longer pairs II–IV, VI, and VII, a copulatory duct without a swollen distal portion, and a non-sclerotized vagina. Of all Cichlidogyrus spp., only C. halli Price & Kirk, 1967 and the Tanganyikan C. sturmbaueri (Vanhove, Volckaert & Pariselle, 2011) and C. rectangulus (Rahmouni, Vanhove & Šimková, 2017) are included in this group. Therefore, the new species is the third representative described from endemic Tanganyikan cichlids displaying this hook configuration. C. salzburgeri sp. nov. differs from its congeners C. buescheri, C. schreyenbrichardorum, and C. vealli isolated from I. loocki, and from the undescribed species previously identified on some representatives of Petrochromis, by the shape of the anchors, specifically the marked bases in the ventral ones (Pariselle et al., 2015b). The dorsal bar in C. salzburgeri sp. nov. with its well-developed auricles is reminiscent of C. muterezii described from S. diagramma (see Van Steenberge et al., 2015). However, C. salzburgeri sp. nov. differs from the latter by (i) the hook pairs (shorter hook pair I and longer pairs II–IV, VI, and VII in C. salzburgeri sp. nov. vs short hook pairs I–IV, VI, and VII in C. muterezii) and (ii) the pincer-like ending in the accessory piece, a feature missing in C. muterezii. Further, according to the comparative morphology of the reproductive organs, C. salzburgeri sp. nov. differs from C. franswittei found on the gills of P. marginatus and P. curvifrons (see Van Steenberge et al., 2015) by (i) the hook pairs (shorter hook pair I and longer pairs II–IV, VI, and VII in C. salzburgeri sp. nov. vs short hook pairs I–IV, VI, and VII in C. franswittei), (ii) the shorter dorsal anchors (26–29 mm in C. salzburgeri sp. nov. vs 31–40 mminC. franswittei), (iii) the shorter copulatory duct (39–42 mminC. salzburgeri sp. nov. vs 47–57 mminC. franswittei), and (iv) the shorter accessory piece (28–30 mminC. salzburgeri sp. nov. vs 31–46 mmin C. franswittei). C. salzburgeri sp. nov. is distinguishable from the newly described species C. masilyai sp. nov. by (i) the longer dorsal bar (35–38 mminC. salzburgeri sp. nov. vs 22–24 mminC. masilyai sp. nov.), (ii) the length of its auricles (21–24 mmin C. salzburgeri sp. nov. vs 8–11 mminC. masilyai sp. nov.), (iii) the differently shaped copulatory duct (straight, large copulatory duct and large distal opening in C. salzburgeri sp. nov. vs thin, slightly curved proximally, straight halfway, folded distally in C. masilyai sp. nov.), and (v) the differently shaped accessory piece (linked to basal bulb, thin proximally, straight, ending pincer-like with one extremity shorter than the

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 20/37 Figure 9 Sclerotized structures of Cichlidogyrus sergemorandi sp. nov. ex Tylochromis polylepis. DA, dorsal anchor; DB, dorsal bar; VA, ventral anchor; VB, ventral bar; I–VII, hooks; MCO, male copulatory organ; He, heel; Ct, copulatory tube; Ap, accessory piece; Vg, vagina. Full-size  DOI: 10.7717/peerj.5604/fig-9

other in C. salzburgeri sp. nov. vs thin and slightly curved in the middle part ending in pincer-like structure in C. masilyai sp. nov.).

Type-host: Petrochromis trewavasae Poll, 1948 (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Tropheini Poll, 1986).

Host accession number: MH298007.

Site of infection: Gills.

Type-locality: Pemba (340′S, 2910′E; caught when snorkelling), DRC, LT.

Prevalence & intensity of infection: one fish specimen infected/two fish specimen examined, four parasite specimens on infected fish.

Holotype: MRAC M.T.38448.

Paratype: MRAC M.T.38449.

Etymology: the specific epithet ‘salzburgeri’ honours the biologist Dr. Walter Salzburger, professor at the University of Basel (Switzerland), for his work on cichlid evolution and in appreciation of his assistance during the fieldtrip.

Cichlidogyrus sergemorandi sp. nov. Fig. 9 urn:lsid:zoobank.org:act:7A4653B3-2C25-4E3A-9D4A-3FA76165C167.

Description. Based on seven specimens fixed in GAP. Body 659 (594–769; n = 4) long, 115 (95–146; n = 4) wide at mid-body. Dorsal anchors with short shaft and more

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 21/37 pronounced guard (approximately two times the length of shaft), slightly bent blade with arched point: a = 24 (23–26; n = 5); b = 19 (18–20; n = 5); c =2–3(n = 5); d =7(6–8; n = 5); e =6–7(n = 5). C-shaped dorsal bar with blunt endings and short auricles: h =4–5 (n = 5); w =3–4(n = 5); x = 18 (16–20; n = 5); y =9(7–10; n = 5). Ventral anchors with shorter shaft than guard and arched point: a = 23 (22–24; n = 4); b =20–21 (n = 5); c =3(2–4; n = 5), d =7(6–8; n = 5); e =9(8–10; n = 5). V-shaped ventral bar: w =4–5 (n = 5); x = 26 (25–27; n = 5). Haptor with seven short hook pairs, hooks V retain their larval size, each hook with erect thumb and shank comprised of two subunits (see above): pair I = 13–14 (n = 5) long, pair II = 18 (17–19; n = 5) long, pair III = 21 (19–22; n = 5) long, pair IV = 22 (21–23; n = 5) long, pair V = 10 (9–11; n = 5) long, pair VI = 22 (21–24; n = 5) long, and pair VII = 22 (20–24; n = 4) long. Male copulatory organ with an elongated basal bulb prolonged into a long, spirally coiled copulatory duct with thick walls, ending distally in a sharp extremity: MCO = 40 (38–44; n = 6); Ct = 45 (42–47; n = 7). Distinct heel, He = 5 (4–6; n = 6). Large accessory piece unattached to the basal bulb and pierced distally by the copulatory duct, Ap = 38 (33–39; n = 7). Vagina pouch-like in shape, variable in size: V = 29 (19–38; n = 6); v =9(7–11; n = 6).

Diagnosis. Regarding the morphology of the hook pairs and the vagina, C. sergemorandi sp. nov. belongs to the same morphological group as C. antoineparisellei sp. nov. (see above). The newly described species possesses a dorsal bar with short auricles, a spirally shaped copulatory duct, and an accessory piece separated from the basal bulb, features observed in many representatives described on tylochromine cichlids such as C. mulimbwai and C. muzumanii, parasites of the same cichlid host from LT (Muterezi Bukinga et al., 2012), and the non-Tanganyikan C. berrebii,C.kothiasi, and C. pouyaudi from T. jentinki (Steindachner, 1894) (Pariselle & Euzet, 1994), and C. chrysopiformis and C. djietoi from T. sudanensis Daget, 1954 (Pariselle, Bitja Nyom & Bilong Bilong, 2014). C. sergemorandi sp. nov. is most reminiscent of C. mulimbwai. The former differs from C. mulimbwai by (i) the shorter dorsal and ventral anchors (respectively, 23–26; 22–24 mminC. sergemorandi sp. nov. vs 32–42; 31–40 mminC. mulimbwai), (ii) the shorter ventral and dorsal bars (respectively, 16–20; 25–27 mminC. sergemorandi sp. nov. vs 33–44; 38–52 mminC. mulimbwai), and (iii) the vagina (no sclerotized vagina in C. mulimbwai unlike in C. sergemorandi sp. nov.). C. sergemorandi sp. nov. differs from C. muzumanii by (i) the first hook pair (thin pair I in C. sergemorandi sp. nov. vs large pair in C. muzumanii), (ii) the shorter dorsal and ventral anchors (respectively, 23–26; 22–24 mminC. sergemorandi sp. nov. vs 44–58; 37–47 mminC. muzumanii), (iii) the shorter dorsal bar and its auricles (respectively, 16–20; 4–5 mminC. sergemorandi sp. nov. vs 45–62; 10–19 mminC. muzumanii), (iv) the shorter ventral bar (25–27 mmin C. sergemorandi sp. nov. vs 47–63 mminC. muzumanii), (v) the shorter copulatory duct distally differently shaped (sharp extremity, 42–47 mminC. sergemorandi sp. nov. vs broad extremity (not mentioned in the original description), 57–68 mminC. muzumanii), (vi) the longer accessory piece (33–39 mminC. sergemorandi sp. nov. vs 17–20 mmin C. muzumanii), and (vii) the vagina (no sclerotized vagina in C. muzumanii vs sclerotized vagina in C. sergemorandi sp. nov.).

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 22/37 Table 2 Overview of the undescribed species of Cichlidogyrus characterized in the present study with their type-host and locality of sampling. Cichlidogyrus spp. Host Type-locality C. sp. ‘Callochromis melanostigma’ Fig. 10A C. melanostigma Kilomoni beach (Boulenger, 1906) (320′S, 2910′E) C. sp. ‘Xenotilapia flavipinnis 1’ Fig. 10B X. flavipinnis Poll, 1985 Pemba (337′S, 299′E) C. sp. ‘Xenotilapia flavipinnis 2’ Fig. 10C C. sp. ‘Interochromis loocki 5’ Fig. 10D I. loocki (Poll, 1949) Pemba (337′S, 299′E) C. sp. ‘Petrochromis orthognathus 2’ Fig. 10E P. orthognathus Matthes, 1959 Pemba (337′S, 299′E) C. sp. ‘Petrochromis orthognathus 3’ Fig. 10F

Previous record: Cichlidogyrus sp. ‘T. polylepis 3’ Muterezi Bukinga et al., 2012.

Type-host: Tylochromis polylepis (Boulenger, 1900) (Perciformes Bleeker, 1859: Cichlidae Heckel, 1840: Tylochromini Poll, 1986).

Host accession number: MH298008.

Site of infection: Gills.

Type-locality: Mulongwe fish market (322′S, 296′E), Uvira, DRC, LT.

Prevalence & intensity of infection: two fish specimens infected/three fish specimens examined, one to five parasite specimens per infected host.

Holotype: MRAC M.T.38444.

Paratype: MRAC M.T.38445.

Etymology: the specific epithet of the new species ‘sergemorandi’ honours the French evolutionary biologist and ecologist Dr. Serge Morand from CNRS and CIRAD (France), and CILM (Laos) for his work on ecology and the evolution of parasites.

Morphological characterisation of the MCO of the undescribed species On the basis of the MCO features, we characterize six new undescribed species, namely C. sp. ‘C. melanostigma’, C. sp. ‘X. flavipinnis 1’, and C. sp. ‘X. flavipinnis 2’ (all from ectodine hosts), and C. sp. ‘I. loocki 5’, C. sp. ‘P. orthognathus 2’, and C. sp. ‘P. orthognathus 3’ (all from tropheine hosts) (Table 2; Fig. 10A–10F). These species could not be formally described because of the lack of material. Further, the haptoral parts of some specimens were not clearly visible for drawings. However, the general shape of the MCO provided sufficient morphological information to distinguish these species that can be considered as new to science. The morphological characterization of the MCO of the undescribed species of Cichlidogyrus recognized on Congolese host specimens did not allow us to confirm the presence/absence of a sclerotized vagina.

Cichlidogyrus sp. ‘C. melanostigma’ Fig. 10A

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 23/37 Figure 10 Sclerotized structures of the undescribed species of Cichlidogyrus characterized in this study. (A) Cichlidogyrus sp. ‘C. melanostigma.’ (B) Cichlidogyrus sp. ‘X. flavipinnis 1.’ (C) Cichlido- gyrus sp. ‘X. flavipinnis 2.’ (D) Cichlidogyrus sp. ‘I. loocki 5.’ (E) Cichlidogyrus sp. ‘P. orthognathus 2.’ (F) Cichlidogyrus sp. ‘P. orthognathus 3.’ He, heel; Ct, copulatory tube; Ap, accessory piece. Full-size  DOI: 10.7717/peerj.5604/fig-10

In the gills of the ectodine C. melanostigma we found a single monogenean species, namely C. sp. ‘C. melanostigma’. Its MCO lacks a heel and possesses an ovoid basal bulb prolonged into a curved copulatory duct ending in a large opening: MCO = 46 (45–47; n = 2); Ct = 47–48 (n = 2). The accessory piece is curved, L-shaped, attached to the basal bulb and ending in a thick hook, Ap = 36–37 (n = 2). Further, a visible second wall lines the surface of the copulatory duct, which confers a twisted appearance. Cichlidogyrus sp. ‘C. melanostigma’ resembles C. banyankimbonai described from S. diagramma regarding the large distal opening of the copulatory duct (see Van Steenberge et al., 2015). However, the MCO of C. sp. ‘C. melanostigma’ lacks a heel, unlike that of C. banyankimbonai. In addition, C. sp. ‘C. melanostigma’ exhibits a differently shaped accessory piece ending in a hook, a feature not observed in C. banyankimbonai. According to the comparative morphology of the MCO, C. sp. ‘C. melanostigma’ is reminiscent of C. discophonum from A. dewindti, both parasites of ectodine cichlids (see Rahmouni, Vanhove & Šimková, 2017). The two species lack a heel and possess a curved copulatory duct, and an accessory piece ending in a hook. However, C. sp. ‘C. melanostigma’ exhibits a longer accessory piece (36–37 mminC. sp. ‘C. melanostigma’ vs 15–22 mmin C. discophonum). Further, the copulatory duct in C. sp. ‘C. melanostigma’ appears double walled and ends in a large opening, while there is a thick proximal part which tapers distally in C. discophonum. Also, the accessory piece in C. sp. ‘C. melanostigma’ is

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 24/37 L-shaped, while C. discophonum possesses an accessory piece composed of two parts twisted distally. Regarding these differences, we consider C. sp. ‘C. melanostigma’ to be a different species from C. discophonum and new to science.

Cichlidogyrus sp. ‘X. flavipinnis 1’ Fig. 10B

The MCO of C. sp. ‘X. flavipinnis 1’ exhibits a small heel and a thick, well curved copulatory duct, MCO = 37 (n = 1); He = 2 (n = 1); Ct = 36 (n = 1). The subterminal opening of the copulatory duct seems to be located at the last third. Such a feature has never been reported in Cichlidogyrus species described so far. Thus, we cannot confirm whether C. sp. ‘X. flavipinnis 1’ really exhibits this characteristic in its copulatory duct, or it is only an artefact due to isolation or fixation procedures. The accessory piece attached to the basal bulb is thick, curved both proximally and distally with a finger-like ending, Ap = 29 (n = 1). Cichlidogyrus sp. ‘X. flavipinnis 1’ is most similar to C. sp. ‘C. melanostigma’. Both species occur on closely related ectodine host species, that is, X. flavipinnis and C. melanostigma. However, C. sp. ‘X. flavipinnis 1’ presents a heel, a feature missing in C. sp. ‘C. melanostigma’, in addition to the differently shaped proximal endings of the accessory piece (finger-like in C. sp. ‘X. flavipinnis 1’ vs thick hook in C. sp. ‘C. melanostigma’). Therefore, we consider C. sp. ‘X. flavipinnis 1’ and C. sp. ‘C. melanostigma’ as two different species.

Cichlidogyrus sp. ‘X. flavipinnis 2’ Fig. 10C

The second undescribed species isolated from X. flavipinnis has a shorter MCO compared to that of C. sp. ‘X. flavipinnis 2’, and a slightly curved copulatory duct with a narrow extremity, MCO = 22 (n = 1); Ct = 24 (n = 1). Heel reduced to inconspicuous, He = 1 (n = 1). The accessory piece is proximally thick and irregularly shaped, attached to the basal bulb by a small additional part, distally with pincer-like ending, Ap = 16 (n = 1). The heel structure in C. sp. ‘C. melanostigma’, C. sp. ‘X. flavipinnis 1’, and C. sp. ‘X. flavipinnis 2’ is absent to inconspicuous. Further, C. sp. ‘X. flavipinnis 2’ is easily distinguishable from C. sp. ‘C. melanostigma’ and C. sp. ‘X. flavipinnis 1’ (i) by its shorter MCO and a dissimilar copulatory duct with a narrow extremity, and (ii) by the differently sized accessory piece and its characteristic pincer-like shape at its extremity, the distal parts of which are finger and hook-like in C. sp. ‘X. flavipinnis 1’ and C. sp. ‘C. melanostigma’, respectively. On the basis of these differences, we consider C. sp. ‘X. flavipinnis 2’ to be different from C. sp. ‘C. melanostigma’ and C. sp. ‘X. flavipinnis 1’ and to represent a new species to science.

Cichlidogyrus ‘I. loocki 5’ Fig. 10D

Specimens of I. loocki harboured a second parasite species and, according to the morphology of the MCO, we consider it as new to science. Cichlidogyrus sp. ‘I. loocki 5’ exhibits a long MCO with a heel attached to the side of an irregularly shaped basal bulb, MCO = 79 (n = 1); He = 6 (n = 1). The copulatory duct of constant width is long, wavy, and well curved proximally, Ct = 104 (n = 1). The accessory piece is separated

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 25/37 proximally into two thin parts, curved halfway with a twisted gutter-like appearance, distally with an additional small outgrowth and an irregular blunt ending, Ap = 64 (n = 1). However, we cannot reliably confirm the blunt shape in the distal part of the accessory piece since we cannot reject the possibility that this feature is an artefact of parasite fixation. According to the morphology of the MCO, C. sp. ‘I. loocki 5’ is most similar to C. georgesmertensi from P. babaulti in having a long and wavy copulatory duct, in addition to a curved accessory piece attached to the basal bulb (see Van Steenberge et al., 2015). However, C. sp. ‘I. loocki 5’ exhibits a heel attached to the side of the basal bulb like in C. masilyai sp. nov., while in C. georgesmertensi, it is located at the bottom of the basal bulb as in most species of Cichlidogyrus. Further, no gutter-like appearance or additional outgrowth in the distal part of the accessory piece were reported for C. georgesmertensi. Cichlidogyrus sp. ‘I. loocki 5’ can be compared to C. vealli (see Pariselle et al., 2015b). Indeed, the new undescribed species shares the host I. loocki with C. vealli, but the two parasites were sampled in different localities (northern lakeshore vs southern tip of the lake in the case of C. vealli). Moreover, in C. sp. ‘I. loocki 5’, the extremity of the MCO is narrower than that of C. vealli. Also, the undescribed species shows an additional small outgrowth at the proximal third of its MCO, a feature missing in its congener. Further, the accessory piece in C. sp. ‘I. loocki 5’ presents a gutter-like structure only in the middle part and a blunt extremity. In C. vealli, this feature exists throughout the accessory piece. On the basis of these differences, we consider C. sp. ‘I. loocki 5’ to be a different species from C. vealli.

Cichlidogyrus sp. ‘P. orthognathus 2’ Fig. 10E

Cichlidogyrus sp. ‘P. orthognathus 2’ possesses a long MCO exhibiting an elongated irregularly shaped basal bulb with long heel, MCO = 55 (53–57; n = 4); He = 7 (6–8; n = 4). The copulatory duct is well curved, Ct = 49 (47–51; n = 4). The accessory piece is linked to the basal bulb by a thin filament, curved in the middle part, distally with an outgrowth ending in a hook, Ap = 38 (37–40; n = 4). Cichlidogyrus sp. ‘P. orthognathus 2’ presents a morphotype similar to that of its undescribed congener C. sp. ‘I. loocki 5’, especially in having the additional small outgrowth in the distal part of the accessory piece. However, the MCO and the copulatory duct are shorter in C. sp. ‘P. orthognathus 2’ than in C. sp. ‘I. loocki 5’. Further, the heel is located differently (see above). Thus, on the basis of the morphological differences listed above, we separated the species C. sp. ‘P. orthognathus 2’ from its congener C. sp. ‘I. loocki 5’.

Cichlidogyrus sp. ‘P. orthognathus 3’ Fig. 10F

Cichlidogyrus sp. ‘P. orthognathus 3’ exhibits a short MCO with a reduced heel, MCO = 33 (31–34; n = 2); He = 2–3(n = 2). The copulatory duct is thick, starting in an ovoid basal bulb, curved halfway with a big distal opening, Ct = 30 (28–31; n = 2). The accessory piece is linked to the basal bulb, thick and spirally coiled ending in a hook, Ap = 21 (20–22; n = 2). The general shape of the MCO of C. sp. ‘P. orthognathus 3’ is

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 26/37 reminiscent of C. raeymaekersi, a species described from the tropeine S. diagramma (see Van Steenberge et al., 2015). C. raeymaekersi shows a short and wide copulatory duct with a poorly developed heel, and a thick spirally coiled accessory piece attached to the basal bulb as observed in C. sp. ‘P. orthognathus 3’. However, the latter possesses a copulatory duct with an ovoid basal bulb with a distal wide part, different from the elongated bulb and the bevelled ending in C. raeymaekersi, which makes C. sp. ‘P. orthognathus 3’ distinct from C. raeymaekersi.

DISCUSSION The cichlid fishes of LT have undergone spectacular diversification, filling a diversity of ecological niches within a short time period, and therefore represent one of the most interesting models of adaptive radiation (Tsuboi et al., 2016). Research on cichlids is nowadays combined with the investigation of their parasite diversity in order to study the speciation of both cichlids and their specific parasites (Vanhove et al., 2015, 2016). Currently, only 32 Cichlidogyrus spp. are known from cichlids inhabiting LT (see the overview by Rahmouni et al. (2017)). The present study, based on morphological characters, increases knowledge of the diversity of host specific monogenean species in the cichlids living the northern sub-basin of the lake. We provide descriptions of seven gill monogenean species parasitizing six Congolese representatives of four cichlid tribes in LT. They are C. adkoningsi sp. nov. on C. frontosa (Cyphotilapiini); C. koblmuelleri sp. nov. on C. schoutedeni (Ectodini); C. habluetzeli sp. nov. on C. frontosa and C. schoutedeni; C. antoineparisellei sp. nov. on I. loocki; C. masilyai sp. nov. on P. orthognathus; C. salzburgeri sp. nov. on P. trewavasae (both Tropheini); and finally C. sergemorandi sp. nov. on T. polylepis (Tylochromini). We characterized also six undescribed parasite species on the basis of the morphology of their MCO. They are C. sp. ‘C. melanostigma’ from C. melanostigma; C. sp. ‘X. flavipinnis 1’ and C. sp. ‘X. flavipinnis 2’ from X. flavipinnis (both Ectodini); C. sp. ‘I. loocki 5’ from I. loocki; and finally C. sp. ‘P. orthognathus 2’ and C. sp. ‘P. orthognathus 3’ from P. orthognathus (all Tropheini). First, we identified the cichlid species using morphology and DNA sequence data from the mitochondrial cyt-b region. Nowadays, DNA barcoding targeting mitochondrial regions such as the COI (cytochrome oxidase I) or cyt-b genes for cichlid fish identification is well established and documented (Kullander et al., 2014; Breman et al., 2016). Molecular analyses confirmed the morphological identification of the cichlids analysed for the presence of monogenean parasites in this study. Then, we morphologically characterized the newly described species of Cichlidogyrus on the basis of the sclerotized parts of their attachment (haptor) and reproductive organs (MCO and sclerotized vagina when visible). The sclerotized structures of dactylogyridean monogeneans have been extensively investigated in various ecological and evolutionary contexts. Several studies have reported the influence of such sclerotized structures on host specificity, parasite specialization, and reproductive isolation among congeners through niche ecology (Šimková & Morand, 2008; Vignon, Pariselle & Vanhove, 2011; Messu Mandeng et al., 2015). Because of the limitations of light microscopy as regards some morphological characters, sclerotized structures are increasingly studied

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 27/37 using enzymatic digestion followed by scanning electron microscopy. In the case of Cichlidogyrus, we can cite recent studies of Fannes, Vanhove & Huyse (2017) and Igeh, Dos Santos & Avenant-Oldewage (2017). Using this method, they redescribed three Cichlidogyrus spp. (C. tiberianus, C. dossoui, and C. philander), and provided morphological details on the haptoral and reproductive hard parts, which are not visible with light microscopy. Regarding our study, most sclerites of C. adkoningsi sp. nov. found on C. frontosa are reminiscent of species previously described from ectodine cichlids (see diagnosis). Despite these similarities, C. adkoningsi sp. nov., which represents the first record of a gill ectoparasite on a Tanganyikan cyphotilapiine, is easily distinguishable from its Tanganyikan congeners by the shape and dimensions of its sclerotized structures (such as the dorsal bar and its very characteristic long auricles, and the MCO with its irregularly shaped heel and S-shaped accessory piece ending in a hook). Similarly, C. habluetzeli sp. nov., a common species found on the same host and on the gills of C. schoutedeni, exhibits the same morphotype as species infecting ectodines, as well as other Cichlidogyrus species described from various Tanganyikan tribes. Morphologically, most of the sclerotized structures of C. habluetzeli sp. nov. are bigger than in similar species. Yet, the dorsal bar (with its small hollow outgrowths on the anterior face and its straight auricles), in addition to the straight copulatory duct, heel, and accessory piece, are structures reminiscent of those of C. aspiralis, C. pseudoaspiralis, C. centesimus (Vanhove, Volckaert & Pariselle, 2011; Rahmouni, Vanhove & Šimková, 2017), C. casuarinus (Pariselle et al., 2015a), and finally C. nshomboi (Muterezi Bukinga et al., 2012). However, some features are present in some species and missing in others. This is the case with the spirally-ornamented wall of the MCO observed exclusively in C. casuarinus, C. centesimus, and C. nshomboi, or the sclerotized vagina present in all the species listed above (including C. habluetzeli sp. nov.) except for C. pseudoaspiralis (Vanhove, Volckaert & Pariselle, 2011; Muterezi Bukinga et al., 2012; Pariselle et al., 2015a; Kmentová et al., 2016b; Rahmouni et al., 2017). Regarding the haptoral configuration, C. habluetzeli sp. nov. with its hook pairs (long pairs I–IV, VI, and VII with large pair I) joins the group that harbours, so far, a single member, C. centesimus. In recent studies on the monogenean diversity of Tanganyikan cichlids, similarities in the haptoral and reproductive organs of Cichlidogyrus spp. parasitizing phylogenetically closely related cichlid hosts are far from unusual. In the present study, for instance, C. habluetzeli sp. nov. and C. koblmuelleri sp. nov. infecting C. schoutedeni are morphologically similar to some congeners of ectodine cichlids. This is also true for the undescribed species C. sp. ‘C. melanostigma’ and C. discophonum from A. dewindti sampled in north-eastern LT. In fact, Rahmouni et al. (2017) reported that two cichlid hosts Eretmodus marksmithi Burgess, 2012 and Tanganicodus irsacae Poll, 1950, both belonging to the Eretmodini, host two morphologically similar monogenean species C. jeanloujustinei and C. evikae, respectively. Moreover, monogenean communities composed of morphologically similar Cichlidogyrus species were reported in Burundese O. nasuta and its congeners from more southern localities. This observation was explained by the large distribution of ectodine hosts in the lake (Vanhove, Volckaert & Pariselle, 2011;

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 28/37 Rahmouni, Vanhove & Šimková, 2017). Also in the parasite fauna of tropheine cichlids, morphologically similar Cichlidogyrus species infect phylogenetically related cichlid species (Vanhove, 2012; Pariselle et al., 2015b; Van Steenberge et al., 2015). As reported by Braga, Araújo & Boeger (2014), the phylogenetic relatedness of the host species has a strong influence on the distribution of monogenean parasites. On the basis of molecular data, recent work on Tanganyikan cichlids placed the Ectodini as a sister group to a clade formed by Cyphotilapiini and two other tribes (Eretmodini Poll, 1986 and Haplochromini), all mouthbrooding lineages (Meyer, Matschiner & Salzburger, 2015). This could explain the presence of C. habluetzeli sp. nov. on both C. frontosa and C. schoutedeni. At the same time, Boulengerochromini and Bathybatini are phylogenetically distant from Cyphotilapiini and Ectodini (Meyer, Matschiner & Salzburger, 2015), although they all share the morphotype of Cichlidogyrus exhibiting a spiral thickening in the wall of the copulatory duct. Possibly, living in at least partially overlapping habitat(s) could allow unrelated cichlids to host similar Cichlidogyrus species. In relation to the unique morphology of the hook pairs observed in C. habluetzeli sp. nov. and its congener C. centesimus, Rahmouni et al. (2017) already suggested the existence of more haptoral groups in Cichlidogyrus than previously reported by Vignon, Pariselle & Vanhove (2011), and proposed a reinvestigation of the structural diversity of the hook pairs in Cichlidogyrus spp. and a redefinition of the ‘boundaries’ between the haptoral groups. In addition to hook morphology, Rahmouni et al. (2017) also discussed the sclerotization of the vagina. On the basis of this study, we find that a few Cichlidogyrus spp. described from Tanganyikan hosts and possessing a sclerotized vagina belong to none of the haptoral groups defined by Vignon, Pariselle & Vanhove (2011) (who did not include species of Cichlidogyrus from LT (Pariselle & Euzet, 2009; Rahmouni, Vanhove & Šimková, 2017)). This is the case for C. habluetzeli sp. nov., C. casuarinus, C. centesimus, and C. nshomboi (Vanhove, Volckaert & Pariselle, 2011; Muterezi Bukinga et al., 2012; Pariselle et al., 2015a). Moreover, only three Tanganyikan species of Cichlidogyrus specifically C. mbirizei from O. tanganicae (Oreochromini) (Muterezi Bukinga et al., 2012) and two of the newly described species (C. antoineparisellei sp. nov. from I. loocki (Tropheini), and C. sergemorandi sp. nov. from T. polylepis (Tylochromini)) exhibit short hook pairs I–IV,VI,andVIIandasclerotizedvagina.Membersofthe Oreochromini and Tylochromini tribes occurinthelakebutcolonizedthelake relatively recently (Meyer, Matschiner & Salzburger, 2015). The tribe Oreochromini was proposed only recently (Dunz & Schliewen, 2013). Therefore, formulating hypotheses on the significance of the hooks and the sclerotized vagina, seen together in Cichlidogyrus spp., is problematic. Rahmouni et al. (2017) has already underlined the necessity to investigate and analyse whether there is a correlation between the reproductive organs (the presence/ absence of a sclerotized vagina) and the haptoral sclerites (the morphology of the hook pairs). Muterezi Bukinga et al. (2012) reported the presence of Cichlidogyrus sp. ‘T. polylepis 3’, in addition to two other Cichlidogyrus species isolated from T. polylepis sampled in the central sub-basin of the lake. Because of the low number of helminth specimens collected and studied, they did not provide a formal description. On the basis of the

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 29/37 original drawing of the hard parts of Cichlidogyrus sp. ‘T. polylepis 3’, we were easily able to identify the species, complete the observations made by Muterezi Bukinga et al. (2012), and formally describe the new species herein as C. sergemorandi sp. nov. The Tropheini is one of the best studied cichlid tribes with respect to ectoparasitic monogeneans. Twelve gill species of Cichlidogyrus were previously recognized on eight cichlid species of this tribe (Gillardin et al., 2012; Pariselle et al., 2015b; Van Steenberge et al., 2015). In this study, I. loocki, P. orthognathus, and P. trewavasae sampled from the northern Congolese lakeshore revealed the presence of C. antoineparisellei sp. nov., C. masilyai sp. nov., and C. salzburgeri sp. nov. The latter two species represent the first formal descriptions of gill monogeneans on representatives of Petrochromis. I. loocki from the Zambian lakeshore was previously studied for monogenean parasites and three species, C. buescheri, C. schreyenbrichardorum, and C. vealli, were described (Pariselle et al., 2015b). The new species of the present study are morphologically highly differentiated from those described by Pariselle et al. (2015b). Our record is strong new evidence of the geographical variation in parasite diversity in a particular cichlid species throughout LT. Indeed, Vanhove, Volckaert & Pariselle (2011) described four Cichlidogyrus spp. from the ectodine host O. nasuta and its two congeners O. ventralis and O. boops sampled along the more southern coastline of LT in DRC, Zambia, and Tanzania, while Rahmouni et al. (2017) reported three different species of Cichlidogyrus found on O. nasuta inhabiting the northern part of the lake. Moreover, Pariselle et al. (2015b) previously compared the three Cichlidogyrus spp. described on I. loocki to parasite specimens belonging to hitherto undescribed species retrieved from Petrochromis spp. from the collection of the MRAC. They provided micrographs of the haptoral hard parts of potentially six Cichlidogyrus spp. isolated from three cichlids (P. fasciolatus Boulenger, 1914, a species phylogenetically closely related to I. loocki; P. famula Matthes & Trewavas, 1960, an intermediate species between the Petrochromis/Interochromis clade and the ‘large’ Petrochromis clade; and finally, P. trewavasae ephippium Brichard, 1989, a member of the ‘large’ Petrochromis)(Yamaoka, 1997). On the basis of these micrographs, we were able to compare the morphology of the dorsal and ventral anchors and bars and identify differences between the newly described species on representatives of Petrochromis and the undescribed species reported by Pariselle et al. (2015b). However, due to the lack of haptoral parts in our specimens (damaged parts), and the lack of reproductive organs in the micrographs of Cichlidogyrus included in Pariselle et al. (2015b), we were not able to clearly state whether Cichlidogyrus spp. isolated from P. orthognathus represents one of the species collected from P. famula, P. fasciolatus, and P. trawavasae ephippium. In contrast, C. masilyai sp. nov. and C. salzburgeri sp. nov. isolated respectively from P. orthognathus and P. trawavasae were easily distinguishable from their congeners reported by Pariselle et al. (2015b) by their typical C-shaped dorsal bar with straight auricles and a dorsal bar with relatively wide auricles, respectively. Regarding the undescribed species of Cichlidogyrus presented herein, resampling C. melanostigma, I. loocki, P. orthognathus, and X. flavipinnis would allow future researchers to collect more parasite specimens, characterize the haptoral parts, and provide formal descriptions.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 30/37 CONCLUSION Lake Tanganyika has an impressive degree of cichlid parasite diversity with numerous species as yet undescribed. Cichlidogyrus includes (to date) 39 species from LT, including the new species described in the present study, and 79 species from other locations. Species of Cichlidogyrus infecting closely related cichlid fishes are often morphologically similar, but taxonomically distinct. Further morphological and molecular studies are necessary to elucidate the origin of the various lineages of Cichlidogyrus in LT and their evolutionary history.

INSTITUTIONAL ABBREVIATIONS MNHN Muséum National d’Histoire Naturelle, Paris, France. MZH Finish Museum of Natural History, Helsinki, Finland. MRAC Royal Museum for Central Africa, Tervuren, Belgium.

ACKNOWLEDGEMENTS The authors would like to thank the following people and institutions for their invaluable support in the realization of this study: Walter Salzburger (University of Basel, Switzerland), Donatien Muzumani Risasi (CRH-Uvira) and Maarten Van Steenberge (MRAC/RBINS) for fish identification; Ludmila Raisingerová and Nikol Kmentová (Masaryk University, Brno, Czech Republic) and Fidel Muterezi Bukinga (Centre de Recherche en Hydrobiologie, Uvira, DRC) for helping with fish dissection, parasite isolation, and fixation; Pascal Masilya Mulungula, Vercus Lumami Kapepula, Théophile Mulimbwa N’sibula, Eugène Bahane Byaragi, Simon Kambale Mukeranya, and all the staff working in the Centre de Recherche en Hydrobiologique in Uvira for their precious help with the co-organisation of the fieldwork and for their scientificinputon Tanganyikan cichlids; Katerina Francová, Evá Řehulková, Ivá Prikrylová, Carlos Mendoza-Palmero, Mária Seifertová and Jirí Vorel (Masaryk University, Brno, Czech Republic) for advice concerning taxonomy and genetic questions; and Kristína Civáňová, Eliška Jirounková, Kristýna Koukalová and Lenka Gettová (Masaryk University, Brno, Czech Republic) for their assistance in the molecular laboratory. We are also grateful to many of the above for providing specimens or hosting the authors during the fieldtrip. Special thanks go to Ondrej Hájek (Masaryk University, Brno, Czech Republic) for making the map with the sampling sites. Finally, the authors would like to thank Matthew Nicholls and Joseph Lennon for English language revision of the manuscript.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding This research was funded by the Czech Science Foundation project no. P505/12/G112— European Centre of Ichtyoparasitology (ECIP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 31/37 Grant Disclosures The following grant information was disclosed by the authors: Czech Science Foundation project no. P505/12/G112—European Centre of Ichtyoparasitology (ECIP).

Competing Interests The authors declare that they have no competing interests.

Author Contributions  Chahrazed Rahmouni conceived and designed the experiments, performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.  Maarten P.M. Vanhove conceived and designed the experiments, analysed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft, co-organised the field trip and the parasite collection.  Andrea Šimková analysed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Animal Ethics The following information was supplied relating to ethical approvals (i.e. approving body and any reference numbers): This study was approved by the Animal Care and Use Committee of the Faculty of Science, Masaryk University in Brno (Czech Republic) (approval number CZ01308).

Field Study Permissions The following information was supplied relating to field study approvals (i.e. approving body and any reference numbers): Sampling was carried out under mission statements 022/MINEURS/CRH-U/2013 and 031/MINRST/CRH-U/2016 from the Centre de Recherche en Hydrobiologie-Uvira. In the absence of relevant animal welfare regulations in the D.R. Congo, the same strict codes of practice enforced within the European Union were applied.

DNA Deposition The following information was supplied regarding the deposition of DNA sequences: GenBank: MH297985–MH298008. New Species Registration The following information was supplied regarding the registration of a newly described species: Publication LSID: urn:lsid:zoobank.org:pub:7076794A-B9EB-4FFC-AC49- 66C304EC5BFB. Cichlidogyrus adkoningsi sp. nov. LSID urn:lsid:zoobank.org:act:526C2A74-E3D6-4357-B1E8-03B08B95CE38. Cichlidogyrus koblmuelleri sp. nov. LSID urn:lsid:zoobank.org:act:473DB764-6798-43EE-8DD8-3B10D1AC1BBF.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 32/37 Cichlidogyrus habluetzeli sp. nov. LSID urn:lsid:zoobank.org:act:EC79CE69-2D88-4D96-A255-AC0E31B76EAE. Cichlidogyrus antoineparisellei sp. nov. LSID urn:lsid:zoobank.org:act:B324AD44-4520-4129-8DBB-8985A981783D. Cichlidogyrus masilyai sp. nov. LSID urn:lsid:zoobank.org:act:4D5AF9E3-9CCE-4D3F-95B4-CCDA7B1A5C91. Cichlidogyrus salzburgeri sp. nov. LSID urn:lsid:zoobank.org:act:612A6D2B-09DD-4902-8640-A4877277E7F3. Cichlidogyrus sergemorandi sp. nov. urn:lsid:zoobank.org:act:7A4653B3-2C25-4E3A-9D4A-3FA76165C167.

Data Availability The following information was supplied regarding data availability: The type-material of the new species described in this study is housed in the invertebrate collection of the Royal Museum for Central Africa (MRAC), Tervuren, Belgium; the Finnish Museum of Natural History (MZH), Helsinki, Finland; and the Muséum National d’Histoire Naturelle (MNHN), Paris, France (see taxonomic summaries for details on repositories and accession numbers).

REFERENCES Barluenga M, Meyer A. 2010. Phylogeography, colonization and population history of the Midas cichlid species complex (Amphilophus spp.) in the Nicaraguan crater lakes. BMC Evolutionary Biology 10(1):326 DOI 10.1186/1471-2148-10-326. Birgi E, Lambert A. 1986. Présence chez un Nandidae (Téléostéen), Polycentropsis abbreviata Boulenger, 1901, du genre Cichlidogyrus (Monogenea, Monopisthocotylea, Ancyrocephalidae). Description de Cichlidogyrus nandidae n. sp. et de Cichlidogyrus euzeti n. sp. Annales de Parasitologie Humaine et Comparée 61(5):521–528 DOI 10.1051/parasite/1986615521. Braga MP, Araújo SBL, Boeger WA. 2014. Patterns of interaction between Neotropical freshwater fishes and their gill Monogenoidea (Platyhelminthes). Parasitology Research 113(2):481–490 DOI 10.1007/s00436-013-3677-8. Breman FC, Loix S, Jordaens K, Snoeks J, Van Steenberge M. 2016. Testing the potential of DNA barcoding in vertebrate radiations: the case of the littoral cichlids (Pisces, Perciformes, Cichlidae) from Lake Tanganyika. Molecular Ecology Resources 16(6):1455–1464 DOI 10.1111/1755-0998.12523. Chakrabarty P. 2004. Cichlid biogeography: comment and review. Fish and Fisheries 5(2):97–119 DOI 10.1111/j.1467-2979.2004.00148.x. Cohen AS, Lezzar KE, Tiercelin AJJ, Soreghan M. 1997. New palaeogeographic and lake-level reconstructions of Lake Tanganyika: implications for tectonic, climatic and biological evolution in a rift lake. Basin Research 9(2):107–132 DOI 10.1046/j.1365-2117.1997.00038.x. Dossou C, Birgi E. 1984. Monogènes parasites d’Hemichromis fasciatus Peters, 1857 (Teleostei, Cichlidae). Annales des Sciences Naturelles. Zoologie et Biologie Animale 6:101–109. Douëllou L. 1993. Monogeneans of the genus Cichlidogyrus Paperna, 1960 (Dactylogyridae: Ancyrocephalinae) from cichlid fishes of Lake Kariba (Zimbabwe) with descriptions of five new species. Systematic Parasitology 25(3):159–186 DOI 10.1007/BF00007007.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 33/37 Dunz AR, Schliewen UK. 2013. Molecular phylogeny and revised classification of the haplotilapiine cichlid fishes formerly referred to as “Tilapia”. Molecular Phylogenetics and Evolution 68(1):64–80 DOI 10.1016/j.ympev.2013.03.015. Fannes W, Vanhove MPM, Huyse T. 2017. Redescription of Cichlidogyrus tiberianus Paperna, 1960 and C. dossoui Douellou, 1993 (Monogenea: Ancyrocephalidae), with special reference to the male copulatory organ. Systematic Parasitology 94(1):133–144 DOI 10.1007/s11230-016-9685-1. Froese R, Pauly D. 2017. Fishbase. World Wide Web electronic publication. Available at http://www.fishbase.org (accessed September 2017). Gillardin C, Vanhove MPM, Pariselle A, Huyse T, Volckaert FAM. 2012. Ancyrocephalidae (Monogenea) of Lake Tanganyika: II: description of the first Cichlidogyrus spp. parasites from Tropheini fish hosts (Teleostei, Cichlidae). Parasitology Research 110(1):305–313 DOI 10.1007/s00436-011-2490-5. Gussev AV. 1983. The method of collecting and processing of fish parasitic monogenean material. Nauka, Leningrad. 48 [in Russian]. Igeh PC, Dos Santos QM, Avenant-Oldewage A. 2017. Redescription of Cichlidogyrus philander (Monogenea, Ancyrocephalidae) using scanning electron microscopy (SEM) and molecular analysis. Parasite 24:49 DOI 10.1051/parasite/2017046. Jorissen MWP, Pariselle A, Huyse T, Vreven EJ, Snoeks J, Volckaert FAM, Manda AC, Kasembele GK, Artois T, Vanhove MPM. 2018. Diversity and host specificity of monogenean gill parasites (Platyhelminthes) of cichlid fishes in the Bangweulu-Mweru ecoregion. Journal of Helminthology 92(4):417–437 DOI 10.1017/S0022149X17000712. Kidd MR, Duftner N, Koblmüller S, Sturmbauer C, Hofmann HA. 2012. Repeated parallel evolution of parental care strategies within Xenotilapia, a genus of cichlid fishes from Lake Tanganyika. PLOS ONE 7(2):e31236 DOI 10.1371/journal.pone.0031236. Kmentová N, Gelnar M, Koblmüller S, Vanhove MPM. 2016a. Deep-water parasite diversity in Lake Tanganyika: description of two new monogenean species from benthopelagic cichlid fishes. Parasites & Vectors 9(1):426 DOI 10.1186/s13071-016-1696-x. Kmentová N, Gelnar M, Mendlová M, Van Steenberge M, Koblmüller S, Vanhove MPM. 2016b. Reduced host-specificity in a parasite infecting non-littoral Lake Tanganyika cichlids evidenced by intraspecific morphological and genetic diversity. Scientific Reports 6(1):39605 DOI 10.1038/srep39605. Koblmüller S, Sefc KM, Sturmbauer C. 2008. The Lake Tanganyika cichlid species assemblage: recent advances in molecular phylogenetics. Hydrobiologia 615(1):5–20 DOI 10.1007/s10750-008-9552-4. Kocher TD. 2004. Adaptive evolution and explosive speciation: the cichlid fish model. Nature Reviews Genetics 5(4):288–298 DOI 10.1038/nrg1316. Konings A. 2015. Tanganyika cichlids in their natural habitat. El Paso: Cichlid Press. Kritsky D, Thatcher DE, Boeger W. 1986. Neotropical monogenea. 8. Revision of Urocleidoides (Dactylogyridae, Ancyrocephalinae). Proceedings of the Helminthological Society of Washington 53:1–37. Kullander SO, Karlsson M, Karlsson M, Norén M. 2014. Chalinochromis cyanophleps, a new species of cichlid fish (Teleostei: Cichlidae) from Lake Tanganyika. Zootaxa 3790(3):425–438 DOI 10.11646/zootaxa.3790.3.2. Malmberg G. 1957. On the occurrence of Gyrodactylus on Swedish fishes. Skrifter utgivna av Södra Sveriges Fiskeriföreningen 1956:19–76 [in Swedish].

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 34/37 Mendlová M, Desdevises Y, Civáňová K, Pariselle A, Šimková A. 2012. Monogeneans of west African cichlid fish: evolution and cophylogenetic interactions. PLOS ONE 7(5):e37268 DOI 10.1371/journal.pone.0037268. Mendoza-Palmero CA, Blasco-Costa I, Hernandez-Mena D, Perez-Ponce De Leon G. 2017. Parasciadicleithrum octofasciatum n. gen., n. sp. (Monogenoidea: Dactylogyridae), parasite of Rocio octofasciata (Regan) (Cichlidae: Perciformes) from Mexico characterised by morphological and molecular evidence. Parasitology International 66(2):152–162 DOI 10.1016/j.parint.2017.01.006. Messu Mandeng FD, Bilong Bilong CF, Pariselle A, Vanhove MPM, Bitja Nyom AR, Agnèse JF. 2015. A phylogeny of Cichlidogyrus spp. (Monogenea, Dactylogyridea) clarifies a host-switch between fish families and reveals an adaptive component to attachment organ morphology of this parasite genus. Parasites & Vectors 8(1):582 DOI 10.1186/s13071-015-1181-y. Meyer BS, Matschiner M, Salzburger W. 2015. A tribal level phylogeny of Lake Tanganyika cichlid fishes based on a genomic multi-marker approach. Molecular Phylogenetics and Evolution 83:56–71 DOI 10.1016/j.ympev.2014.10.009. Mizelle J. 1936. New species of trematodes from the gills of Illinois fishes. American Midland Naturalist 17(5):785–806 DOI 10.2307/2420687. Muschick M, Indermaur A, Salzburger W. 2012. Convergent evolution within an adaptive radiation of cichlid fishes. Current Biology 22(24):2362–2368 DOI 10.1016/j.cub.2012.10.048. Musilová N, Řehulková E, Gelnar M. 2009. Dactylogyrids (Platyhelminthes: Monogenea) from the gills of Labeo (Teleostei: Cyprinidae) from West Africa. Zootaxa 2241:47–68 DOI 10.5281/zenodo.190572. Muterezi Bukinga F, Vanhove MPM, Van Steenberge M, Pariselle A. 2012. Ancyrocephalidae (Monogenea) of Lake Tanganyika: III: Cichlidogyrus infecting the world’s biggest cichlid and the non-endemic tribes Haplochromini, Oreochromini and Tylochromini (Teleostei, Cichlidae). Parasitology Research 111(5):2049–2061 DOI 10.1007/s00436-012-3052-1. NCBI Resource Coordinators. 2017. Database resources of the National Center for Biotechnology Information. Nucleic Acids Research 45(D1):D12–D17 DOI 10.1093/nar/gkw1071. Paperna I. 1960. Studies on monogenetic trematodes in Israel. 2 Monogenetic trematodes of cichlids. Bamidgeh 12:20–33. Paperna I, Thurston JP. 1969. Monogenetic Trematodes collected from cichlid fish in Uganda; including the description of five new species of Cichlidogyrus. Revue de Zoologie et de Botanique Africaines 79:15–33. Pariselle A, Bilong Bilong CF, Euzet L. 2003. Four new species of Cichlidogyrus Paperna, 1960 (Monogenea, Ancyrocephalidae), all gill parasites from African mouthbreeder tilapias of the genera Sarotherodon and Oreochromis (Pisces, Cichlidae), with a redescription of C. thurstonae Ergens, 1981. Systematic Parasitology 56(3):201–210 DOI 10.1023/B:SYPA.0000003807.27452.bd. Pariselle A, Boeger WA, Snoeks J, Bilong Bilong CF, Morand S, Vanhove MPM. 2011. The monogenean parasite fauna of cichlids: a potential tool for host biogeography. International Journal of Evolutionary Biology 2011:471–480 DOI 10.4061/2011/471480. Pariselle A, Euzet L. 1994. Three new species of Cichlidogyrus Paperna, 1960 (Monogenea: Ancyrocephalidae) parasitic on Tylochromis jentinki (Steindachner, 1895) (Pisces, Cichlidae) in West Africa. Systematic Parasitology 29(3):229–234 DOI 10.1007/BF00009678. Pariselle A, Euzet L. 1996. Cichlidogyrus Paperna, 1960 (Monogenea, Ancyrocephalidae): gill parasites from West African Cichlidae of the subgenus Coptodon Regan, 1920 (Pisces),

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 35/37 with descriptions of six new species. Systematic Parasitology 34(2):109–124 DOI 10.1007/BF00009685. Pariselle A, Euzet L. 2003. Four new species of Cichlidogyrus (Monogenea: Ancyrocephalidae), gill parasites of Tilapia cabrae (Teleostei: Cichlidae), with discussion on relative length of haptoral sclerites. Folia Parasitologica 50(3):195–201 DOI 10.14411/fp.2003.035. Pariselle A, Euzet L. 2009. Systematic revision of dactylogyridean parasites (Monogenea) from cichlid fishes in Africa, the Levant and Madagascar. Zoosystema 31(4):849–898 DOI 10.5252/z2009n4a6. Pariselle A, Bitja Nyom AR, Bilong Bilong CF. 2014. Four new species of Cichlidogyrus (Monogenea, Ancyrocephalidae) from Sarotherodon mvogoi and Tylochromis sudanensis (Teleostei, Cichlidae) in Cameroon. Zootaxa 3881(3):258–266 DOI 10.11646/zootaxa.3881.3.4. Pariselle A, Muterezi Bukinga F, Van Steenberge M, Vanhove MPM. 2015a. Ancyrocephalidae (Monogenea) of Lake Tanganyika: IV. Cichlidogyrus parasitizing species of Bathybatini (Teleostei, Cichlidae): reduced host-specificity in the deepwater realm? Hydrobiologia 748(1):99–119 DOI 10.1007/s10750-014-1975-5. Pariselle A, Van Steenberge M, Snoeks J, Volckaert F, Huyse T, Vanhove M. 2015b. Ancyrocephalidae (Monogenea) of Lake Tanganyika: does the Cichlidogyrus parasite fauna of Interochromis loocki (Teleostei, Cichlidae) reflect its host’s phylogenetic affinities? Contributions to Zoology 84:25–38. Rahmouni I, Řehulková E, Pariselle A, Berrada Rkhami O, Šimková A. 2017. Four new species of Dactylogyrus Diesing, 1850 (Monogenea: Dactylogyridae) parasitising the gills of northern Moroccan Luciobarbus Heckel (Cyprinidae): morphological and molecular characterisation. Systematic Parasitology 94(5):575–591 DOI 10.1007/s11230-017-9726-4. Rahmouni C, Vanhove MPM, Šimková A. 2017. Underexplored diversity of gill monogeneans in cichlids from Lake Tanganyika: eight new species of Cichlidogyrus Paperna, 1960 (Monogenea: Dactylogyridae) from the northern basin of the lake, with remarks on the vagina and the heel of the male copulatory organ. Parasites & Vectors 10(1):591 DOI 10.1186/s13071-017-2460-6. Řehulková E, Mendlová M, Šimková A. 2013. Two new species of Cichlidogyrus (Monogenea: Dactylogyridae) parasitizing the gills of African cichlid fishes (Perciformes) from Senegal: morphometric and molecular characterization. Parasitology Research 112(4):1399–1410 DOI 10.1007/s00436-013-3291-9. Salzburger W, Mack T, Verheyen E, Meyer A. 2005. Out of Tanganyika: genesis, explosive speciation, key-innovations and phylogeography of the haplochromine cichlid fishes. BMC Evolutionary Biology 5(1):17 DOI 10.1186/1471-2148-5-17. Šimková A, Morand S. 2008. Co-evolutionary patterns in congeneric monogeneans: a review of Dactylogyrus species and their cyprinid hosts. Journal of Fish Biology 73(9):2210–2227 DOI 10.1111/j.1095-8649.2008.02064.x. Snoeks J. 2000. How well known is the ichthyodiversity of the large East African lakes? Advances in Ecological Research 31:17–38 DOI 10.1016/S0065-2504(00)31005-4. Sturmbauer C, Hainz U, Baric S, Verheyen E, Salzburger W. 2003. Evolution of the tribe Tropheini from Lake Tanganyika: synchronized explosive speciation producing multiple evolutionary parallelism. Hydrobiologia 500(1–3):51–64 DOI 10.1023/a:1024680201436. Takahashi T, Koblmüller S. 2011. The adaptive radiation of cichlid fish in Lake Tanganyika: a morphological perspective. International Journal of Evolutionary Biology 2011:620754 DOI 10.4061/2011/620754.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 36/37 Takahashi T, Koblmüller S. 2014. A new species of Petrochromis (Perciformes: Cichlidae) from Lake Tanganyika. Ichthyological Research 61:252–264 DOI 10.1007/s10228-014-0396-9. Takahashi T, Sota T. 2016. A robust phylogeny among major lineages of the East African cichlids. Molecular Phylogenetics and Evolution 100:234–242 DOI 10.1016/j.ympev.2016.04.012. Tsuboi M, Kotrschal A, Hayward A, Buechel SD, Zidar J, Lovlie H, Kolm N. 2016. Evolution of brain-body allometry in Lake Tanganyika cichlids. Evolution: International Journal of Organic Evolution 70(7):1559–1568 DOI 10.1111/evo.12965. Vanhove MPM. 2012. Species flocks and parasite evolution towards a co-phylogenetic analysis of monogenean flatworms of cichlids and gobies. D. Phil. thesis Belgium: Leuven University. Vanhove M, Hablützel PI, Pariselle A, Šimková A, Huyse T, Raeymaekers JAM. 2016. Cichlids: a host of opportunities for evolutionary parasitology. Trends in Parasitology 32(10):820–832 DOI 10.1016/j.pt.2016.07.002. Vanhove MPM, Pariselle A, Van Steenberge M, Raeymaekers JAM, Hablützel PI, Gillardin C, Hellemans B, Breman FC, Koblmüller S, Sturmbauer C, Snoeks J, Volckaert FAM, Huyse T. 2015. Hidden biodiversity in an ancient lake: phylogenetic congruence between Lake Tanganyika tropheine cichlids and their monogenean flatworm parasites. Scientific Reports 5(1):13669 DOI 10.1038/srep13669. Vanhove MPM, Van Steenberge M, Dessein S, Volckaert FAM, Snoeks J, Huyse T, Pariselle A. 2013. Biogeographical implications of Zambezian Cichlidogyrus species (Platyhelminthes: Monogenea: Ancyrocephalidae) parasitizing Congolian cichlids. Zootaxa 3608(5):398–400 DOI 10.11646/zootaxa.3608.5.8. Vanhove MPM, Volckaert FAM, Pariselle A. 2011. Ancyrocephalidae (Monogenea) of Lake Tanganyika: I: four new species of Cichlidogyrus from Ophthalmotilapia ventralis (Teleostei: Cichlidae), the first record of this parasite family in the basin. Zoologia (Curitiba, Impresso) 28(2):253–263 DOI 10.1590/S1984-46702011000200016. Van Steenberge M, Pariselle A, Huyse T, Volckaert F, Snoeks J, Vanhove MPM. 2015. Morphology, molecules, and monogenean parasites: an example of an integrative approach to cichlid biodiversity. PLOS ONE 10(4):e0124474 DOI 10.1371/journal.pone.0124474. Vignon M, Pariselle A, Vanhove MPM. 2011. Modularity in attachment organs of African Cichlidogyrus (Platyhelminthes: Monogenea: Ancyrocephalidae) reflects phylogeny rather than host specificity or geographic distribution. Biological Journal of the Linnean Society 102(3):694–706 DOI 10.1111/j.1095-8312.2010.01607.x. Wanek KA, Sturmbauer C. 2015. Form, function and phylogeny: comparative morphometrics of Lake Tanganyika’s cichlid tribe Tropheini. Zoologica Scripta 44(4):362–373 DOI 10.1111/zsc.12110. Yamaoka K. 1997. Trophic ecomorphology of Tanganyikan cichlids. In: Kawanabe H, Hori M, Nagoshi M, eds. Fish communities in Lake Tanganyika. Kyoto: Kyoto University Press, 25–56.

Rahmouni et al. (2018), PeerJ, DOI 10.7717/peerj.5604 37/37 Study C

Intraspecific morphological variation in Cichlidogyrus (Monogenea) parasitizing two cichlid hosts from Lake Tanganyika exhibiting different

dispersal capacities

Rahmouni, C., Van Steenberge, M., Vanhove, M.P.M. and Šimková, A. (2020)

Hydrobiologia; https://doi.org/10.1007/s10750-020-04429-1

Hydrobiologia

https://doi.org/10.1007/s10750-020-04429-1 (0123456789().,-volV)( 0123456789().,-volV)

ADVANCES IN CICHLID RESEARCH IV

Intraspecific morphological variation in Cichlidogyrus (Monogenea) parasitizing two cichlid hosts from Lake Tanganyika exhibiting different dispersal capacities

Chahrazed Rahmouni . Maarten Van Steenberge . Maarten P. M. Vanhove . Andrea Sˇimkova´

Received: 24 February 2020 / Revised: 25 September 2020 / Accepted: 30 September 2020 Ó Springer Nature Switzerland AG 2020

Abstract As parasites depend on their hosts and play used to confirm parasite conspecificity. Dorsal and a significant role in their ecology and evolution, we ventral anchors of the attachment organ of parasite hypothesized an association between the host dispersal specimens were used to evaluate variability in shape. capacity and the intraspecific variability of their host- Geomorphometric analyses revealed that populations specific parasites. We investigated the morphological of C. milangelnari, which parasitize poorly dispersing variability of the gill monogeneans Cichlidogyrus cichlids, are more differentiated than populations of C. gistelincki and C. milangelnari parasitizing the Tan- gistelincki infecting well-dispersing hosts. Both ganyika cichlids ‘Ctenochromis’ horei and Cypri- anchors showed significant shape variation between chromis microlepidotus, respectively. The profound populations of C. milangelnari.InC. gistelincki, ecological and behavioural differences between these anchors were highly similar in comparisons of popu- host species allowed us to assume that the former is a lations from nearby and from distant locations. good and the latter a poor disperser. Specimens of monogeneans were collected from cichlids inhabiting Keywords Attachment organ Á Cichlids Á different locations at the northern end of Lake Cyprichromini Á Fish dispersal Á Gill parasites Á Tanganyika. Sequences of the 28S rDNA gene were Monogeneans Á Tropheini

Guest editors: S. Koblmu¨ller, R. C. Albertson, M. J. Genner, K. M. Sefc & T.Takahashi / Advances in Cichlid Research IV: Behavior, Ecology andEvolutionary Biology

C. Rahmouni (&) Á M. Van Steenberge Á M. Van Steenberge Á M. P. M. Vanhove M. P. M. Vanhove Á A. Sˇimkova´ Laboratory of Biodiversity and Evolutionary Genomics, Department of Botany and Zoology, Faculty of Science, Department of Biology, University of Leuven, Louvain, Masaryk University, Brno, Czech Republic Belgium e-mail: [email protected] M. P. M. Vanhove M. Van Steenberge Centre for Environmental Sciences, Research Group Operational Directorate Taxonomy and Phylogeny, Royal Zoology: Biodiversity & Toxicology, Hasselt University, Belgian Institute of Natural Sciences, Brussels, Belgium Diepenbeek, Belgium

M. Van Steenberge M. P. M. Vanhove Section Vertebrates, Ichthyology, Royal Museum for Zoology Unit, Finnish Museum of Natural History, Central Africa, Tervuren, Belgium University of Helsinki, Helsinki, Finland 123 Hydrobiologia

Introduction attachment organ, intraspecific geographic variation seems to be especially present in the shape of the Monogenea van Beneden, 1858 is a cosmopolitan anchors (Vignon & Sasal, 2010; Rodrı´guez-Gonza´lez group of flatworms (or Platyhelminthes) parasitizing et al., 2017; Kmentova´ et al., 2020a, b). mainly aquatic vertebrates. They particularly attach to Cichlidogyrus Paperna, 1960 is the most common, fish gills, fins and scales, but some of them also infect most species-rich and most host-specific gill flatworm the eyes, nostrils or internal organs. They are highly genus known from African cichlids (Pariselle et al., diverse with an estimated 25 000 species and excep- 2011), with over 25 new species discovered only tionally host-specific (Cribb et al., 2002; Theisen et al., during the past 2 years (Rahmouni et al., 2017, 2018; 2017). In contrast to other parasitic flatworms that Jorissen et al., 2018a, b; Geraerts et al. 2020). require one or more intermediate hosts to complete Considering the host specificity, direct life cycle, their lifecycle, monogeneans are characterized by a limited dispersal ability, and vicariance of monoge- single-host life cycle, a feature that considerably neans, the biogeographical distribution of Cichlido- reduces barriers that could preclude to infect their gyrus species seems to follow the patterns of their hosts (Gussev, 1995; Huyse et al., 2003). cichlid hosts (Pariselle et al., 2011). Therefore, these The posterior end of all monogeneans bears a parasites were proposed as a tool to better understand highly characteristic structure, the attachment organ, the adaptive radiations driving rapid speciation in also called haptor. It comprises sclerotized hard parts cichlid assemblages (see the review by Vanhove et al., such as marginal hooks, connective bars, clamps or 2016). Few studies have focused on the morphological anchors. Unsurprisingly, the haptor exhibits huge evolution of the hard parts of the attachment organ in differentiation within the group (Roberts & Janovy, species of Cichlidogyrus. Mendlova´ et al. (2012) 2009). The various forms of attachment organ struc- investigated species of Cichlidogyrus of West African tures have been interpreted as adaptations to the host cichlids and suggested a link between phylogeny and species that have influenced the specialization of these morphological adaptation of these host-specific para- parasites and considerably contributed to their host sites, whereas Messu Mandeng et al. (2015) suggested ˇ ´ specificity (Simkova et al., 2006; Olstad et al., 2009; an adaptive component to the haptoral morphology of Bueno-Silva et al., 2011). Monogeneans are nowadays species of this genus. considered one of the best model systems for address- Lake Tanganyika (LT) is one of the main hotspots ing fundamental ecological and evolutionary ques- for cichlid adaptive radiations in African freshwaters ˇ ´ tions related to fish–parasite interactions (Simkova and represents an important model system to under- et al., 2001; Olstad et al., 2009; Bueno-Silva & stand biological diversity and mechanisms of diversi- Boeger, 2019). Their simple life cycle, species diver- fication (Salzburger, 2018; Meyer et al., 2019). This sity and host specificity make them the first choice for lake contains a highly diverse assemblage of approx- investigating diversity and speciation in parasites of imately 250 species of cichlids that are subdivided into ˇ ´ closely related hosts (Pariselle et al., 2003,Simkova 12 to 16 tribes (Poll, 1986; Takahashi, 2003, 2014), ´ ˇ ´ et al., 2004; Mendlova et al., 2012;Simkova et al., which are supported by phylogenetic analyses (Kobl- 2013). A lot of consideration has been given to the mu¨ller et al., 2008; Takahashi & Sota, 2016). Species shape variation of the monogenean haptoral sclerites of two tribes, (Cyprichromini Poll, 1986; Tropheini (see for instance Rohde & Watson, 1985a, b; Huyse & Poll, 1986) were selected for this study. We investi- ´ Volckaert, 2002; Jarkovsky et al., 2004; Olstad et al., gated Cyprichromis microlepidotus (Poll, 1956) 2009; Khang et al., 2016). Intraspecific variation in the (Cyprichromini) and ‘Ctenochromis’ horei (Gu¨nther, shape and size of the haptoral hard parts was 1894) (Tropheini) and their respective gill monoge- previously reported in a few monogenean groups, of neans, C. milangelnari Rahmouni et al., 2017a and which dactylogyrids (Vignon & Sasal, 2010; Khang Cichlidogyrus gistelincki Gillardin et al., 2012. The ´ et al., 2016; Kmentova et al., 2016a, b, 2020b) and two cichlid species differ strongly in life history traits, ´ diplectanids (Vignon & Sasal, 2010; Kmentova et al., behaviour, geographical distribution and dispersal 2020b) were investigated using a geomorphometric capacity. ‘Ctenochromis’ horei is widely distributed ´ approach Kmentova et al., 2020b. Compared to the throughout LT, and the most common cichlid species marginal hooks and the connective bars of the living in shallow intermediate and vegetated habitats 123 Hydrobiologia

(Konings, 2015). Although belonging to the endemic of LT cichlids have been carried out (Vanhove et al., LT tribe Tropheini, ‘C.’ horei has a generalist 2011, 2015; Kmentova´ et al., 2016a, b). morphology that deviates from most other tropheines, As a high richness of host species could lead to a which are more specialized. It has a very broad dietary high richness of parasites, we would expect that the range (Muschick et al., 2011) and a broad ecological extraordinary diversity of cichlids in LT would bring tolerance. Besides its preferences for vegetated about a high parasite diversity and diversification (see patches, this cichlid is also commonly observed at the review by Vanhove et al., 2016). Additionally, other habitats along the Lake’s shoreline, including monogeneans could also diversify within cichlid rocky coasts (Sturmbauer et al., 2008). Given its broad hosts. Such diversification would be hampered by ecological tolerance, ‘C.’ horei encounters few barri- dispersal and gene flow between populations of host- ers for dispersal. Hence, it also occurs out of LT in the specific monogeneans, which, in turn, depends on the Lukuga (Kullander & Roberts, 2011), Malagarasi and dispersal of the fish hosts (Criscione & Blouin, 2004). Rusizi Rivers (De Vos et al., 2001; Konings, 2015). Gene flow caused by dispersal among subpopulations Additionally, in contrast to most other mouthbrooding of fish hosts affects genetic variation (Pettersen et al., cichlids, neither males nor females of ‘C.’ horei 2015), whereas low gene flow contributes to high possess well-defined territories. This is because the levels of genetic differentiation within parasite species hierarchy between males, rather than the defence of (Maze´-Guilmo et al., 2016). We hypothesize in the mating territories, determines the mating system of the present study that cichlid dispersal capacity in LT species, at least in the North of the Lake (Ochi, 1993). would drive the diversity of their parasite assem- Finally, this species does not harbour any known blages, and that limited dispersal ability precluding differentiation into colour morphs, and exhibits low gene flow between cichlid populations could promote intraspecific genetic divergence (Van Steenberge the differentiation of their monogenean assemblages et al., 2015). In view of the above, we consider the (Gre´goir et al., 2015). Hitherto, relatively little species to be a good disperser. Cyprichromis attention has been given to the effect of cichlid host microlepidotus, however, shows only a weak dispersal dispersal capacity on genetic and morphological ability. Although species of Cyprichromis have differentiations within Cichlidogyrus species. Such evolved several adaptations allowing them to live research has, until now, only been carried out on and in the open water with a capacity to catch intraspecific phenotypic variability in a Tanganyikan pelagic prey, they remained strongly dependent on monogenean that infects a few species of deep-water deep rocky shores for shelter (Konings, 2015). As this cichlids (Kmentova´ et al., 2016b). habitat is distributed in a patchy way along the In this study, we hypothesize that there is a link shoreline of LT, populations of Cyprichromis are between host dispersal capacity and the intraspecific geographically isolated. Therefore, species of Cypri- diversity in gill monogeneans. More specifically, we chromis contain many colour variants that evolved due hypothesized that, because of low gene flow in to the geographic isolation. This specifically holds for parasites, cichlid species with limited dispersal capac- C. microlepidotus, which only occurs at the Lake’s ity will harbour more morphologically differentiated northern half, and which contains several geograph- monogenean populations in terms of haptoral mor- ically isolated (colour) variants. phology (measured by shape variation in the anchors), Cyprichromine cichlids were investigated for than populations of well-dispersing cichlids. monogenean parasites only recently by Rahmouni et al. (2017) who described the first species of Cichlidogyrus infecting a member of this tribe: C. Materials and methods microlepidotus. In contrast, among the endemic cich- lids living in LT, Tropheini is the group that was most Fish and parasite collections extensively studied for their gill monogenean fauna, with over 15 nominal species of Cichlidogyrus Cichlid hosts were sampled from the northern part of recognized (see the overview published by Rahmouni LT (Fig. 1a). Twelve specimens of ‘C.’ horei (Fig. 1b) et al. (2017) and Rahmouni et al. (2018)). In addition, and fifteen specimens of C. microlepidotus (Fig. 1c) only a few molecular studies on monogenean parasites were collected from the Burundese and Congolese 123 Hydrobiologia shorelines in 2013 and 2016. The following localities Mukuruka, and only 22 km between Nyaruhongoka were sampled for ‘C.’ horei (Burundi): Magara (3° 440 and Kalundo (Fig. 1a). The protocols used for dis- S, 29° 190 E; n = 4), Mukuruka (4° 140 S, 29° 330 E; secting the cichlid fish, as well as for isolating, fixing n = 1) and Nyaruhongoka (3° 410 S, 29° 200 E; n = 7). and drawing gill-infecting monogeneans (Fig. 1d and Specimens of C. microlepidotus were sampled from 1e), follow Rahmouni et al. (2017, 2018). Basic Nyaruhongoka (n = 3) and Kalundo (3° 260 S, 29° 070 epidemiological data, i.e. prevalence, mean abun- E; n = 12) (see Fig. 1a). The geographical distance dance, minimum and maximum intensity of infection, between localities was calculated using Geographic were calculated for each monogenean species accord- Distance Matrix Generator software v. 1.2.3 (Ersts, ing to Bush et al. (1997). Host nomenclature follows 2014). Only 3.5 km separates Magara and Nyaruhon- FishBase (Froese & Pauly, 2019), except with respect goka, while 79.4 km separates Magara and Mukuruka. for the use of ‘’ (single quotation) in ‘C.’ horei, where There is 82.5 km between Nyaruhongoka and Konings (2015) is followed. This notation is used as

Fig. 1 Design of the study. a Map of northern Lake Tanganyika longicirrus Paperna, 1965), e sclerotized structures of Cichli- with sampling localities at the Burundese and Congolese dogyrus gistelincki (DA, dorsal anchor; VA, ventral anchor; DB, shorelines (edited with http://www.simplemappr.net and Pho- dorsal bar; VB, ventral bar; HI-HVII, marginal hooks; MCO, toshop v. 13.0), b ‘Ctenochromis’ horei, c Cyprichromis male copulatory organ; He, heel; Ap, accessory piece), f loca- microlepidotus (photos Radim Blazˇek, Burundi 2013), d whole tion of the nine analysed landmarks on the right anchor (example view of Cichlidogyrus sp. (exemplified by Cichlidogyrus DA of Cichlidogyrus gistelincki) 123 Hydrobiologia

Takahashi (2003) showed that ‘C.’ horei is not closely Geomorphometrics related with the nominal species of the genus: Ctenochromis pectoralis Pfeffer, 1893. All applicable Variation in anchor shape between parasite popula- institutional, national and international guidelines for tions of various localities was analysed using land- the care and use of animals were followed. Sampling mark-based (LM) geometric morphometrics. We was carried out under mission statements 022/MINE- analysed dorsal and ventral anchors (DA and VA), URS/CRH-U/2013 and 031/MINRST/CRH-U/2016 two sclerotized structures of the posterior haptoral from the Centre de Recherche en Hydrobiologie- apparatus of the worms (Fig. 1e). The number of DA Uvira. In the absence of relevant animal welfare and VA analysed (nDA:nVA) for parasite specimens regulations in the D.R. Congo or Burundi, the same collected from ‘C’. horei per locality was as follows: strict codes of practice enforced within the European (17:17, Magara); (16:16, Mukuruka); and (30:32, Union were applied. Nyaruhongoka). Parasite specimens were collected from C. microlepidotus from two opposite locations Molecular characterization and genetic analysis and nDA:nVA per locality was as follows: (27:27, Nyaruhongoka) and (10:7, Kalundo). We used only To confirm the conspecificity of parasites infecting the monogenean specimens wholly body mounted on respective host species, a fragment of the 28S rDNA slides with a drop of glycerine ammonium picrate region was amplified and sequenced for 18 parasite (GAP) (Malmberg, 1957). Anchors were then pho- specimens collected from all sampling localities and tographed by using an Olympus BX51 phase-contrast host species. Ribosomal DNA regions such as 28S are microscope, under magnifications 20X and 40X, and highly conservative, which makes them suitable and using Olympus Stream Image Analysis v. 1.9.3 commonly used for species recognition in flatworms software. Voucher specimens of parasite species from (Vanhove et al., 2013; see for instance the studies of each sampling locality were deposited in the Muse´um Sˇimkova´ et al., 2006; Mendlova´ et al., 2012; National d’Histoire Naturelle (MNHN, Paris, France) Mendlova´ &Sˇimkova´, 2014; Messu Mandeng et al., under the accession numbers (Cichlidogyrus gis- 2015). These 18 specimens were cut into half using telincki: MNHN HEL1195-1202; Cichlidogyrus fine needles under a dissecting microscope during the milangelnari: MNHN HEL1203-05 and MNHN fieldtrip. The reproductive organs were fixed on slides HEL1223). The anchor shape variables were obtained (see below), whereas the other half of the body was using nine homologous LMs based on the studies of placed in 96% ethanol for DNA extraction. As this half Vignon & Sasal (2010) and Kmentova´ et al. (2016) contained the haptor with sclerotized anchors, spec- (Fig. 1f). Landmark terminology follows Rodrı´guez- imens used for molecular analyses could not be used Gonza´lez et al. (2015): (LM1) anchor point; (LM2) for the morphological part of the study. The universal inner point base; (LM3) inner shaft base; (LM4) most primers C1 (F: 50-ACCCGCTGAATTTAAGCAT-30) convex point base; (LM5) most proximal point of and D2 (R: 50-TCCGTGTTTCAAGACGG-30) (Has- inner root; (LM6) notch between inner and outer roots; souna et al., 1984) were used, following the protocol (LM7) mean point of outer root; (LM8) outer shaft published in Rahmouni et al. (2017). Sequences were base; and (LM9) outer point base. Dorsal and ventral edited using the SequencherÒ software v. 5.0 (Gene anchors were aligned by their vertical axis, which is Codes Corporation, Ann Arbor, MI USA), aligned defined by LM2 and LM9. Digitalization of the LMs using the ClustalW algorithm (Thompson et al., 1994) was performed using tpsDig2 software (Rohlf, 2006). implemented in MEGA X (Kumar et al., 2018) and The LM coordinates were forwarded to MorphoJ v. deposited in GenBank under accession numbers: 1.06 (Klingenberg, 2011). We performed a Procrustes MK860914-16. Uncorrected p-distances were calcu- fit by aligning the coordinates using Generalized lated between specimens and populations, using the Procrustes Analysis (GPA). The Procrustes method same software. removes all information related to size and orientation and superimposes LM configurations to achieve an overall best fit. We generated covariance matrices for each of the two parasite species, which were used in further analyses, i.e. principal component analyses 123 Hydrobiologia

(PCA) and canonical variate analyses (CVA). Princi- each respective host species was confirmed. The pal component analyses were used to visualize the sequence of the partial 28S rDNA was 682 bp long variation in the datasets, whereas canonical variate for C. gistelincki and 591 bp long for C. milangelnari. analyses were performed to investigate whether No variability was observed within the sequences anchor shape could differentiate between a priori obtained from specimens of C. gistelincki. However, defined groups (i.e. localities) (Klingenberg & Mon- the 28S rDNA sequences of C. milangelnari showed teiro, 2005). The latter analysis computes the axes of weak differentiation among the populations from variance by minimizing differences within groups and localities on opposite lake shores (a single nucleotide; maximizing differences between groups. We tested 0.2%). Thus, two 28S rDNA sequences representing whether different populations from the same species each of the populations of C. milangelnari and a single differed morphologically by computing the Procrustes sequence for C. gistelincki were deposited in GenBank distances between specimens and by using a permu- (see accession numbers in Materials and methods tation test (Good, 2001) with 10 000 randomizations section). The genetic distance between C. milangel- (significance level a = 0.05). For this, p-values were nari and C. gistelincki ranged from 3% to 3.2%. adjusted using Holm–Bonferroni correction (Holm, 1979). Phenotypic change patterns in DA and VA for Anchor shape variation in populations of C. the main axes of PCA and CVA were visualized in milangelnari parasitizing C. microlepidotus MorphoJ with respect to a consensus using a wire- frame scheme (Klingenberg, 2011). Results from the PCA performed on the DA and VA datasets of populations of C. milangelnari are shown in Fig. 2. Regarding the DA (Fig. 2a), PC1 explained Results 38%, and PC2 18.1% of the variation. Concerning the VA (Fig. 2b), 35.9% of the variation was explained by Species identification and genetic characterization PC1 and 17.5% by PC2. Samples from the two localities overlapped in the scatter plots for DA and The morphological identification of species of Cich- VA. However, specimens from Nyaruhongoka had, on lidogyrus was based on their haptoral and reproductive average, higher values for PC2 in the DA dataset and sclerites using the original descriptions. Specimens lower values for PC1 in the VA dataset. The changes collected from ‘C.’ horei and C. microlepidotus were along the first PC corresponded with a DA having a assigned to C. gistelincki (Fig. 1e) and C. milangel- slightly broader and more pronounced inner root, and a nari, respectively. Only a single species of dactylo- broader, more curved shaft base, and a more elevated gyrid monogenean species was found on the gills of convex point. The second PC corresponded with a DA ‘C.’ horei, as was also the case in the study of Gillardin having a broader and more pronounced inner root, a et al. (2012). The same held for the gills of C. deeper notch, and more reduced outer root and outer microlepidotus, in accordance with Rahmouni et al. shaft base (Fig. 2a). For the VA dataset, specimens (2017). These parasite species have never been with high values for PC1 had a VA with a narrower recorded from any other cichlid hosts so far and are, and more pronounced inner root, and a thinner, shorter thus, considered as strict specialists (Mendlova´ & shaft base. The highest contribution to PC2 was a Sˇimkova´, 2014). All specimens of ‘C.’ horei were change in the inner root that had a broader and more infected by C. gistelincki (100%), the mean abundance pronounced shape (Fig. 2b). was 18.3 ± 8.2 and the intensity of infection ranged The frequencies of the distribution of the samples from 4 to 33 monogeneans per infected host. Cichli- across the CV axes are represented in Fig. 2c,d. In the dogyrus milangelnari parasitized 10 out of 15 spec- case of the DA, CVA almost completely separated the imens (66.6%), the mean abundance was 7.6 ± 12.2 two populations (Fig. 2c), whereas a complete sepa- and the intensity of infection ranged from 1 to 39 ration was obtained in the VA dataset (Fig. 2d). The monogeneans per infected host. From three to five shape changes in DA along the CV corresponded with specimens from each sampling locality were success- a thinner base and a more pronounced outer root fully sequenced. Using the partial sequences of 28S (Fig. 2c). For VA, the CV corresponded with having a rDNA, the conspecificity of all monogeneans from more pronounced distance between the roots with a 123 Hydrobiologia

Fig. 2 Geomorphometric analyses on DA and VA of Cichli- blue, and target shapes (changes) associated with extreme values dogyrus milangelnari. Scatter plots of the PCA of DA (a) and (value ?0.1) in dark blue. Scatter plots of the CVA of DA VA (b) datasets, shape changes next to each PC are shown by (c) and VA (d), shape changes next to CV axes are shown by the wireframes with starting shapes (consensus, value 0) in light wireframes associated with extreme values (?4) longer and slightly broader shaft (Fig. 2d). The Anchor shape variation in populations of C. permutation tests using Procrustes distances revealed gistelincki parasitizing ‘C.’ horei significant differences in shape for both DA and VA (Table 1). The differences remained significant after Principal component analyses were performed on Holm–Bonferroni correction. morphometrical landmarks of the DA (Fig. 3a) and

Table 1 Matrix of Procrustes distances and p-values from for C. milangelnari are represented in the right side of the permutation test with 10 000 randomizations (in bold) among table with the left column for DA and right column for VA. localities using the DA (left side of the diagonal) and VA (right *Indicates a statistically significant p-value after Holm–Bon- side of the diagonal) observations of C. gistelincki. Distances ferroni correction Species C. gistelincki C. milangelnari Localities Magara Mukuruka Nyaruhongoka Magara Kalundo

Mukuruka 0.0214 – 0.0246 0.0295 – – 0.2866 – 0.0577 0.0383 –– Nyaruhongoka 0.0235 0.0331 – 0.0218 0.0320 0.0299 0.1248 0.0022* – 0.1469 0.0179* 0.038*

123 Hydrobiologia

VA (Fig. 3b) of C. gistelincki (Fig. 3). The first two anchor base, resulting from a shift of the latter. A high PC axes accounted for 44.7% of the total DA shape value for PC2 corresponded to anchors with a broader variation (27.9% and 16.8%) and for 45.4% of the total base and a more upturned inner and outer point base, VA shape variation (26.1% and 19.3%). On the scatter with a more conspicuous inner root in terms of length plots of both datasets, there was a complete overlap and angle that forms the notch (Fig. 3a). For VA, between groups. Variations in the shape of anchors higher values for PC1 corresponded with a more associated to each of the PCs are shown next to their reduced point associated to the inner and outer roots corresponding axis (Fig. 3a, b). For the DA, a high with redressed point base and narrow base. Variations value for PC1 mostly corresponded with reduced inner related to VA along PC2 corresponded to anchors with roots with a relatively reduced distance between the relatively longer points, thinner and more curved inner shaft base and the most convex point of the

Fig. 3 Geomorphometric analyses on DA and VA of Cichli- target shapes (changes) associated with extreme values (?0.1) dogyrus gistelincki. Scatter plots of the PCA of DA (a) and VA in dark blue. Scatter plots of the CVA of DA (c) and VA (d), (b) datasets, shape changes next to each PC are shown by shape changes next to CV axes are shown by the wireframes wireframes with starting shapes (consensus) in light blue, and associated with extreme values (?5) 123 Hydrobiologia shafts with broader inner roots and more pronounced Discussion outer roots (see Fig. 3b). Canonical variate analyses only partially separated We investigated shape variation of the anchors in the the samples among localities for both haptoral scle- attachment organ of C. gistelincki and C. milangel- rites and a considerable amount of overlap remained. nari, monogeneans that parasitize two endemic Tan- For DA (Fig. 3c), CV1, explaining 69.8% of the total ganyika cichlids that are expected to differ in dispersal shape variation, partially separated monogenean pop- capacities, ‘C.’ horei and C. microlepidotus, respec- ulations of Nyaruhongoka and Mukuruka. Meanwhile, tively. We collected specimens of ‘C.’ horei—a CV2, explaining 30.1% of the variation, partially tropheine, which disperses easily in the Lake, and C. separated those parasites of Magara from those of the microlepidotus, a cyprichromine, which shows other two localities. For VA (Fig. 3d), CV1 (60.9% of restricted dispersal ability. Our goal was to investigate variation) partially separated populations of the association between the host’s dispersal capacity Nyaruhongoka and Magara from Mukuruka. The and the morphological differentiations in cichlid- second CV (39% of variation), partially separated specific monogeneans, i.e. species of Cichlidogyrus. populations from Magara and Nyaruhongoka. For both We specifically studied the shape variability of the anchors, CV axes corresponded with different shape haptoral sclerites, as these are linked to adaptations to variations than those observed for PC axes. The first the fish host (Sˇimkova´ et al., 2006; Olstad et al., 2009; CV axis on the DA plot described anchors having a Bueno-Silva et al., 2011). We hypothesized that, less curved shaft and longer inner root with a deeper because of the limited gene flow, cichlids with notch. Along the second CV axis, specimens of C. restricted dispersal capacity will harbour morpholog- gistelincki showed a DA with thinner base and slightly ically more differentiated parasite populations com- shorter inner root and wider outer root, as well as a pared to good dispersers. This morphological thinner shaft with a more pronounced shaft point approach focused on landmark-based data by evalu- (Fig. 3c). For the VA, variations along CV1 corre- ating intraspecific anchor shape variations. sponded to a reduced notch, a thinner base with wider Host dispersal is assumed to drive the genetic shaft at its base, and a narrower shaft point. Along structure and the diversity of parasites (Maze´-Guilmo CV2, anchors mainly displayed a thinner base with a et al., 2016). The results obtained from the morpho- relatively more reduced inner root and notch between logical data of C. milangelnari populations infecting the roots (Fig. 3d). C. microlepidotus agreed with our hypothesis. Geo- The pairwise Procrustes distances, as well as the morphometric results obtained for C. milangelnari results of the permutation tests, are shown in Table 1. reflected differentiation between two relatively distant Surprisingly, the smallest Procrustes distance for the populations. Using a geomorphometric approach, DA dataset was found between the populations differences in the shape of their anchors (DA and from Mukuruka and Magara. In the VA dataset, VA) were found between specimens from Nyaruhon- samples from Nyaruhongoka and Magara were the goka and Kalundo. In contrast, geomorphometric most similar in shape. The highest distances were patterns did not reveal differentiation among the found between the Mukuruka and Nyaruhongoka studied populations of C. gistelincki infecting ‘C.’ populations for the DA, and between the Magara and horei, a well-dispersing tropheine. It should be noted, Mukuruka populations for the VA dataset. However, however, that populations of C. microlepidotus stem the difference in morphology was only significant for from opposite sides of the Rusizi River, a known the DA between the Mukuruka and Nyaruhongoka barrier for many cichlid species, whereas those of ‘C.’ populations and for the VA only between the Magara horei all originate from the same side. However, as and Mukuruka populations. Only the p-value of the ‘C.’ horei thrives in vegetated areas and is even found first comparison (Magara and Nyaruhongoka) more upstream in this river (De Vos et al., 2001), we remained significant after Holm–Bonferroni adjust- do not expect this to influence our results. Although ment for multiple testing. one significant difference in shape was revealed, geomorphometric data of anchors of C. gistelincki showed profound shape overlap among distant and neighbouring populations for both the DA and VA 123 Hydrobiologia datasets (see Results section). The lack of a clear Conclusion geographical trend in the shape of haptoral structures of C. gistelincki can also be explained by other factors Despite the estimation of a high diversity of cichlid that are known to influence morphological diversifi- monogeneans in LT, studies on the intraspecific cation in monogeneans, such as historical and local morphological and genetic variability of these cich- environmental factors (Ergens & Gelnar, 1985;Da´vi- lid-specific parasites remain scarce. Only few studies dova´ et al., 2005; Bueno-Silva & Boeger, 2019). As focused on intraspecific differences in the haptoral we only used a genetic marker that is highly conserved hard parts of gill-infecting monogeneans. Here, we in monogenean species, we cannot say whether the showed that higher morphological differentiation is patterns of morphological variation in anchors of C. found in host-specific monogenean species that infect gistelincki and C. milangelnari are also reflected in the a poorly dispersing cichlid than in those that infect a genomes of these monogenean parasites. Hence, good disperser. This indicates that the ecology of a additional sampling, supplemented by the analyses host lineage influences diversification and therefore of multi-locus data, would help us in the future to potentially speciation of its parasite fauna. investigate the population structure of each of the studied cichlid species and their monogeneans across Acknowledgements The authors are grateful to many geographical scales. colleagues and friends who assisted with the sample collections during the fieldtrip in Burundi in 2013 and in the In our study, the fifth landmark, which corre- Democratic Republic of the Congo in 2016. We thank C. sponded to the inner root, was the most variable in all Sturmbauer and S. Koblmu¨ller (University of Graz, Austria), W. analyses. In monogeneans, anchors are often sup- Salzburger (University of Basel, Switzerland), D. Muzumani ported by other sclerotized structures of the haptoral Risasi (CRH-Uvira) for their precious help with cichlid identification; M. Reichard and R. Blazˇek (Czech Academy of apparatus such as ventral and dorsal bars or accessory Sciences, Czech Republic) for their help with fish collection, sclerites (Roberts & Janovy, 2009). In Cichlidogyrus, dissection and providing us the photographs of cichlid species the inner roots of the anchors are more closely situated during the fieldtrip; A. Meyer (University of Konstanz, to the bars than the outer roots. Possibly, the high Germany), G. Banyankimbona (University of Burundi) and the Schreyen-Brichard family (Burundi/Belgium) for the sample variation in the inner roots is due to morphological collection and fish identification; P. Masilya Mulungula, T. changes in the other sclerotized parts of the haptor. Mulimbwa N’sibula, V. Lumami Kapepula, E. Bahane Byaragi, The study of Rodrı´guez-Gonza´lez et al. (2015) S. Kambale Mukeranya and F. Muterezi Bukinga (CRH-Uvira) supported this hypothesis as they connected shape for co-organizing the field work and their scientific contribution on Tanganyikan cichlids; Sˇ. Masˇova´, V. Micha´lkova´,E. variability in the DA and VA displayed by species of Rˇ ehulkova´,I.Prˇikrylova´, L. Raisingerova´ and N. Kmentova´ Ligophorus Euzet & Suriano, 1977 with that of the (Masaryk University, Brno, Czech Republic) for their help with dorsal and ventral bars. So far, there are no detailed fish dissection, parasite isolation and fixation. The first author ˇ ´ studies focusing on the functional role of the sclero- thanks L. Starhova Serbina (Masaryk University, Brno, Czech Republic) for stimulating discussions and constructive tized structures of the attachment organ in Cichlido- suggestions and critics related to the geomorphometry. The gyrus. Therefore, further studies are necessary to study kind help of K. Civa´nˇova´, E. Jirounkova´, K. Koukalova´, and shape variability in other cichlid gill flatworms using especially M. Seifertova´ (Masaryk University, Brno, Czech cichlid gill flatworms. However, we did not investi- Republic), who provided the necessary background in genetics and an unconditional assistance in the molecular laboratory, is gate the pattern of the shape variation in other haptoral acknowledged. Finally, the authors would like to thank E. sclerites as, compared to anchors, the marginal hooks Verheyen, T. Backeljau, and all the colleagues working at the and the connective bars are less suited for a study of Royal Belgian Institute of Natural Sciences (RBINS, Brussels), two-dimensional landmarks. The marginal hooks are M. Parrent from the Royal Museum for Central Africa (RMCA, Tervuren), T. Artois, M.W.P. Jorissen, L. Steenaerts and T. Van commonly prone to modifications from the flattening Dijck (Hasselt University, Diepenbeek, Belgium) for their kind and/or the fixation processes. Similarly, the connec- hospitality and for the precious help they provided during the tive bars are thick, which make them less easily stay in Belgium. This research was funded by the Czech Science flattened and more exposed to distortion during Foundation project no. P505/12/G112—European Centre of Ichtyoparasitology (ECIP) and C.R., M.V.S. and M.P.M.V. fixation and mounting (Vignon & Sasal, 2010). were further supported by standard project GA19-13573S. The funder had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.

123 Hydrobiologia

References sympatric cichlids of Lake Tanganyika. Freshwater Biol- ogy 60: 323–335. Bueno-Silva, M. & W. A. Boeger, 2019. Rapid divergence, Gussev, A. V., 1995. Some pathways and factors of monoge- molecular evolution, and morphological diversification of nean microevolution. Canadian Journal of Fisheries and coastal host-parasite systems from southern Brazil. Para- Aquatic Sciences 52: 52–56. sitology 146: 1313–1332. Hassouna, N., B. Michot & J. P. Bachellerie, 1984. The com- Bueno-Silva, M., W. A. Boeger & M. R. Pie, 2011. Choice plete nucleotide sequence of mouse 28S rRNA gene. matters: incipient speciation in Gyrodactylus corydori Implications for the process of size increase of the large (Monogenoidea: Gyrodactylidae). International Journal for subunit rRNA in higher eukaryotes. Nucleic Acids Parasitology 41: 657–667. Research 12: 3563–3583. Bush, A. O., K. D. Lafferty, J. M. Lotz, A. W. Shostak, et al., Holm, S., 1979. A simple sequentially rejective multiple test 1997. Parasitology meets ecology on its own terms: mar- procedure. Scandinavian Journal of Statistics 6: 65–70. golis et al. revisited. Journal of Parasitology 83: 575–583. Huyse, T. & F. A. M. Volckaert, 2002. Identification of a host- Cribb, T. H., L. A. Chisholm & R. A. Bray, 2002. Diversity in associated species complex using molecular and morpho- the monogenea and digenea: does lifestyle matter? metric analyses, with the description of Gyrodactylus Interntional Journal of Parasitology 32: 321–328. rugiensoides n. sp. (Gyrodactylidae, Monogenea). Inter- Criscione, C. D. & M. S. Blouin, 2004. Life cycles shape par- national Journal of Parasitology 32: 907–919. asite evolution: comparative population genetics of salmon Huyse, T., V. Audenaert & F. A. M. Volckaert, 2003. Speciation trematodes. Evolution 58: 198–202. and host-parasite relationships in the parasite genus Gy- Da´vidova´, M., J. Jarkovsky´, I. Matejusova´ & M. Gelnar, 2005. rodactylus (Monogenea, Platyhelminthes) infecting gobies Seasonal occurrence and metrical variability of Gyro- of the genus Pomatoschistus (Gobiidae, Teleostei). Inter- dactylus rhodei Zitnan 1964 (Monogenea, Gyrodactyli- national Journal for Parasitology 33: 1679–1689. ˇ dae). Parasitology Research 95: 398–405. Jarkovsky´, J., S. Morand, A. Simkova´ & M. Gelnar, 2004. De Vos, L., J. Snoeks & D. T. van den Audenaerde, 2001. An Reproductive barriers between congeneric monogenean annotated checklist of the fishes of Rwanda (East Central parasites (Dactylogyrus: Monogenea): attachment appara- Africa), with historical data on introductions of commer- tus morphology or copulatory organ incompatibility? Par- cially important species. Journal of East African Natural asitology Research 92: 95–105. History 90: 41–68. Jorissen, M. W. P., A. Pariselle, T. Huyse, E. J. Vreven, J. Ergens, R. & M. Gelnar, 1985. Experimental verification of the Snoeks, E. Decru, T. Kusters, S. W. Lunkayilakio, F. effect of temperature on the size of hard parts of haptor Muterezi Bukinga, T. Artois & M. P. M. Vanhove, 2018a. of Gyrodactylus katharineri Malmberg, 1964 (Monoge- Six new dactylogyrid species (Platyhelminthes, Monoge- nea). Folia Parasitologica 32: 377–380. nea) from the gills of cichlids (Teleostei, Cichliformes) Ersts, P. J., 2014. Geographic Distance Matrix Generator (ver- from the Lower Congo Basin. Parasite 25: 64. sion 1.2.3). American Museum of Natural History. Centre Jorissen, M. W. P., A. Pariselle, T. Huyse, E. J. Vreven, J. for Biodiversity and Conservation [online]. Accessed 26 Snoeks, F. A. M. Volckaert, A. Chocha Manda, G. Kape- Dec 2018. pula Kasembele, T. Artois & M. P. M. Vanhove, 2018b. Euzet, L. & D. M. Suriano, 1977. Ligophorus n. g. (Monogenea, Diversity, endemicity and host-specificity of monogenean Ancyrocephalidae) parasite des Mugilidae (Te´le´oste´ens) gill parasites (Platyhelminthes) of cichlids in the Bang- en Me´dite´rrane´e. Bulletin du Muse´um National d’Histoire weulu-Mweru ecoregion. Journal of Helminthology 92: Naturelle. 3rd ser. n° 472, Zool. 320: 797–82 417–437. Froese, R. & D. Pauly, 2019. Fishbase. World Wide Web Khang, T. F., O. Y. M. Soo, W. B. Tan & L. H. S. Lim, 2016. Electronic Publication. Accessed 02 Oct 2019. Monogenean anchor morphometry: systematic value, Geraerts, M., F. Muterezi Bukinga, M. P. M. Vanhove, A. phylogenetic signal, and evolution. PeerJ 4: e1668. Pariselle, A. Chocha Manda, E. Vreven, T. Huyse & T. Klingenberg, C. P., 2011. MorphoJ: an integrated software Artois, 2020. Six new species of Cichlidogyrus (Monoge- package for geometric morphometrics. Molecular Ecology nea, Platyhelminthes) from the gills of cichlids (Teleostei, Resources 11: 353–357. Cichliformes) from the Lomami River Basin (DRC: Mid- Klingenberg, C. P. & L. R. Monteiro, 2005. Distances and dle Congo). Parasites & Vectors 13: 187. directions in multidimensional shape spaces: implications Gillardin, C., M. P. M. Vanhove, A. Pariselle, T. Huyse & F. for morphometric applications. Systematic Biology 54: A. M. Volckaert, 2012. Ancyrocephalidae (Monogenea) of 678–688. Lake Tanganyika: II: description of the first Cichlidogyrus Kmentova´, N., M. Gelnar, S. Koblmu¨ller & M. P. M. Vanhove, spp. parasites from Tropheini fish hosts (Teleostei, Cich- 2016a. First insights into the diversity of gill monogeneans lidae). Parasitology Research 110: 305–313. of ‘Gnathochromis’ and Limnochromis (Teleostei, Cichli- Good, P., 2001. Permutation Tests: A Practical Guide to dae) in Burundi: do the parasites mirror host ecology and Resampling Methods for Testing Hypotheses, 2nd ed. phylogenetic history? Peer J 4:e1629 Springer-Verlag, New-York. Kmentova´, N., M. Gelnar, M. Mendlova´, M. Van Steenberge, S. Gre´goir, A. F., P. I. Hablu¨tzel, M. P. M. Vanhove, A. Pariselle, J. Koblmu¨ller & M. P. M. Vanhove, 2016b. Reduced host- Bamps, F. A. M. Volckaert & J. A. M. Raeymaekers, 2015. specificity in a parasite infecting non-littoral Lake Tan- A link between host dispersal and parasite diversity in two ganyika cichlids evidenced by intraspecific morphological and genetic diversity. Scientific Reports 6: 39605.

123 Hydrobiologia

Kmentova´, N., S. Koblmu¨ller, M. Van Steenberge, J. A. M. Ochi, H., 1993. Mate monopolization by a dominant male in a Raeymaekers, T. Artois, E. L. R. De Keyzer, L. Milec, F. multi-male social group of a mouthbrooding cichlid, Cte- Muterezi Bukinga, T. Mulimbwa N’sibula, P. Masilya nochromis horei. Japanese Journal of Ichthyology 40: Mulungula, G. Ntakimazi, F. A. M. Volckaert, M. Gelnar 209–218. & M. P. M. Vanhove, 2020a. Weak population structure Olstad, K., L. Bachmann & T. A. Bakke, 2009. Phenotypic and recent demographic expansion of the monogenean plasticity of taxonomic and diagnostic structures in gyro- parasite Kapentagyrus spp. infecting clupeid fishes of Lake dactylosis-causing flatworms (Monogenea, Platy- Tanganyika. International Journal for Parasitology. https:// helminthes). Parasitology 136: 1305–1315. doi.org/10.1016/j.ijpara.2020.02.002. Pariselle, A., S. Morand, M. R. Deveney & L. Pouyaud, 2003. Kmentova´, N., S. Koblmu¨ller, M. Van Steenberge, T. Artois, F. Parasite species richness of closely related hosts : historical Muterezi Bukinga, T. Mulimbwa N’sibula, D. Muzumani scenario and ‘genetic’ hypothesis. In Combes, C. & J. Risasi, P. Masilya Mulungula, M. Gelnar & M. P. M. Jourdane (eds.), Taxonomie, Ecologie et Evolution des Vanhove, 2020b. Failure to diverge in African Great Me´tazoaires Parasites (livre hommage a` Louis Euzet). Lakes: the case of Dolicirroplectanum lacustre gen. nov. Presses Universitaires de Perpignan, Perpignan: 147–163. comb. nov. (Monogenea, Diplectanidae) infecting latid Pariselle, A., W. A. Boeger, J. Snoeks, C. F. Bilong Bilong, S. hosts. Journal of Great Lakes Research. https://doi.org/10. Morand & M. P. M. Vanhove, 2011. The monogenean 1016/j.jglr.2019.09.022. parasite fauna of cichlids: a potential tool for host bio- Koblmu¨ller, S., K. M. Sefc & C. Sturmbauer, 2008. The Lake geography. International Journal of Evolutionary Biology Tanganyika cichlid species assemblage: recent advances in 2011: 471–480. molecular phylogenetics. Hydrobiologia. 615: 5–20. Pettersen, R. A., T. A. Mo, H. Hansen & L. A. Vøllestad, 2015. Konings, A., 2015. Tanganyika Cichlids in Their Natural Genetic population structure of Gyrodactylus thymalli Habitat, 3rd ed. Cichlid Press, El Paso, Texas. (Monogenea) in a large Norwegian river system. Para- Kullander, S. O. & T. R. Roberts, 2011. Out of Lake Tan- sitology 142: 1693–1702. ganyika: endemic lake fishes inhabit rapids of the Lukuga Poll, M., 1986. Classification des Cichlidae du lac Tanganika: River. Ichthyological Exploration of Freshwaters 22: tribus, genre et epe`ces. Academie Royale de Belgique 355–376. Memoires de la Classe des Sciences 45: 1–163. Kumar, S., G. Stecher, M. Li, C. Knyaz & K. Tamura, 2018. Rahmouni, C., M. P. M. Vanhove & A. Sˇimkova´, 2017a. MEGA X: molecular evolutionary genetics analysis across Underexplored diversity of gill monogeneans in cichlids computing platforms. Molecular Biology and Evolution from Lake Tanganyika: eight new species of Cichlidogyrus 35: 1547–1549. Paperna, 1960 (Monogenea: Dactylogyridae) from the Kuusela, J., M. S. Zietara & J. Lumme, 2007. Hybrid origin of northern basin of the lake, with remarks on the vagina and baltic salmon-specific parasite Gyrodactylus salaris:a the heel of the male copulatory organ. Parasites & Vectors model for speciation by host switch for hemiclonal 10: 591. organisms. Molecular Ecology 16: 5234–5245. Rahmouni, I., E. Rˇ ehulkova´, A. Pariselle, O. B. Rkhami & A. Malmberg, G., 1957. On the occurrence of Gyrodactylus on Sˇimkova´, 2017b. Four new species of Dactylogyrus Dies- Swedish fishes. Skr. utgivna av So¨dra Sveriges Fisk 1956: ing, 1850 (Monogenea: Dactylogyridae) parasitising the 19–76. [in Swedish]. gills of northern Moroccan Luciobarbus Heckel (Cyprini- Maze´-Guilmo, E., S. Blanchet, K. D. Mccoy & G. Loot, 2016. dae): morphological and molecular characterisation. Sys- Host dispersal as the driver of parasite genetic structure: a tematic Parasitology 94: 575–591. paradigm lost? Ecology Letters 19: 336–347. Rahmouni, C., M. P. M. Vanhove & A. Sˇimkova´, 2018. Seven Mendlova´, M. & A. Sˇimkova´, 2014. Evolution of host speci- new species of Cichlidogyrus Paperna, 1960 (Monogenea: ficity in monogeneans parasitizing African cichlid fish. Dactylogyridae) parasitizing the gills of Congolese cich- Parasites & Vectors 7: 69. lids from northern Lake Tanganyika. PeerJ 23: e560. Mendlova´, M., Y. Desdevises, K. Civa´nˇova´, A. Pariselle & A. Roberts, L. & J. Janovy, 2009. Foundations of Parasitology, 8th Sˇimkova´, 2012. Monogeneans of west African cichlid fish: ed. Mc Graw Hill, United States. evolution and cophylogenetic interactions. PLoS One 7: Rodrı´guez-Gonza´lez, A., R. Mı´guez-Lozano, C. Llopis-Be- e37268. lenguer & J. A. Balbuena, 2015. Phenotypic plasticity in Messu Mandeng, F. D., C. F. Bilong Bilong, A. Pariselle, M. haptoral structures of Ligophorus cephali (Monogenea: P. M. Vanhove, A. R. Bitja Nyom & J. F. Agne`se, 2015. A Dactylogyridae) on the flathead mullet (Mugil cephalus): a phylogeny of Cichlidogyrus spp. (Monogenea, Dactylo- geometric morphometric approach. International Journal gyridea) clarifies a host-switch between fish families and for Parasitology 45: 295–303. reveals an adaptive component to attachment organ mor- Rodrı´guez-Gonza´lez, A. G., V. Sarabeev, & J. A. Balbuena, phology of this parasite genus. Parasites & Vectors 8: 582. 2017. Evolutionary morphology in shape and size of hap- Meyer, B. S., P. I. Hablu¨tzel, A. K. Roose, M. J. Hofmann, W. toral anchors in 14 Ligophorus spp. (Monogenea: Dactyl- Salzburger & J. A. M. Raeymaekers, 2019. An exploration ogyridae). PLoS ONE 12(5): e0178367. of the links between parasites, trophic ecology, morphol- Rohde, K. & N. Watson, 1985a. Morphology, microhabitats and ogy, and immunogenetics in the Lake Tanganyika cichlid geographical variation of Kuhnia spp. (Monogenea: Poly- radiation. Hydrobiologia 832: 215–233. opisthocotylea). International Journal for Parasitology 15: Muschick, M., A. Indermaur & W. Salzburger, 2011. Conver- 569–586. gent evolution within an adaptive radiation of cichlid Rohde, K. & N. Watson, 1985b. Morphology and geographical fishes. Current Biology 22: 2352–2368. variation of Pseudokuhnia minor n.g., n.comb. 123 Hydrobiologia

(Monogenea: Polyopisthocotylea). International Journal 1968): an endoparasite of croakers (Teleostei: Sciaenidae) for Parasitology 15: 557–567. from Indonesia. PLoS ONE 12: e0184376. Rohlf, F. J., 2006. TpsDig2, digitize landmarks and outlines, Thompson, J. D., D. G. Higgins & T. J. Gibson, 1994. CLUS- Version 2.10. Department of Ecology and Evolution, State TAL W: improving the sensitivity of progressive multiple University of New York at Stony Brook. sequence alignment through sequence weighting, position- Salzburger, W., 2018. Understanding explosive diversification specific gap penalties and weight matrix choice. Nucleic through cichlid fish genomics. Nature Reviews Genetics Acids Research 22: 4673–4680. 19: 705–717. Van Steenberge, M., A. Pariselle, T. Huyse, F. Volckaert, J. Sˇimkova´, A., Y. Desdevises, M. Gelnar & S. Morand, 2001. Snoeks & M. P. M. Vanhove, 2015. Morphology, mole- Morphometric correlates of host specificity in Dactylo- cules, and monogenean parasites: an example of an inte- gyrus species (Monogenea) parasites of European cyprinid grative approach to cichlid biodiversity. PLoS ONE 10: fish. Parasitology 123: 169–177. 0124474. Sˇimkova´, A., S. Morand, E. Jobet, M. Gelnar & O. Verneau, Vanhove, M. P. M., J. Snoeks, F. A. M. Volckaert & T. Huyse, 2004. Molecular phylogeny of congeneric monogenean 2011. First description of monogenean parasites in Lake parasites (Dactylogyrus): a case of intrahost speciation. Tanganyika: the cichlid Simochromis diagramma (Tele- Evolution 58: 1001–1018. ostei, Cichlidae) harbours a high diversity of Gyrodactylus Sˇimkova´, A., O. Verneau, M. Gelnar & S. Morand, 2006. species (Platyhelminthes, Monogenea). Parasitology 138: Specificity and specialization of congeneric monogeneans 364–380. parasitizing cyprinid fish. Evolution 60: 1023–1037. Vanhove, M. P. M., B. Tessens, C. Schoelinck, U. Jondelius, D. Sˇimkova´, A., Serbielle, C., Pariselle, A., Vanhove, M. P. M. T. J. Littlewood, T. Artois & T. Huyse, 2013. Problematic & S. Morand, 2013. Speciation in Thaparocleidus barcoding in flatworms: a case-study on monogeneans and (Monogenea: Dactylogyridae) parasitizing Asian Pan- rhabdocoels (Platyhelminthes). ZooKeys 30: 355–379. gasiid catfishes. Biomedical Research International, 2013: Vanhove, M. P. M., A. Pariselle, M. Van Steenberge, J. A. M. Article ID 353956. Raeymaekers, P. I. Hablu¨tzel, C. Gillardin, B. Hellemans, Sturmbauer, C., C. Fuchs, G. Harb, E. Damm, D. Duftner, M. F. C. Breman, S. Koblmu¨ller, C. Sturmbauer, J. Snoeks, F. Maderbacher, M. Koch & S. Koblmu¨ller, 2008. Abun- A. Volckaert & T. Huyse, 2015. Hidden biodiversity in an dance, depth distribution and territorial areas of rock- ancient lake: phylogenetic congruence between Lake dwelling Lake Tanganyika cichlid fish species. Hydrobi- Tanganyika tropheine cichlids and their monogenean flat- ologia 615: 57–68. worm parasites. Scientific Reports 5: 13669. Takahashi, T., 2003. Systematics of Tanganyikan cichlid fishes Vanhove, M. P. M., P. I. Hablu¨tzel, A. Pariselle, A. Sˇimkova´,T. (Teleostei: Perciformes). Ichthyological Research 50: Huyse & J. A. M. Raeymaekers, 2016. Cichlids: a host of 367–382. opportunities for evolutionary parasitology. Trends in Takahashi, T., 2014. Greenwoodochromini Takahashi from Parasitology 32: 820–832. Lake Tanganyika is a junior of Limnochromini Vignon, M. & P. Sasal, 2010. The use of geometric morpho- Poll (Perciformes: Cichlidae). Journal of Fish Biology metrics in understanding shape variability of sclerotized 84:929–936. haptoral structures of monogeneans (Platyhelminthes) with Takahashi, T. & T. Sota, 2016. A robust phylogeny among insights into biogeographic variability. Parasitology major lineages of the East African cichlids. Molecular International 59: 181–191. Phylogenetics and Evolution 100: 234–242. Theisen, S., H. W. Palm, S. H. Al-Jufaili & S. Kleinertz, 2017. Publisher’s Note Springer Nature remains neutral with Pseudempleurosoma haywardi sp. nov. (Monogenea: regard to jurisdictional claims in published maps and Ancyrocephalidae (sensu lato) Bychowsky & Nagibina, institutional affiliations.

123 Study D

Conservative divergent evolution in a gill monogenean parasitizing distant cichlid lineages of Lake Tanganyika: Cichlidogyrus nshomboi (Monogenea:

Dactylogyridae) from representatives of Boulengerochromini and

Perissodini

Rahmouni, C., Vanhove, M.P.M. and Šimková, A. and Van Steenberge, M. (2021)

submitted to Evolutionary Biology

Study E

Molecular phylogeny and speciation patterns in host-specific monogeneans

(Cichlidogyrus, Dactylogyridae) parasitizing cichlids in Lake Tanganyika

Rahmouni, C., Vanhove, M.P.M., Koblmüller, S. and Šimková, A. (2021)

submitted to International Journal for Parasitology