Monogenea Hlubinných Cichlid Jezera Tanganika: Snížená Specifita Nebo Skrytá Diverzita? Diplomová Práce Nikol Kmentová

MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BOTANIKY A ZOOLOGIE

Monogenea hlubinných cichlid jezera Tanganika: snížená specifita nebo skrytá diverzita? Diplomová práce Nikol Kmentová

Vedoucí práce: Maarten Vanhove, Ph.D. Brno 2015 Bibliografický záznam

Autor: Bc. Nikol Kmentová Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie

Název práce: Monogenea hlubinných cichlid jezera Tanganika: snížená specifita nebo skrytá diverzita?

Studijní program: Ekologická a evoluční biologie

Studijní obor: Zoologie

Vedoucí práce: Maarten Vanhove, Ph.D.

Akademický rok: 2014/2015

Počet stran: 86+2

Klíčová slova: Cichlidogyrus, cichlidy, Bathybatini, hostitelské spektrum, vnitrodruhová variabilita, fenotypová plasticita, fylogenetika, genový tok, demografická historie

Bibliographic Entry

Author Bc. Nikol Kmentová Faculty of Science, Masaryk University Department of botany and zoology

Titleof Thesis: Monogenean parasites from deepwater Lake Tanganyika cichlids: reduced specificity or hidden diversity? Degreeprogramme: Ecological and evolutionary biology

Fieldof Study: Zoology

Supervisor: Maarten Vanhove, Ph.D.

AcademicYear: 2014/2015

NumberofPages: 86+2

Keywords: Cichlidogyrus, cichlids, Bathybatini, host range, intraspecific variation, phenotypic plasticity, phylogeny, gene flow, demographic history

Abstrakt

V této diplomové práci se věnujeme žábrohlístům vyskytujících se na hlubinných druzích cichlid jezera Tanganika. Toto jezero je jedním z nejdůležitějších míst pro studium biologické rozmanitosti, jakož i procesů a mechanimů evoluce. Je překvapivé, jak málo informací bylo doposud zjištěno o zdejší parazitické fauně. Jedna z předchozích studií naznačuje snížení hostitelské specifity třídy Monogenea (Cihlidogyrus, Dactylogyridae) v hloubokých vodách. Naším cílem bylo otestovat uváděný pokles tohoto faktoru a zodpovědět následující otázky: je tento zdánlivý pokles hostitelské specifity způsoben na první pohled nezřetelnými speciačními procesy, nebo skutečným snížením hostitelské preference? Jak daleko toto hostitelské rozmezí zasahuje? Jaká je genetická struktura a demografická historie této parazitické populace? V této práci jsme se zaměřili a dále analyzovali žábrohlísty parazitující na druzích reprezentující čtyři různé kmeny cichlid (Bathybatini, Trematocarini, Benthochromini, Limnochromini). Vzorky pocházely z mnoha lokalit z různých částí jezera. Hypotézy byly testovány pomocí tří různých technik zahrnující morfometrické a geomorfologické metody s využitím vícerozměrných statistických přístupů společně s genetickou charakterizací zahrnující oblasti ribosomální a mitochondriální DNA. Celkem bylo shromážděno 849 žábrohlístů z 9 hostitelských druhů. Tři různé druhy patřící do rodu Cichlidogyrus byly rozpoznány na základě morfometrických i genetických analýz všech oblastí ribosomální DNA. Hostitelské spektrum druhu C. casuarinus je rozšířeno mezi druhy kmene Bathybatini. Na druhou stranu, morfometrické stejně jako geomorfologické analýzy ukazují jistou míru diferenciace tohoto druhu ovlivněnou hostitelským druhem a geografickým původem jedinců. Tento pozorovaný jev se zdá být díky jeho nezávislosti na genetické struktuře populace s největší pravděpodobností způsoben fenotypovými změnami při ontogenetickém vývoji parazita. Výsledky analýzy týkající se demografické historie druhu C. casuarinus naznačují jeho nedávný populační nárůst. Další studie jsou zapotřebí ke kompletnímu odhalení vztahů hostitelsko- parazitického systému, diverzity a evoluční historie žábrohlístů obývající hlubinné vody jezera Tanganika. Abstract In this thesis we focus on the monogenean fauna of cichlid species prefering deepwater habitat in Lake Tanganyika. The lake is one of the most important study areas in the fields of biodiversity and of evolutionary biology. Surprisingly, the knowledge about the lake’s parasite is still premature. A previous study indicated a reduction of monogenean host specifity (Cichlidogyrus, Dactylogyridae) in the deepwater habitat. We verify whether the seemingly low host-specificity is caused by cryptic speciation or by a real decrease in host preference. We also assessed how broad this host range is. Finally, we examined the genetic structure and demographic history of this parasite population. In our study we examined and analysed monogeneans from host species representing four different cichlid tribes (Bathybatini, Trematocarini, Benthochromini, Limnochromini). Samples originated from many localities throughout the lake. Hypotheses were tested by three different techniques: morphometrics and geomorphometrics using multivariate statistical approaches together with genetic characterization of ribosomal as well as mitochondrial DNA markers. Finally, 849 parasitic individuals from 9 host fish species were collected. Three different species belonging to Cichlidogyrus were distinguished by both morphometric and genetic characterization in all obtained ribosomal DNA regions. The host range of C. casuarinus encompasses almost the whole Bathybatini tribe. On the other hand, our morphometric as well as geomorphometric analyses show some kind of differentiation of C. casuarinus influenced by host preference and geographical origin. This observed pattern most likely seems to be caused by phenotypic changes during ontogenetic development because of its independence to genetic population structure. Analyses of parasite demographic history indicate recent population expansion of C. casuarinus. Other investigations are needed to further clarify monogenean diversity, host-parasite relationships and the evolutionary history of monogeneans occurring in deepwater in Lake Tanganyika.

Poděkování

Na tomto místě bych chtěla vyjádřit vděčnost všem skvělým lidem, kteří mě během vzniku této diplomové práci podporovali. Zcela zásadní vliv nejen na mou práci jako takovou, ale také motivaci měl školitel Maarten M. P. Vanhove, Ph.D., kterému bych touto cestou chtěla poděkovat za jeho úsilí a trpělivost při mém vedení. Velmi si cenním také poskytnutých rad a nenahraditelných zkušeností doc. Milana Gelnara, Ph.D., a tímto mu děkuji za jeho ochotu a vlídnost. Dále nemohu opomenout pomoc Moniky Mendlové, Ph.D., Kristíny Civáňové, Ph.D. a Mgr. Elišky Šrámové při zpracování vzorků určených k molekulární analýze. Neméně důležitá byla pro mne také přítomnost a ochota Boženy Koubkové, Ph.D., Evy Řehulkové, Ph.D. a Katky Francové, Ph.D. při řešení jakéhokoli problému týkajícího se morfologických analýz. Velké díky patří také Maartenovi Van Steenberge, Ph.D. za nesčetné konzultace zaměřené na geomorfologické analýzy a dále také Stephanovi Koblmüller, Ph.D. za objasnění problematiky genetické struktury populací. Nedílnou součástí byla také spolupráce s Royal Museum for Central Africa, Tervuren, jmenovitě Tine Huyse, Ph.D., Prof. Jos Snoeks, Ph.D. a Miguël Parrent, kterým tímto děkuji nejen za poskytnutí vzorků, ale také za možnost strávit nějaký čas v jejich společnosti v těchto historických prostorách. Mé díky patří také pracovnímu kolektivu z instituce Royal Belgian Institute for Natural Sciences. Důležitým prvkem ovlivňujícím mé pracovní nasazení a tím také konečné podoby této diplomové práce byla nikdy nekončící přívětivost a tolerance všech pracovníků a spolužáků na katedře parazitologie, kterým jsem vděčná za skvělou atmosféru. V neposlední řadě bych tímto chtěla také poděkovat své rodině a přátelům za psychickou podporu, bez které bych si předešlé dva roky nedokázala představit. Tato práce byla realizována za finanční podpory grantu ECIP číslo P505/12/G112.

Prohlášení Prohlašuji, že jsem svoji diplomovou práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány.

Brno 4. měsíce 2015 ……………………………… Jméno Příjmení 1. CONTENTS

1. CONTENTS ...... 9 2. INTRODUCTION ...... 10

2.1 Literature review ...... 10

2.1.1 Lake Tanganyika ...... 10 2.1.2 Cichlids (Teleostei, Cichlidae) ...... 12 2.1.3 Monogenea (Platyhelminthes: Neodermata) ...... 20 2.1.4 Goals ...... 29

3. MATERIAL AND METHODS ...... Chyba! Záložka není definována.

3.1 Study area ...... Chyba! Záložka není definována. 3.2 Number of specimens ...... Chyba! Záložka není definována. 3.3 Sample collecting methods ...... Chyba! Záložka není definována. 3.4 Data analyses ...... Chyba! Záložka není definována.

3.4.1 Morphometrics ...... Chyba! Záložka není definována. 3.4.2 Geomorphometrics analysis ...... Chyba! Záložka není definována. 3.4.3 Genetic characterization ...... Chyba! Záložka není definována. 3.4.4 Alignment and phylogenetic analysisChyba! Záložka není definována. 3.4.5 Analyses of population structure and demographic historyChyba! Záložka není definována.

4. RESULTS ...... Chyba! Záložka není definována.

4.1 Cichlidogyrus species found ...... Chyba! Záložka není definována. 4.2 Genetic characterization ...... Chyba! Záložka není definována. 4.3 Phylogenetic analysis ...... Chyba! Záložka není definována. 4.4 Analyses of intraspecific variation ...... Chyba! Záložka není definována.

4.2.1 Principal component analysis and analysis of varianceChyba! Záložka není definována. 4.2.2 Geomorphometrics ...... Chyba! Záložka není definována. 4.2.3 Population structure and historical demography recontructionChyba! Záložka není definována.

5. DISCUSSION ...... Chyba! Záložka není definována. 6. LITERATURE ...... Chyba! Záložka není definována. 7. APPENDIX ...... Chyba! Záložka není definována.

2. INTRODUCTION The Class Monogenea van Beneden, 1858 is the most diverse group of Platyhelminthes with more than 3,500 already described species. It is widespread worldwide mainly in aquatic environments (BUCHMANN & BRESCIANI 2006; ROHDE 2011). Most of the these ectoparasitic flatworms are located on the body or gills mainly of freshwater and marine fishes but some of them have also adopted an endoparasitic lifestyle inside fishes, turtles, amphibians and one mammalian species (ROHDE 2011). Their phylogenetic history is supposed to be linked with co- evolutionary processes such as host switching, adaptive radiation mechanisms and also sympatric speciation (ROHDE 1996; LITTLEWOOD et al. 1997; BAKKE et al. 2002). Cichlids (Teleostei, Cichlidae) are infected by several monogenean genera of which Cichlidogyrus Paperna, 1960 is the most species-rich one. This genus displays variation in host specifity and contains generalist but also strictly specialist species (PARISELLE et al. 2013; MENDLOVÁ & ŠIMKOVÁ 2014). Lake Tanganyika contains unique species flocks of vertebrate as well as invertebrate taxa and cichlids overshadow in diversity all other families there. While several aquatic parasite taxa are reported there (COULTER 1991) only a few host taxa have been investigated in the deepwater environment with only one report about the monogenean part of the story (PARISELLE et al. 2015a). The ecological, behavioural and phylogenetic diversity of (Tanganyika) cichlids (KOCHER 2004) makes them ideal models for investigating parasite speciation mechanisms and the influence of host ecology on parasite diversity (POUYAUD et al. 2006). This fact makes research on monogeneans’ evolutionary mechanisms even more interesting and applicable there.

2.1 Literature review 2.1.1 Lake Tanganyika Lake Tanganyika is situated in the western part of the African Great Rift Valley. This lake is the oldest of the African Great Lakes (the other ones being Lake Malawi and Lake Victoria) and it has started to form around 20 million years ago initially as extensive swampland resulting in deep-lake conditions between 6 and 12 million years

10 ago (LOWE-MCCONNELL 2009). It is also the second deepest lake in the World with 1,463 metres depth at its deepest point and with 570 metres mean depth (COHEN et al. 1993; COHEN et al. 1997a; SMITH 1998; KONINGS 2005). Oxygen penetrates to 213 metres and the water temperature is stable, ranging from 26°C to 27°C (SMITH 1998). Lake Tanganyika occupies an area of 32,600 km2 and the water volume is 18,880 km3 (COHEN et al. 1997a). The water is quite turbid in the rainy season but the visibility is normally more than 20 metres (KONINGS 2005). Increased water turbidity (e.g. by eutrophication or erosion in Lake Victoria) can lead to decline of fish diversity (SEEHAUSEN et al. 1997). There are also other problems and conservation risks because of the rapid human population growth which is connected with over-fishing, increased turbidity, sedimentation and pollution (LOWE-MCCONNELL 2009). Donohue et al. (2003) performed an experiment in situ in Lake Tanganyika and recorded a significant reduction of species numbers in benthic algivorous cichlids and the benthic invertebrate community after sediment addition. Congo on the western side and Tanzania on the eastern side possess the largest parts of Lake Tanganyika’s 2000 km long coastlines. On its northeastern side, the lake is lined by Burundi and on the southern part by Zambia (SMITH 1998; KONINGS 2005). We can distinguish three major basins: Zongwe, Kalemie and Kigoma (KONINGS 2005). The lake is currently an open basin which flows into the Congo through the Lukuga River, the lake’s only outflow (COHEN et al. 1997a). Permanent but seasonally variable tributaries flow into the lake, such as the Malagarasi River (Tanzania), the Rusizi River (Burundi) and the Lufubu River (Zambia). There are also other small rivulets of temporary character (Fig. 1) (COHEN et al. 1997a; KONINGS 2005). The Rusizi River is also an important source of nutrients (CRAIG et al. 1974). In the past, the lake water level fluctuated and the changes were induced by both climatic and geomorphic factors (SCHOLZ & ROSENDAHL 1988; TIERCELIN & MONDEGUER 1991; COHEN et al. 1993 1997a 1997b; LEZZAR et al. 1996; ALIN & COHEN 2003; SCHOLZ et al. 2003; KONINGS 2005). It is suggested the division of the lake into three separated basins 1 million years ago influenced the evolutionary history of the lake’s species fauna (DANLEY et al. 2012). Alin & Cohen (2003) studied the level history by ostracode-inferred water-depth reconstruction and they refined the dating of lowstands during the late Holocene and other droughts during the Little ice age (1580 ±

15 AD, 1730 ± 35 AD, and 1800 ± 30 AD). The lowest water level in recent decades (772,5 – 776,7 metres above sea level) was recorded during the 19th century and the highest values (784 metres above sea level) in 1878 AD (HABERYAN a HECKY 1987; COHEN et al. 1997b; BIRKETT et al. 1999).

Fig. 1: Map of Lake Tanganyika, Cohen et al. 1997, edited. Lake Tanganyika is an important study area for evolutionary biologists because it shows the greatest diversity of adaptations and speciation mechanisms throughout the Great Lakes of Africa (SMITH 1998). Cichlidae overshadows in diversity all other animal families in the lake, but we can find a high level of endemicity and radiation also in non-cichlid taxa. The lake is home to species assemblages from other fish families (Latidae, Mastacembelidae and Mochokidae) (SALZBURGER et al. 2002, SALZBURGER et al. 2014) and also invertebrate taxa (SNOEKS 2000).

2.1.2 Cichlids (Teleostei, Cichlidae)

Systematic classification and distribution

Cichlid fishes belong to the Class Osteichtyes, Order Perciformes and Family Cichlidae and they are closely related to the Embiotocidae and Pomacentridae. These

12 three groups have an unusually structured set of pharyngeal jaws which enable them to specialize on different food sources as one of the synapomorphies of the Labroidei clade (BARLOW 2000). A total evidence phylogeny of Cichlidae was performed by Farias et al. (2000). This classification is based on molecular data from the 16S rRNA region and illustrates relationships between Malagasy/Indian, African and Neotropical lineages (FARIAS et al. 2000). Sparks & Smith (2004) used nuclear and mitochondrial characters of major cichlid lineages and confirmed the cichlids’ monophyly. More recent phylogenetic analyses of Wainwright et al. (2012) indicate polyphyly of the Labroidei clade and grouped Cichlidae, Embiotocidae, Pomacentridae and other lineages to a new clade named Ovalentaria. There are two main hypotheses which explain cichlid distribution; the vicariance model and the dispersal model. The vicariance model requires a vicariant event as a result of the breakup of Gondwana (165 to 121 million years ago (MYA)) while the dispersal model assumes cichlids originated 57-76 MYA when they evolved near Madagascar and secondarily colonized their present-day range by marine migration (FRIEDMAN et al. 2013). Basal lineages are present in India, Madagascar and West Africa (GENNER et al. 2007; PARISELLE et al. 2011). Cichlid fishes are found mainly in tropical freshwaters, but some of them can penetrate and survive in marine systems (BARLOW 2000; TURNER 2007). It is one of the most diverse vertebrate family with more than 2,000 described species. Its distribution ranges from Central and South America, across Africa, Iran, the Middle East and Madagascar to India and Sri Lanka, but most of them are concentrated in the Neotropics and in Africa (CHAKRABARTY 2004). In most of these areas we can notice independent rapid radiation processes and similar adaptations (parallel evolution) (SCHLIEWEN et al. 1994; MCKAYE et al. 2002). Cichlids provide most of the proteins for many African communities and also an important part of the meat in some parts of Central and South America (BARLOW 2000). Tilapias (Cichlidae, “Tilapiini”) are the most widely cultivated food fish in the tropics, but as non-indigenous species mean a serious danger for the native environment because of diseases, competition with native species and excrement contamination (COWARD & LITTLE 2001).

Evolution and main characteristics

Lake Tanganyika shows the deepest fish diversity (FRYER & ILES 1972) which has probably evolved during the late Tertiary (OWEN et al. 1990). Species radiation and speciation in the cichlid family have probably occurred in a short period of time (KOCHER 2004). For many years it was thought, that new species can arise only due to geographic and reproductive barriers among populations, a process called allopatric speciation (MAYR 1963). The huge influence in cichlid evolution could also have had the founder effect as a specific type of allopatric speciation (MAYR 1963). Allopatric speciation cannot explain the origin of all species and the primary origin of reproductive incompatibility might be sexual antagonism between male and female individuals (KOCHER 2004). This is the principle of sympatric speciation, when new species originate from a common ancestor while inhabiting the same geographic region (MAYR 1963). Positive correlation between fitness and mate-choice traits seems to be the starting point for such speciation (KOCHER 2004). Lande (1982) proposed a hypothesis about the co-evolution of male traits and female preferences. Reproductive isolation is provided mainly by male colour patterns and different ecological habitats (TURNER 2007). Schliewen et al. (1994) provided proof of sympatric speciation in a cichlid community. They showed the existence of monophyletic species flocks which had evolved in Lake Barombi Mbo and Bermin (Cameroon) after a single colonization event without geographic barriers, lake level fluctuations and in isolation from river systems (SCHLIEWEN et al. 1994). A final natural speciation process is parapatric speciation which is characterized by gene exchange among adjoining populations (MAYR 1963, KOCHER 2004). There is still a gap in our understanding of cichlid evolutionary processes. We have not identified most of the genes responsible for phenotypic differences and speciation (KOCHER 2004). Studies about cichlid adaptation mechanisms could provide important information that will be generally applicable in evolutionary biology (KOCHER 2004). Most cichlid tribes prefer a certain type of biotope (KONINGS 2005). The huge diversity in feeding strategies and sources (algae, invertebrates, plankton, fishes…) in the cichlid community is made possible by the variable arrangement of the muscles and jaws; it depends especially on the upper pharyngeal jaws (LIEM 1973). We can

14 distinguish three categories in cichlid feeding strategies: manipulation (the fish bites into the food source), suction feeding (fish creates an inward jet of water to carry the prey into the mouth cavity) and raw feeding (fish hide out and engulf its prey). Most cichlids use a combination of manipulation and suction feeding (BARLOW 2000). One of the most important aspect of cichlids is their complex communication capacities which include sounds but most of the signals are visual (they vastly outstrip chameleons) (BARLOW 2000). Cichlids have a well-developed colour vision with a trichromatic system which plays an important role in their diverse mating behaviour. Colour morph and species numbers increase with the breadth of the light spectrum in the water and a reduced effectiveness of colour signals influences the cichlid mating system. Increased water turbidity can hence lead to a reduction of their species diversity (SEEHAUSEN et al. 1997). Two types of remarkable parental care occur in this fish family, mouth brooders and substrate brooders (KOCHER 2004). Mouth brooders (almost always the female) incubate the fertilized eggs in their mouths. Substrate brooders hide their eggs in rocky habitat or inside empty shells and guard them (STURMBAUER & MEYER 1993, DUPONCHELLE et al. 2008). An interesting phenomenon in cichlids is breeding in social groups when a dominant pair produces most of the eggs and the other members take care of them and defend the territory (TURNER 2007). Migration of sub-adult males is important for gene flow in populations. On the other hand, female individuals tend to stay in their native territory (e.g. Pseudotropheus zebra (Boulenger, 1899) in Lake Malawi) (KNIGHT et al. 1999). Aggressive behaviour is generally observed among members of the same species (TURNER 2007). However Hori et al. (1993) studied rocky littoral cichlid communities in Lake Tanganyika and discovered feeding relationships which result in interspecific territoriality. It is also evident that behavioural differences between morphologically similar species enable their coexistence in the same area (HORI et al. 1993).

Cichlids in Lake Tanganyika

Lake Tanganyika contains around 250 cichlid species in 53 genera (SNOEKS 2000, TAKAHASHI 2003), which have usually been divided by Poll (1986) into 12 tribes but a revised classification based on internal and external morphological features by

Takahashi (2003) (Fig. 2) recognizes 16 tribes. The most recent study based on nuclear and mitochondrial markers recognised the existence of 15 cichlid tribes (Fig. 3) (MUSCHICK et al. 2012). Their origin seems to be polyphyletic, they probably have evolved from several ancestral lineages (NISHIDA 1991, SALZBURGER et al. 2002, KOBLMÜLLER et al. 2008). Dates indicate that the lake was colonized independently by every major cichlid lineage (LOWE-MCCONNELL 2009). There are currently more than 200 described endemic species in the lake (KOBLMÜLLER et al. 2008). Cichlid evolution in this lake has been determined by level fluctuations throughout history. When it was divided into three smaller basins separated cichlid fish populations have evolved into distinct colour variants and new species by allopatric but also sympatric speciation (SMITH 1998). Lake Tanganyika accommodates the most diverse cichlid flock in Africa in terms of genetics, morphology and behavior (SALZBURGER et al. 2002, SALZBURGER 2009).

Fig. 2: Classification of Lake Tanganyika cichlids (TAKAHASHI 2003).

Fig. 3: Classification of Lake Tanganyika cichlids (MUSCHICK et al. 2012).

Deepwater cichlids in Lake Tanganyika The primary lacustrine radiation of deepwater species in Lake Tanganyika was dated back to 5-6 million years ago (TIERCELIN & MONDEGUER 1991). Cichlid species richness decreases with deeper water; most of their diversity is found in littoral habitats. This situation is caused by three main factors: reduction of niche diversity, the fact that the short-wave length blue light spectrum in the depths does not promote diversification mechanisms and also the absence of strong geographic barriers (SEEHAUSEN et al. 1997, SEEHAUSEN et al. 1999, KNIGHT et al. 2004, MAAN et al. 2004). There is some kind of mystery about their ecology because it is almost impossible to observe them in their natural environment. One of the deepwater groups of Lake Tanganyika cichlids is an endemic tribe called Bathybatini Poll 1986, with currently 17 recognised species in three genera (Fig. 4) (TAKAHASHI 2003, KIRCHBERGER et al. 2012). Phylogenetic relationships in

Bathybatini are still unclear. Poll (1986) proposed Bathybatini and Trematocarini as separate tribes. This result was also confirmed by Koblmüller et al. 2005. In contrast Stiassny (1981) and Takahashi (2003) recognised Hemibates Regan, 1920 as a sister group to Bathybates Boulenger, 1898 and also to Trematocarini. The split between the genera Trematocara, Bathybates and Hemibates is assumed to date back to 8,3-9,9 MYA (KOBLMÜLLER et al 2005). The radiation of this ancestral lineage is therefore supposed to have occurred before the primary lacustrine radiation.

Fig. 4: Phylogenetic relationships of the Bathybatini based on AFLP loci (KIRCHBERGER et al. 2012). There is only little infomation about this tribe, which is found throughout the lake (KONINGS 1998). All species are maternal mouthbrooders and (KUWAMURA 1997) lay large eggs (7 mm) (KONINGS 1998, TAKAHASHI 2003). They are also characterized by sexual colour dimorphism. Males have species-specific monochromatic dark stripes and blotches on a silver background while females are silvery coloured without any special patterns (KOBLMÜLLER et al. 2005, KIRCHBERGER et al. 2012). Representatives of Bathybates are large (maximum size between 25 and 40 cm) piscivorous predators mainly of pelagic clupeids (B. fasciatus

Boulenger 1901, B. leo Poll 1956), benthic cichlids (B. graueri Steindachner, 1911, B. vittatus Boulenger, 1914, B. ferox Boulenger, 1898) and small clupeids (B. minor Boulenger 1906). The prey of B. horni Steindachner 1911 is unknown. They usually descend into depths of 150 – 200 metres (MARÉCHAL & POLL 1991, KONINGS 2005, KIRCHBERGER et al. 2012). Bathybates minor feeds on sardine shoals and is characterized by surprise attack on its prey. It figures as an ancestral lineage of the genus Bathybates of which the split can be dated to 5-6 MYA (similar to the primary lacutrine radiation in the lake) while the specialization of the rest of the Bathybates species is dated to 2,5-3 MYA (KOBLMÜLLER et al. 2005). Bathybates fasciatus and B. leo are usually found in open water together with their prey (sardines). Hemibates is a monotypic genus: its only species H. stenosoma has two distinct colour-variants which occur in sympatry (KONINGS 1998). It has a lake-wide distribution and migrates to the shallow waters during night where it preys on other cichlids (KONINGS 1998). It is also found in depths between 100 and 200 metres feeding on fish and shrimps and produces some of the largest known cichlid eggs (MARÉCHAL & POLL 1991). Trematocara belongs to the separate tribe Trematocarini and comprises nine benthic and bathypelagic species which feed on invertebrates, fish eggs and phytoplankton. Trophic differentiation between them has been observed. They have an extensive sensory system on the head (KONINGS 1998). Trematocara species can be found usually between 70 and 200 metres of depth, but they also migrate to the littoral at night following their prey (COULTER 1991). There are several more deep-water dwelling taxa, whose species richness is lower than in the shallow habitat. Only little is known about their lifestyle. Examples belong to genera such as Limnochromis Boulenger, 1901, Baileychromis (Bailey & Stewart, 1977), Reganochromis Boulenger, 1901, Plecodus Boulenger, 1898, Xenochromis Boulenger, 1899 and Benthochromis (Poll, 1948) (KONINGS 1998).

2.1.3 Monogenea (Platyhelminthes: Neodermata) The class Monogenea is one of the most species-rich group of parasitic flatworms (ROHDE 2011). It is estimated that 50% of all metazoan parasites occuring in Europe belong to this class (GELNAR et al. 1994, DUŠEK et al. 1998).

Systematic classification and characterisation Traditional nomenclature classifies Monogenea, together with the classes Trematoda Rudolphi, 1808 and Cestoda van Beneden, 1849, under the group Neodermata within the phylum Platyhelminthes. Monogeneans are commonly divided into the subclasses Monopisthocotylea and Polyopisthocotylea. There is also an alternative which defines the subclasses Polyonchoinea (equivalent to Monopisthocotylea), Polystomatoinea and Oligonchoinea (BOEGER & KRITSKY 1997). Littlewood et al. (1999) confirmed the monophyly of monogeneans after phylogenetic analysis of the complete 18S and two partial 28S rDNA gene sequences. The subclass Monopisthocotylea is formed by 6 orders (Capsalidea, Gyrodactylidea, Dactylogyridea, Monocotylidea, Lagarocotylidea and Montchadskyellidea). The other subclass contains 4 orders (Chimaericolidea, Diclybothriidea, Mazocraeidea and Polystomatidea) (CAIRA & LITLEWOOD 2013). However, the deeper phylogenetic structure within subclasses is still under discussion (ŠIMKOVÁ et al. 2003). Monogeneans together with parasitic copepods are the most diverse ectoparasitic group of fishes (WHITTINGTON & CHISHOLM 2008). Almost all species infect the gills, skin or fins of freshwater and marine fishes but some of them have adopted an endoparasitic lifestyle in the mouth cavity, foregut, stomach and urinary bladder of fish but also turtles and amphibians. Oculotrema hippopotami Stunkard, 1924, which parasitises the conjunctival sac of hippopotamus, is the only monogenean known from a mammalian host (ROHDE 2011). Gill monogeneans tend to select specific microhabitats on the gills (ROHDE 1977). The high species number is probably a consequence of co-evolution between host and parasite, as a result of host switching, adaptive radiation mechanisms and also sympatric speciation because of e.g. microhabitat choice (ROHDE 1996, LITTLEWOOD et al. 1997, BAKKE et al. 2002).

The body structure The body of monogeneans is dorsoventrally flattened and usually reaches a size between 0.15 mm and 20 mm, with a maximum of 30 mm (PUGACHEV et al. 2009). The surface of adults is formed by a neodermis without cilia. Monogeneans are able to move in a leech-like manner. Their digestive system consists of an anterior sucker or suckers, a pharynx and a blind ending caecum, usually with a number of side branches. Monopisthocotyleans are epithelial feeders whereas Polyopisthocotyleans are blood

21 feeders (BUCHMANN & BRESCIANI 2006). The excretory system is made up from numerous flame bulbs and two separate excretory pores. It is an example of a protonephridial system. The nervous system is formed by a ladder with longitudinal connectives and transverse commissures (ROHDE 2011). Different types of proprioceptors, photoreceptors, rheoreceptors and tangoreceptors are also present. Almost all larval monopisthocotyleans have two pairs of pigmented eyes while polyopisthocotylean larvae possess only one pair of eyes. The muscles are generally formed by longitudinal and transversal fibres below the worm’s surface. These flatworms are hermaphroditic organisms and are equipped with an ovary and one, two or more testes. Vitellaria, an ootype, seminal receptacle, cirrus and a vagina may be present too (BUCHMANN & BRESCIANI 2006) (Fig. 5). The most important attachement organ is the opisthaptor in their posterior part which contains sclerotized structures (hooks, clamps or suckers) (PAPERNA 1996). The opisthaptor is characteristic of species groups or lineages while the morphology of the male copulatory organ (MCO) is very important for species-level diagnosis (ROHDE 2011). The pathogenicity of monogeneans is influenced by the manner in which the haptor attaches to the host (WHITTINGTON & CHISHOLM 2008). The anterior part (prohaptor) is important especially for locomotion, feeding and reproduction and is usually formed by 2 – 6 suckers (BUCHMANN & BRESCIANI 2006, PUGACHEV et al. 2009).

Fig. 5: The body structure of Dactylogyridae, exemplified by a representative of Actinocleidus (Monopistocotylea, Dactylogyridae) according to BEVERLY-BURTON 1984 (edited).

Life cycle Monogeneans are monoxenous parasites (i.e. only one host in their life cycle) with two different ways of reproduction. Most species are oviparous with up to 20 intrauterine eggs. However, Gyrodactylidae contains viviparous species. Gyrodactylids are also unique in displaying progenetic polyembryony with a second and often third generation inside one embryo (PAPERNA 1996, CABLE & HARRIS 2002). The time of hatching of the eggs is influenced by light intensity, chemicals in fish mucus or mechanical disturbances. The egg with operculum and one or more filaments, develops into a cilliated larva called oncomiracidium, which searches a suitable host. This larva has pigmented eyes, anterior glands, a pharynx, a gut and a complete excretory and nervous system (PUGACHEV et al. 2009). Its development time is influenced by water temperature (WHITTINGTON & CHISHOLM 2008). Reproductive barriers between

23 species are made up by niche segregation and complicated copulatory organs (BUCHMANN & BRESCIANI 2006).

Evolutionary history Monogeneans have probably evolved from turbellarians several hundred MYA (KEARN 1994). Parasite evolution is connected with co-evolutionary processes: cospeciation, intrahost speciation, host switching, failure to diverge and sorting events (RONQUIST 1997, CHARLESTON 1998, PAGE & CHARLESTON 1998, JOHNSON et al. 2003). Direct transfer of parasites during host contacts, which facilitates host switching, has played an important role in the diversification of gyrodactylids (KEARN 1994). Poulin (2005) studied parasitic organisms producing eggs in freshwater systems and there was no significant correlation between parasite prevalence and phylogenetic distance of the host. It is supposed that parasite speciation is results from a combination of phylogenetic and also ecological determinants (POULIN 2005). Littlewood et al. 1997 observed that congeneric monogenean species (polystomes in this case) infecting the same site on different host species are phylogenetically more closely related than congeneric species which are located on the same host species. It meanssome kind of allopatric speciation took place in the evolutionary history of these monogeneans (LITTLEWOOD et al. 1997). In the case of dactylogyrideans, a different scenario probably occurred, with primarily intrahost speciation in congeneric parasite species (i.e. species belonging to the same genus) as a result of niche segregation (ŠIMKOVÁ et al. 2004). The direct life cycle of monogeneans and the relatively long life span of their hosts probably enabled sympatric speciation events (BROOKS & MCLENNAN 1993). It is supposed that host-body size is an important factor facilitating intrahost speciation (POULIN 2002). There is evident positive correlation between this feature and the species richness of monogenean communities (SASAL et al. 1997). The evolutionary success of this parasitic group is based on opisthaptor diversity and its adaptability to different hosts and infection sites. It is supposed that the long co- evolutionary history between Monogenea and fishes resulted in harmonious balance and that’s why infections caused by these flatworms are usually insignificant under natural conditions (WHITTINGTON & CHISHOLM 2008). Due to differences in host immunity, new host-parasite combinations produced by anthropogenic introductions

24 may inflict strong pathologies and potentially cause mortality. One example is the vanishing of ship sturgeon, Acipenser nudivetris Lovetsky, 1828, in the Aral Sea. This was caused by Nitzschia sturionis (Abildgaard, 1794) (Monopisthocotylea, Capsalidae) which was introduced there together with its host Acipenser stellatus Pallas, 1771 in 1936-1937 (ZHOLDASOVÁ 1997).

Monogeneans and cichlids Currently there are 13 known monogenean genera infecting cichlid species (Tab. 1), but only six of them have been observed on African cichlid fishes: Urogyrus Bilong Bilong, Birgi & Euzet, 1994, Enterogyrus Paperna, 1963, Onchobdella Paperna, 1968, Scutogyrus Pariselle & Euzet, 1995, Cichlidogyrus Paperna, 1960 and Gyrodactylus von Nordmann, 1832 (PARISELLE & EUZET 2009, PARISELLE et al. 2011, VANHOVE et al. 2011a). Ectoparasitic Cichlidogyrus, Scutogyrus and Onchobdella are located on the gills, endoparasitic Enterogyrus and Urogyrus infect the stomach and urinary bladder, respectively. There is a clear difference between the worldwide distribution patterns of ectoparasitic and endoparasitic genera (Tab. 1). In contrast to endoparasites, ectoparasitic genera seem to be endemic to each continent. Unlike endoparasites they could not have survived marine conditions during cichlid migration - one of the mechanisms potentially explaining cichlid intercontinental distribution (see above). As such, parasites also could be usable to fill gaps in our understanding of cichlid evolution and phylogeny (NIEBERDING & OLIVIERI 2006, PARISELLE et al. 2011).

Tab. 1: Current knowledge about the distribution of monogenean genera from cichlid fishes (adapted from PARISELLE et al. 2011). Ectoparasite genera Endoparasite genera Madagascar Insulacleidus Rakotofiringa and Euzet 1983 Asia Ceylonotrema Gussev 1963 Enterogyrus Sclerocleidoides Agarwal, Yadav and Kritsky 2001

West Africa Cichlidogyrus, Onchobdella, Scutogyrus, Gyrodactylus Enterogyrus, Urogyrus East Africa Cichlidogyrus, Scutogyrus, Gyrodactylus Enterogyrus, Urogyrus Levant Cichlidogyrus, Scutogyrus, Gyrodactylus Enterogyrus Iran Gyrodactylus South Gussevia Kohn and Paperna 1964, America Gyrodactylus, Sciadicleithrum Kritsky, Tchather and Boeger 1989, Trinidactylus Hanek, Molnar and Fernando 1974, Tucuranella Mendoza-Franco, Scholz and Rozkošná 2010

Cichlidogyrus (Monopisthocotylea, Dactylogyridae) is the most species rich genus with 101 species recorded from 86 different host species. It also displays huge variability in its species richness per host (PARISELLE & EUZET 2009, VANHOVE et al. 2011b, MUTEREZI BUKINGA et al. 2012, GILLARDIN et al. 2012, ŘEHULKOVÁ et al. 2013, PARISELLE et al. 2013, PARISELLE et al. 2014, PARISELLE et al. 2015a, PARISELLE et al. 2015b, VAN STEENBERGE et al. 2015). Tilapia cessiana Thys van den Audenaerde, 1968 is registered as a host for only one Cichlidogyrus species. On the other hand 20 Cichlidogyrus species were described on Tilapia guineensis (Günther, 1862). According to Mendlová & Šimková 2014 the host specifity of Cichlidogyrus parasitizing African cichlid fish is significantly influenced by fish phylogeny and form of parental care. There has never been observed a Cichlidogyrus species which infects cichlids with different parental care systems (i.e. substrate brooders as well as mouthbrooders) (POUYAUD et al. 2006). Mendlová & Šimková 2014 also suggest a negative correlation between host longevity and parasite host specifity. This would mean that generalist Cichlidogyrus tend to occur on long- lived cichlids (MENDLOVÁ & ŠIMKOVÁ 2014). However, the research about monogenean parasites of cichlids is still in its early stages. Molecular analyses focused on West African cichlids indicate that the species diversity of monogenean parasites

26 infecting cichlid hosts may be underestimated because of the existence of cryptic species (PARISELLE & EUZET 2003, POUYAUD et al. 2006, PARISELLE & EUZET 2009, ŘEHULKOVÁ et al. 2013). Parasites could also help us to understand the phylogenetic and biogeographic position of cichlids (MUTEREZI BUKINGA et al. 2012). The main morphological differences between these monogenean species are found in the opisthaptor and copulatory organs (POUYAUD et al. 2006). Specialists have an attachment apparatus well-adapted to their hosts (ROHDE 1979). Jarkovský et al. (2004) confirmed more similarities between the haptors within specialist infracommunities (parasite communities from single host individuals) than within generalist infracommunities. While the morphology of the haptor seems to be characteristic to major lineages within a genus, morphological characterisation of the copulatory organs is important for species-level identification in the case of Dactylogyridae (POUYAUD et al. 2006). There are only a few studies focusing on the monogenean fauna of Lake Tanganyika (PAPERNA 1973, VANHOVE et al. 2011a, VANHOVE et al. 2011b, MUTEREZI BUKINGA et al. 2012, GILLARDIN et al. 2012, FANNES et al. 2015, GRÉGOIR et al. 2015, PARISELLE et al. 2015a, PARISELLE et al. 2015b, VAN STEENBERGE et al. 2015). The first description was performed by Paperna (1973), when he found Ancyrocephalus limnotrissae Paperna 1973 on the clupeid Limnothrissa miodon (Boulenger, 1906). Vanhove et al. (2011a) revealed three new Gyrodactylus parasite species on Simochromis diagramma (Günther, 1983): Gyrodactylus sturmbaueri, G. thysi and G. zimbae. In the same year Vanhove et al. (2011b) examined gills from Ophthalmotilapia ventralis (Boulenger, 1898), O. nasuta (Poll & Matthes, 1962) and O. boops (Boulenger, 1901) and found four monogenean species which show evident morphological differences to each other: Cichlidogyrus vandekerkhovei, C. makasai, C. sturmbaueri and C. centesimus. The next year, Gillardin et al. (2012) described for the first time Cichlidogyrus spp. parasites from Tropheini fish hosts: Limnotilapia dardennii (Boulenger, 1899) is infected by C. steenbergei, Ctenochromis horei (Günther, 1894) figures as a host for C. gistelincki and Gnathochromis pfefferi (Boulenger, 1898) provides a living environment to C. irenae. Since then, nine new Cichlidogyrus species were reported from representatives of the Tropheini and

27 described by Pariselle & Vanhove (2015). Cichlidogyrus buescheri, C. schreyenbrichardorum and C. vealli infect Interochromis loocki (Poll, 1949) (PARISELLE et al. 2015b), while C. banyankimbonai, C. muterezii and C. raeymaekersi are found on Simochromis diagramma (Günther, 1894) (VAN STEENBERGE et al. 2015). Pseudosimochromis babaulti (Pellegrin, 1927) harbours C. georgesmertensi, while C. franswittei is hosted by P. curvifrons (Poll, 1942) and P. marginatus (Poll, 1956) and C. frankwillemsi by P. curvifrons alone (VAN STEENBERGE et al. 2015). Another recent study focused on Dactylogyridae of Lake Tanganyika recorded five new species: Cichlidogyrus gillardinae from Astatotilapia burtoni (Günther, 1894), C. mbirizei from Oreochromis tanganicae (Günther, 1894), C. nshomboi from Boulengerochromis microlepis (Boulenger, 1899), and C. mulimbwai and C. muzumanii from Tylochromis polylepis (Boulenger, 1900). These cichlid species are members of non-endemic tribes Haplochromini, Oreochromini, Tylochromini and Boulengerochromini. The phylogenetic position of the tribe Boulengerochromini is still unclear (MUTEREZI BUKINGA et al. 2012). To conclude, a recent study provided the first view on the monogenean fauna of deepwater hosts (Bathybates fasciatus, B. minor, B. vittatus and Hemibates stenosoma). A single Cichlidogyrus species (Cichlidogyrus casuarinus Pariselle, Muterezi Bukinga and Vanhove 2015) seemingly exists on these hosts. It is an indication of reduced host specifity in this environment; hovewer, only a limited number of deepwater host species were examined (PARISELLE et al. 2015). Cichlidogyrus casuarinus, C. nshomboi and C. centesimus are also unique among the whole known Cichlidogyrus fauna because of their unusual spirally coiled thickening of the wall of the copulatory tube (VANHOVE et al. 2011b, MUTEREZI BUKINGA et al. 2012, PARISELLE et al. 2015a). Ectoparasitic monogenean species belonging to Cichlidogyrus show relatively strong host specifity (PAPERNA 1973, MUTEREZI BUKINGA et al. 2012, GILLARDIN et al. 2012, ŘEHULKOVÁ et al. 2013). In previous studies in marine systems it was suggested the reduction of specificity is probably correlated with lower host availability in the deepwater realm (ROHDE 1980, JUSTINE et al. 2012, SCHOELINCK et al. 2012).

2.1.4 Goals The present study focuses on exploring the monogenean fauna from deepwater cichlids in Lake Tanganyika. The obtained information including morphological and genetic data will serve to further investigate the preliminary observations by Pariselle et al. (2015a) suggesting the broad host range of Cichlidogyrus species in deepwater hosts. These authors’ study was based on a limited host and geographical range and only morphological results were included. In this thesis the apparent decrease of the Cichlidogyrus host specifity in the deepwater habitat will be tested for more host species and on a broader geographical scale. Multivariate statistic approaches of morphological characters and genetic characterisation by means of established markers with different rates of molecular evolution will be used to answer the following questions: 1) Is the apparently broad host range of Cichlidogyrus casuarinus infecting bathybatine cichlids caused by ongoing speciation or by a real decrease in host preference? 2) How broad is the host range of this parasite species among deepwater cichlids in Lake Tanganyika? 3) What is the demographic history of this deepwater Cichlidogyrus species?