Acanthobothrium Blanchard, 1948 from the Northwest Atlantic and Their Phylogenetic Relationships with Freshwater Lineages

Yu Golfetti

Acanthobothrium Blanchard, 1948 from the Northwest Atlantic and their phylogenetic relationships with freshwater lineages.

São Paulo 2018

Yu Golfetti

Acanthobothrium Blanchard, 1948 from the Northwest Atlantic and their phylogenetic relationships with freshwater lineages.

Acanthobothrium Blanchard, 1948 do Noroeste Atlântico e seu relacionamento ﬁlogenético com linhagens de água doce.

Dissertação apresentada ao Instituto de Biociências da Universidade de São Paulo, para a obtenção de Título de Mestre em Ciências, na Área de Zoologia. Orientador: Fernando P. de Luna Marques

São Paulo 2018 Golfetti, Yu Acanthobothrium Blanchard, 1948 from the Northwest Atlantic and their phylogenetic relationships with freshwater lineages. 99 páginas Dissertação (Mestrado) - Instituto de Biociências da Universidade de São Paulo. Departamento de Zoologia.

1. Coevolução

2. Neotropical

3. Especﬁcidade

4. Biogeograﬁa

5. Tamanho amostral

I. Universidade de São Paulo. Instituto de Biociências. De- partamento de Zoologia.

Comissão Julgadora:

Prof. Dr. Prof. Dr. Nome Nome

Prof. Dr. Fernando P. de Luna Marques À todos aqueles que ainda lutam "Tem dias que a vida é um ato de coragem"

Se tiver que ser na bala vai - Vanguart Acknowledgements

Apoio ﬁnanceiro CAPES e CNPQ (processo 130093/2018-1)

Apoio técnico e cientíﬁco Laboratório de Helmintologia Evolutiva, Dpto. de Zoologia, IB-USP Laboratório de Sistemática Molecular, Dpto. de Zoologia, IB-USP

Agradecimentos especiais:

É de extrema importância que eu inicie meus agradecimentos com minha família. Familía essa que sempre está ao meu lado, vem me aceitando e que por mais desavenças que tenhamos, vem sendo meu pilar desde sempre. Mais especíﬁcamente eu queria agradecer aos meus avós Elio e Sonia, além de minha Tia Carla, por me apoiarem incondicionalmente desde de meu início de vida até hoje. Família, amo vocês.

Não posso deixar de agradecer a família Tiradentes. Nunca imaginei, isso em 2003, que o escotismo seria algo de tanta importância dentro da minha vida. É no Tiradentes que tenho tido algumas de minhas maiores realizações de vida. Dentre essas realizações, poder ensinar o que um dia me foi passado e ver os jovens crescendo e se tornando melhores cidadãos é gratiﬁcante e emocionante.

Eu tenho muitos amigos, muitos desses eu conheci dentro da USP. Agradeço de coração à Isabela Rodrigues (Tonks), Kléber Mathubara (Fanta), Giulia Magri, Ana Perticarrari, Thomas Creedy, Rachel Montesinos, Yu Oliveira, Ariadne Vilaça, Edgard Lopes, Carol Tieko, Gabrielle Rizatto e ao BacoWeb por todas as risadas, cervejas, conversas, cervejas, choros compartilhados, cervejas e amor que me deram.

Obrigada aos irmãos escoteiros de vida Jacqueline Monteiro, Gilmar Lago, Maria Angélica (Keka), Jéssica Cardana, Ricardo Correia, Luli Brunelli e Geovanna Kerkhoven, com quem eu sei que posso sempro posso contar, seja com o abrigo, ouvidos, abraços, broncas e o mais puro de todos os sentimentos.

Aos pés que não deixam esse tripé cair. Eu agradeço aquelas que me trouxeram luz quando eu só via escuridão. Aquelas que mesmo longe não deixam de estar comigo. Obrigada Camila Cobra e Beatriz Dinardi.

Eu queria agradecer à Laura Muniz e à Mariana Gonçalves. Agradecer por terem dividido o sentimento mais bonito comigo mesmo quando eu era apenas cacos. Obrigada por terem tentado juntar meus cacos. Os cacos estão se colando e hoje eu estou aqui, muito, por causa de vocês.

Obrigada àquelas que me abrigaram para onde eu não tinha para onde ir. Aquelas que dividiram sua casa, seu espaço, comigo e me ensinaram que estender a mão é tão fácil, basta tentar. Priscila Mendes e Jacque Garutti, obrigada! Por tudo, de verdade. As lindas Thayna, Déa, Maju, Bia e Jenny, que nessa reta ﬁnal me ensinaram que dividir é muito mais que dar, é um receber diário.

Ao pessoal da Arquibancada meus eternos amores. Durante meu mestrado acabei me afastando um pouco de vocês, me encontrando com alguns nesses 3 anos, porém o sentimento e todo apoio que me deram está guardado no fundo do peito. Sinto muita saudades de todos. Não posso também esquecer dos amigos que ﬁz durante a organização do V e VI CVZoo. Espero que todos se sintam altamente abraçados e amados nesse momento.

Em especial eu tenho muito a agradecer ao Jonathan Lawley e a Brittany Damron. Acho que se não fossem pelos dois eu não estaria aqui, literalmente falando. Foram eles que seguraram minha mão em minhas crises de pânico, minhas crises depressivas, escutaram minhas reclamações da vida e enxugaram minhas lágrimas... muitas vezes. Mas não só isso, foram eles que me deram os melhores conselhos, acreditaram em mim, paravam tudo que tinham para fazer apenas para me ver melhor. Eu não tenho palavras para agradecer a vocês o que ﬁzeram por mim, vocês me salvaram e por mais que eu ainda chore as vezes e continue sentindo dor, eu consigo lembrar de todo o apoio que me deram e assim levantar.

Eu amo vocês todos meus amigos.

Também não posso deixar de citar as pessoas que me proporcionaram a chance de chegar a USP e estar apresentando hoje essa tese. Sabrina, Madalena, Kaká, Francis, Ana e Aline, sinto muito a falta de vocês. Rogério, Renatinho, Amanda e Joaber... meus quatro orientadores. Os melhores orientadores que um pessoa pode ter nesse mundo. Eu consegui. Sem vocês eu não conseguiria. Vocês me ensinaram tanto. Eu agradeço a cada dia minha vida ter cruzado com a de vocês. À vocês minha eterna devocção e admiração, como aquela que um dia conheceram e que hoje tem a esperança de um dia tentar lhes alcançar.

Não posso deixar de agradecer aos técnicos Enio, Phillip, Bia e Sabrina, sem vocês metade das minhas coisas não teriam sido realizadas e eu continuaria sem saber onde colocar minhas amostras hahahah. Porém não posso deixar de agradecer ao Manu por todo apoio na parte de bancada molecular, fazendo mil e uma reanálises, testando primer e protocolo. Manu, parte dessa tese é sua e ainda te devo um bolo de chocolate.

Não poderia deixar de agradecer aos amigos do LHE. Obrigada Finn, Gigi, Pyro, Lilian e Luana (mesmo que só por um mês) por todas as conversas descompromissadas, risadas, cafés, recadinhos na lousa, preocupações comigo no whatsapp e por me escutarem. Obrigada ao Bjorn por ﬁnanciar a coleta dos hospedeiros ao qual sem esse estudo não teria sido realizado, por me puxar a orelha pelo inglês mal falado e os papos sobre NFL. Obrigada a Nati por toda a parte laboratorial ensinada, por todas as dicas de vida, por me fazer pensar 3 vezes antes de fazer e por todos os momentos dividos. Obrigada Veu pela revisão do inglês da minha tese, por aceitar ser da minha banca, por todos os papos lindos e loucos sobre o espaço e “de onde viemos e para onde vamos” que tivemos (e olha que foram muitos nesse pouco tempo que nos conhecemos). Obrigada Bruna. Não sei se consigo falar mais algo além de obrigada por tudo. eu sei que eu te enchi o saco em muita coisa, durante muito tempo, e tu sempre esteve ali. Obrigada por parar muitas das suas coisas, muitas vezes, para me ajudar com medições, bichos, bibliograﬁa, para me consolar, me ver reclamar das coisa, chorar, conversar sobre a vida ou rir um pouco. Quando eu crescer quero ser igual a você.

Fernando, meu orientador. Queria agradecer primeira mente por ter me aceito como sua orientanda e ter me dado a chance de conhecer tanta coisa e presenciar o que desde o início da graduação eu ansiava. Eu sei que não foi das melhores orientandas, eu sei que dei trabalho, e quando eu estava melhorando, vieram os problemas. Sua compreensão comigo foi crucial para esse dia ter chego. Desculpa não ter conseguido me dedicar tanto nesse ﬁnal, porém fui até onde meus limites de corpo e mente me autorizavam. Obrigada pelos ensinamentos, pelas chances de te questionar, pelas conversas e brigas (porque não?)... Você me fez crescer demais.

Por ﬁm, eu queria agradecer a todos aqueles que lutam, resistem, caem, se levantam e lutam novamente. Sua luta é minha luta.

Obrigada!

. Table of Contents

Introduction1 General goals of the study...... 4

Material and Methods4 Collection Biological Material...... 5 Morphological Data...... 5 Molecular Data...... 7

Results 10 Host Identiﬁcation...... 10 Molecular data...... 10 Morphological data...... 10 Phylogenetic Analysis of Acanthobothrium ...... 16 Taxonomic Actions...... 19 Acanthobothrium n. sp. 1...... 19 Acanthobothrium n. sp. 2...... 28 Acanthobothrium n. sp. 3...... 32 Acanthobothrium n. sp. 4...... 40

Discussion 46 Hosts Identity...... 46 Patterns of host speciﬁcity and parasite distribution...... 47 Phylogeny of Acanthobothrium ...... 49

Conclusions 53

References 60

Resumo 61

Abstract 63

Appendices 65 A.1 Script in R used for Primary Component Analyses (PCA)...... 66 A.2 DNA extraction protocol with Ammonium Acetate...... 67 A.3 DNAdvance Extraction ProtocolTM ...... 68 A.4 Puriﬁcation Protocol with AMPURE...... 69 A.5 Dasyatidae nucleotide sequences for MT-ATP6, MT-CYB and MT-ND2 for terminals used as outgroups...... 70 A.6 Cestode nucleotide sequences acquired from previous studies...... 71 A.7 GenBank nucleotide sequences of cestodes for MT-RNR2 and LSU regions.. 89 A.8 Original hosts nucleotide sequences...... 90 A.9 Original Acanthobothrium nucleotide sequences...... 96 List of Tables

1 Morphometric data obtained from specimens of Hypanus cf. guttatus from Es- tuary of Bay of Marajó, Colares, Pará, Brazil...... 13 2 Valid species of Acanthobothrium found in Northwest Atlantic and Neotropical freshwater river systems...... 22 3 Morphometric data for new species of Acanthobothrium described in present study...... 26 4 Prevalence for species of Acantobothrium ...... 48 List of Figures

1 Phylogenetic relationships among host individuals - Concatenated nucleotide data 11 2 General PCA...... 16 3 Phylogenetic hypothesis for Acanthobothrium ...... 18 4 New Acanthobothrium spp...... 23 5 Acanthobothrium n. sp. 1 morphology...... 24 6 Acanthobothrium n. sp. 1 SEM...... 25 7 Acanthobothrium n. sp. 2 morphology...... 31 8 Acanthobothrium n. sp. 3 morphology...... 35 9 Acanthobothrium n. sp. 3 SEM...... 36 10 Acanthobothrium n. sp. 3 cross sections...... 37 11 Scatter plot of attributes variation of Acanthobothrium n. sp. 3...... 38 12 Variation on bothridial morphology for Acanthobothrium n. sp. 3...... 39 13 Acanthobothrium n. sp. 4 morphology...... 43 14 Acanthobothrium n. sp. 4 SEM...... 44 15 Acanthobothrium n. sp. 4 cross sections...... 45 A.16 Hosts phylogenetic relationship - MT-ND2...... 93 A.17 Hosts phylogenetic relationship - MT-CYB...... 94 A.18 Hosts phylogenetic relationship - MT-ATP6...... 95

Introduction

Studies on metazoan parasitic fauna have been historically neglected (Windsor, 1995), despite 1 1 recent estimates suggesting that the diversity of parasitic lineages may account for 3 to 2 of the global biota (Poulin, 2014). The documentation of this diversity is important because it has the potential of revealing the historical and ecological events that could account for the diversification of these neglected groups (Adamson & Caira, 1994; Bush et al., 2001). However, in order to study the evolutionary processes responsible for the diversification of parasite lineages, the taxonomy and systematics of their hosts needs to be well developed. In addition, understanding the evolutionary history of associated parasites and their hosts makes co-evolutionary inference possible, which would allow us to elucidate and understand implicit historical processes such as patterns of distribution and host specificity (Bush et al., 2001; Page & Charleston, 1998). In the recent years, the documentation of that cestodes parasites of Neotropical freshwater stingrays (Myliobatiformes: Potamotrygonidae) has challenged the current paradigm of host specificity attributed to most cestode genera hosted by elasmobranchs (Cardoso Jr., 2010; Marques & Reyda, 2015; Reyda & Marques, 2011; Trevisan et al., 2017). The paradigm of specific cestodes species to a single host species, and exclusively to their occurrence area, has been questioned. Freshwater potamotrygonids host cestodes that vary widely in their degree of specificity, ranging from taxa that are found in more than 19 hosts species such as Acanthobothrium quinonesi Mayes, Brooks & Thorson, 1978 (Cardoso Jr., 2010), to cestodes that have been reported from a single species of freshwater stingrays. However, there are reports that some cestode lineages known to be restricted to marine batoids can infect freshwater stingrays in estuarine areas (Trevisan & Marques, 2017). It is unclear whether the patterns observed in potamotrygonids are the result of the complex history of colonization of freshwater habitats and diversification - in this case unique or particular to this system - or if our understanding of the specificity of marine assemblages is a taxonomic and/or sampling artifact (Reyda & Marques, 2011; Trevisan & Marques, 2017). In general, the cestode lineages found in freshwater potamotrygonids appear to be restricted to Neotropical river systems. However, unpublished data (Marques, com. pess.) reveal occurrences of Rhinebothroides Mayes, Brooks Thorson, 1981, a genus known only from Golfetti, Y. Dissertação de Mestrado, 2018 freshwater stingrays, in the marine stingray Hypanus guttatus Bloch & Schneider from Orinoco Delta and Caulobothrium Baer, 1948, a phyllobothridean genus that is restricted to marine batoids, in Potamotrygon yepezi Castex & Castello in Lake Maracaibo. Also, the marine Anindobothrium anacolum Marques, Brooks & Lasso, 2001 was found infecting the freshwater potamotrygonid Potamotrygon yepezi (Trevisan et al., 2017). These examples, provide some evidence that cross infections may occur in areas where the distribution of marine and freshwater rays overlap. In addition, a species of Rhinebothrium, R. jaimei Marques & Reyda, 2015, putatively related to marine species of Rhinebothrium, was described from Potamotrygon orbignyi Castelnau in the Bay of Marajó, the mouth of the Amazon basin. These findings make it difficult to assess if the cestode distribution and specificity in marine/estuarine environments are in fact different from the patterns observed in freshwater or if they are simply the result of poor sampling of estuarine regions. To date, the number of studies made of elasmobranch cestodes from estuarine regions are scarce. Cestodes that parasitize elasmobranchs have been studied from the American continent and adjacent marine region since the middle of 19th’ century. These studies mainly focused on documenting the diversity of cestode fauna and did not provide any analyses of the observed infection patterns. The majority of the studies in Brazil focused on cestodes infecting freshwater stingrays (Potamotrygoninae). The origin of Potamotrygoninae appears to have imposed a distinct pattern of diversification in its parasitic fauna. This unique group of freshwater batoids, now represented by about 30 species, distributed into four genera (Carvalho & Lovejoy, 2011; Carvalho & Ragno, 2011; Fontenelle et al., 2014; Froese & Pauly, 2018; Loboda & Carvalho, 2013; Rosa et al., 2008) share a common ancestor with the clade represented by two amphi-American species of Styracura (Carvalho et al., 2016). The freshwater potamotrygonids colonized the river systems of South America during marine incursions of the Paleogene period, more precisely between the lower Miocene and middle Eocene (22.5-46.0 mya; see Carvalho et al., 2004; Lovejoy et al., 1998; Marques, 2000). During this process of colonization, there is evidence that some typically marine cestodes lineages (e.g., Acanthobothrium Blanchard, 1948, Anindobothrium Marques, Brooks & Lasso, 2001 and Rhinebothrium Linton, 1889) accompanied the colonization and diversification of their hosts into inland waters, at the same time that new endemic lineages emerged during these events (e.g., Paroncomegas Campbell, Marques & Ivanov, 1999, Potamotrygonocestus Brooks & Thorson, 1976 and Rhinebothroides. Freshwater potamotrygonids, although generally credited as species restricted to river systems, tolerate low salinity (Thorson et al., 1983, 1978), and several species can be found in the estuaries of the largest Neotropical river basins (e.g., the Orinoco Delta , Maracaibo Lake and Bay of Marajó). In these environments, potamotrygonids and marine stingrays, mainly of the family Dasyatidae, share the same space and some food resources (Almeida et al., 2009;

2 Figueiredo, 1977; Lins, 2008). Sympatric distribution and shared trophic items are necessary, though not sufﬁcient, to allow for the shared occurrence of cestode species. This is because a cestode infection is acquired via food chain when the hosts consumes a food item that is infected with the larval stages of cetodes (Bush et al., 2001; Caira et al., 2012-2018). In this context, despite the advances in cestode studies in batoids of the West Atlantic and the Caribbean Sea, the relationship between marine and freshwater hosts that overlap in at least some of their geographic distribution and how their parasitic fauna is related to one another has been poorly explored. The estuarine region of the Bay of Marajó, State of Pará, Brazil, is an interesting area to study the cestode fauna of dasyatids. Bay of Marajó is characterized by having a large estuary with great variation in salinity. The tides and salinity levels are controlled by the discharge of the Amazonian rivers, which ﬂuctuates according to annual rainfall variation (Almeida et al., 2009). In this area, freshwater potamotrygonids co-occur with tree marine species of dasyatids - H. guttatus, Fontitrygon colarensis (Santos, Gomes & Charvet-Almeida) and Fontitrygon geijskesi Boeseman (Last et al., 2016; Marques & Reyda, 2015; Santos et al., 2004). Although the cestode fauna of the freshwater potamotrigonids species reported from this area are relatively well known (Bueno, 2010; Cardoso Jr., 2010; Marques & Reyda, 2015; Reyda & Marques, 2011), there is no documentation of the cestodes parasitizing the marine stingrays species, especially the dasyatids (Cardoso Jr., 2010; Machado, 2012; Marques & Reyda, 2015). In addition, there are no records of cestodes for F. colarensis and F. geijskesi. Only a few cestode genera, including Acanthobothrium, Rhinebothroides and Rhinebothrium, have been studied in estuarine areas (Bueno, 2010; Cardoso Jr., 2010; Machado, 2012; Marques & Reyda, 2015). From these, Acanthobothrium is a good candidate for analyzing species sharing and the relationships between marine and freshwater cestodes, and with their hosts. In addition, there is no known species of Acanthobothrium from batoids that occur off the Brazilian coast. Acanthobothrium is distributed worldwide and represent the most diverse genus of the Onchobotriidae, representing almost 76% of the species in the family (Caira et al., 2017; Campbell & Beveridge, 2002; Machado & Marques, 2012), with more than 195 nominal species described (Caira et al., 2012-2018). Members of this genus parasitize several species of Elasmobranchii: rays, skates (Myliobatiformes, Rajiformes, Rhinopristiformes and Torpediniformres) and sharks (Charchaniformes, Orectolobiformes, Heterodontiformes and Squaliformes) (Caira et al., 2017). However, most of the diversity of Acanthobothrium is found parasitizing batoids, as the genus has been reported for 21 of the 25 families of rays (Caira et al., 2017). Members of Acanthobothrium can be recognized by the morphology of their scolex. All species posses a scolex that is divided into four bothridia (large sucker-like structures), each of which is further subdivided by two horizontal septa resulting in three loculi.

3 Golfetti, Y. Dissertação de Mestrado, 2018

Each bothridia exhibits a pair of bipronged hooks, and an apical muscular pad bearing an apical sucker (Campbell & Beveridge, 2002; Machado & Marques, 2012). Recently five new species of Acanthobothrium were discovered in the Caribbean Sea (Trevisan, 2016), all of which were hosted by Styracura schmardae - a host previously studied by Brooks (1977). Trevisan (2016) recovered Acanthobothrium sp. 9 as sister-taxon to the freshwater lineage Acanthobothrium sp. 2 sensu Cardoso Jr., 2010. This clade is nested within a larger clade of marine species of Acanthobothrium from Senegal (Machado, 2012; Trevisan, 2016). This phylogenetic pattern could be interpreted as evidence of an evolutionary processes that resulted from multiple entries of marine cestodes taxa, such as Acanthobothrium, into the South American freshwater system. The Bay of Marajó, is one of the estuaries in the North of South America where conditions of co-existence of marine and freshwater stingrays could make this entries events possible to have occurred. Studies of the cestode fauna of batoid species that occur in estuaries have the potential to help elucidate the specific patterns of distribution and diversification of these parasites and their implications on historical patterns of association in both the marine and the freshwater systems.

General goals of the study

The lack of documentation of the cestode fauna of Dasyatidae stingrays from Bay of Marajó prohibits any further studies on their relationships with freshwater cestode lineages, which could possibly reveal a secondary invasion of Acanthobothrium into the South American freshwater system were the motivation for this study. Within this context, the goals of the present study are:

1. Describe the Acanthobothrium assemblage of Hypanus guttatus, Fontitrygon geijskesi and Fontitrygon colarensis from Bay of Marajó, and 2. Study the patterns of distribution and host speciﬁcity of Acanthobothrium.

4 Material and Methods

Collection of Biological Material

Specimens were collected between October 6th and 12th of 2016 in the Bay of Marajó, municipality of Colares, State of Pará, Brazil (0°55’ 53.8”S, 48°17’39.5”W), during the dry season, when marine waters flows into estuary bringing in marine stingrays. Stingrays were collected by fishermen using longlines. While still alive, the rays were anaesthetized, photographed with a scale, and then euthanized by cervical incision. Spiral intestines of Fontitrygon geijskesi (n=22) and Hypanus cf. guttatus (n=59) were collected (the specimens not recognized as F. geijskesi collected were initially assigned to H. cf. guttatus, see Results and Discussion), removed from hosts after an abdominal longitudinal incision and washed with a saline solution and cut in half. One half and the sift were fixed in hot 10% formalin solution and transferred to ETOH 70% for storage after 48 hours. The other half of the spiral intestine was fixed in ETOH 100% and kept at -20°C temperature. Of those collected hosts, 27 specimens of H. guttatus and 13 specimens of F. geijskesi were examined for cestodes.

Morphological Data

Specimen Preparation for light microscopy (permanent mounts):

All intestines and sift were examined under a stereoscope microscope for Acanthobothrium in the laboratory. Specimens selected for morphological analysis were serially hydrated, stained with Delaﬁeld’s hematoxylin, destained in acid an ethanol solution (1% HCl), followed by basic ethanol solution (1% NaOH), dehydrated by an ethanol series to 100%, cleared in methyl salicilate and mounted on glass slides with Canada’s balsam.

Specimen Preparation for Scanning Electron Microscopy (SEM):

Scoleces of selected specimens were removed from the strobila, hydrated in ethanol series, submerged in 1.5% osmium tetroxide for 12˜ hours, completely dehydrated using Golfetti, Y. Dissertação de Mestrado, 2018

Hexamethyldisilazane (HMDS) and ﬁxed on metallic supports (stubs). The strobila were mounted as SEM voucher, as described in the section above. Scoleces were examined with a Zeiss Sigma VP scanning electron microscope at the Instituto de Biociências – Universidade de São Paulo (IB-USP) for the identiﬁcation of microtriches. The terminology employed in describing microtriches follows Chervy (2009).

Specimen Preparation for Histological Sections:

Selected specimens had their mature proglottids cross-sectioned for further anatomical studies. For each individual worm, scolex and immature proglottids were mounted as described in the section above to serve as vouchers. Mature proglottids were prepared following conventional techniques: dehydrated by an ethanol series to 100%, cleared in xylene, included in parafﬁn medium and sectioned to 7휇 thickness using Leica RM 2025 retracting rotary microtome, placed in glass slides, stained in eosin and hematoxylin, and permanently mounted with Canada’s balsam.

Morphometric Data:

Morphological hook data and nomenclature follow Ghoshroy & Caira (2001). The hook handle base was named A for lateral hooks and A’ for medial hooks, inner or abaxial prong (distance from the extremity of the most internal prong in bothridia middle line to the anteriormost point of hook internal curve) B and B’, outer or abaxial prong (distance from the extremity of the most external prong in bothridia middle line to the anteriormost point of hook internal curve) C and C’, total hook length (from the apical portion of the handle to the extremity of the outer prong) D and D’. Morphometric data were obtained using the program Fiji (Schindelin et al., 2012) and compiled with WormBox (Vellutini & Marques, 2011–2014) plugin. Testes counts and measurement of total worm length were made under a light microscope with a standardized ocular ruler. All measurements provided in the descriptions are given in micrometers (휇m), unless otherwise stated and followed by the number of specimens from which the measurements were taken between parentheses. Loculi lengths ratio were calculated in reference to anterior loculus length (min-max). For the stingrays, it was considered all morphometric variables used by Santos et al. (2004) and Santos & Charvet-Almeida (2007) to distinguish F. colarensis from H. guttatus. These included preoral length (POL), disk width (DW), the ratio between preoral length and Disk width (POL/DW), right eye diameter (RED), left eye diameter (LED), eyes average diameter (EAD), rigth eye diameter for preoral length (RED/POL), left eye diameter for preoral length (LED/POL), and eye average diameter for preoral length (EAD/POL). Specimens were divided into male and female to account for the sexual dimorphism between F. colarensis specimens

6 (Santos et al., 2004; Santos & Charvet-Almeida, 2007). Were analyzed using a Principal Components Analyses (PCA) the variables: POL/DW (%), RED/POL (%), LED/POL (%), EAD/POL (%) and sex. PCA was performed using a script for R (Appendix A.1).

Ecological Index:

Prevalence index (P%) were calculated according to Bush et al. (1997).

Molecular Data

DNA Extraction and ampliﬁcation of host tissue:

For molecular identification of hosts, an abdominal muscle portion was taken of each host and fixed in ETOH 100% in the field. An aliquot of each muscle sample was was used for

DNA extraction using the Ammonium Acetate (푁퐻4퐶퐻3퐶푂2) protocol (Appendix A.2). The extractions were quantified using NanoDrop 2000 (Thermo Scientific) and amplified using Polymerase Chain Reaction (PCR). Each PCR cycle had initial denaturation for 5 minutes at 95°C, 35 cycles of denaturations for 30 seconds at 95°C, annealing for 30 seconds at specific temperature depending on primers (see below), 1 minute to 1 minute 10 seconds extension at 72°C, and a final extension for 7 minutes at 72°C. The amplification and sequencing were performed with the follow primer sets: cytochrome b (MT-CYB) with primers CB1-5’F 3’ – CCA TCC AAC ATC TCC ACT TGA TGA AA-5’ and CB3-3’R 3’ – GGC AAA TAG GAA RTA TCA TTC – 5’ at 57°C, ATP synthase membrane subunit 6 (MT-ATP6) with primers COX2MODF 3’ – CGG ACA GTG TTC AGA AAT CTG TGG – 5’ and COX3MODR 3’ – GGT CAT GGG CTG GGG TCA ACT ATG – 5’ at 49°C, and for NADH: ubiquinone oxidoreductase core subunit 2 (MT-ND2) with primers ILEMF 5 –AAG GAG CAG TTT GAT AGA GT – 5’ and ASNMR 3’ – AAC GCT TAG CTG TTA ATT AA – 5’ at 55°C.

DNA Extraction and ampliﬁcation of Acanthobothrium:

Cestodes fixed in ETOH 100% were split into 3 sections: scolex, posterior portion and middle portion. Scolex and posterior portion were mounted as hologenophores vouchers (sensu Pleijel et al., 2008), as described above. DNA was extracted from the middle portions of strobila. Extractions were performed using Agentcourt DNAdvanded – Nucleic Acid Isolation Kit (Beckman Coulter), following manufacturer’s instructions (Appendix A.3) and quantified using NanoDrop 2000 (Thermo Scientific). Polymerase Chain Reaction was used to amplify the D1-D3 regions of the nuclear 28S cytoplasmatic ribosomal RNA (LSU) and mitochondrially encoded 16S RNA (MT-RNR2).

7 Golfetti, Y. Dissertação de Mestrado, 2018

Amplifications for cestode specimens were performed in a 25 휇l volume containing 1 휇l of DNA, 200 mM Tris-HCl (pH 8.4), 50 mM Kcl, 200 1M dNTPs, 1.0-3.0 nM MGCl2, 0.4 1M of each primer, 1U of Taq DNA polymerase recombinant (Fermantas, Thermo Scientific). For each PCR cycle included initial denaturation for 5 minutes at 95°C, 35 cycles of denaturations for 30 seconds at 95°C, annealing for 30 seconds at specific gene temperature (see below), 1 minute to 1:10 minutes for extension at 72°C, and final extension for 7 minutes at 72°C. The amplification and sequencing were performed with the follow primer sets: LSU with LSU-5F 3’ – TAG GTC GAC CCG CTG AAY TTA AGC A – 5´ and LSA-1500R 3’– TGC CTT TTG CAT CAT GCT –5’ at 58°C, MT-RNR2 with 16S-F 3’ – TGC CTT TTG CAT CAT GCT – 5’, Cyclo-16SR 3’ – AAT AGA TAA GAA CCG ACC TGG – 5’ at 55°C. PCR products were purify using an Agencourt AMPuret XP DNA Purification and Cleanup kit (Beckman Coulter) (Appendix A.4). All PCR products, for parasites and hosts, were purified using an Agencourt AMPuret XP DNA Purification and Cleanup kit (Beckman Coulter). Products were subsequently cycle-sequenced directly from both forward and reverse directions using ABI Big-Dye Sequence Terminator version 3.1, cleaned with ethanol precipitation, and sequenced on an ABI Prism Genetic Analyzer (3131XL) automated sequencer (Applied Biosystems/ThermoFisher). Contiguous sequences were assembled using the package Consed/PhredPhrap (Ewing & Green, 1998; Ewing et al., 1998; Gordon et al., 1998, 2001). Finally, sequences were aligned using MAFFT with options –maxiterate 1000 –globalpair (v7.271; Katoh et al., 2002) and edited in BioEdit (version 7.1.3.0; Hall, 1999) to remove leading and trailing gaps resulted from differential sequencing.

Phylogenetic inference:

The phylogenetic analyses of host sequences considered partial sequences of 18 terminals as outgroups, 14 sequences for mitochondrially encoded NADH ubiquinone oxidoreductase core subunit 2 (MT-ND2) (Hypanus dipterurus (Jordan Gilbert) [1], Hypanus americanus (Hildebrand Schroeder) [2], Hypanus sabinus (Lesueur) [1], Fontitrygon margaritella (Compagno Roberts) [1], Fontitrygon margarita (Günther) [1], Bathytoshia brevicaudata (Hutton) [1] and Bathyoshia centroura (Mitchill) [7]), four sequences for cytochrome b (MT- CYB) (Neotrygon kuhlii (Müller Henle) [1], Dasyatis brevis (Garman) [1], Hemitrygon akajei (Müller Henle) [1] and H. dipterurus [2]), and three sequences for ATP synthase membrane subunit 6 (MT-ATP6) (N. kuhlii [1], D. brevis [1] and H. akajei [1]) A.5). As ingroup, it was considered 43 specimens sequences of dasyatids from Bay of Marajó: 43 sequences for MT- ND2 (H. cf. guttatus [29] and F. geijskesi [14]), 39 sequences for MT-CYB (H. cf. guttatus [27] and F. geijskesi [12]) and 40 sequences for MT-ATP6 ((H. cf guttatus [26] and F. geijskesi [14])

8 Sequences were concatenated using SequenceMatrix (Vaidya et al., 2010) and the phylogenetic analysis was performed with TNT (Goloboff et al., 2008, 2008-2018) with 100 replicates of TNT’s new technology searches (i.e., xmult). Nodal support of selected nodes was estimated using the Goodman-Bremer values (Bremer, 1988, 1994; Goodman et al., 1982; Grant & Kluge, 2008a). The phylogenetic analyses of cestode sequences included 86 specimens collected for this study and 137 terminals from the most recent phylogenetic analysis of the genus by Trevisan (2016) (see details in Appendix A.6). The dataset included members of Acanthobothrium from all major body waters of the world. The outgroup was composed by Onchoproteochephalidea sequences obtained from NCBI GenBank (n=10). Also, Acanthobothrium sequences were acquired from NCBI GenBank to compose the ingroup (n=10) (Appendix A.7). Sequences of MT-RNR2 and LSU were submitted to phylogenetic analysis by direct optimization (DO, Wheeler, 1996) as implemented in POY (version 5.1.1; Varón et al., 2010) using parsimony as the optimality criteria. Direct optimization was selected because these regions require alignment. Prior to analyses, nucleotide sequences were visualized and partitioned in BioEdit (version 7.1.3.0; Hall, 1999). Initial tree searches included 10 iterations of two independent searches for 1 h 30min using the command search [i.e., search(maxtime:0:01:30)] assuming equal weights for all character transformations and no gap opening cost (gap opening:0). This search was conducted in a 10 X 2.83 GHz Intel ® CoreTM2 Quad Processor Q9550 computer cluster. After compiling candidate trees by DO, re-diagnosis was performed using iterative pass alignment (DO/IP; Wheeler, 2003a). Finally, the results of POY were veriﬁed by performing a phylogenetic analysis of the implied alignment (sensu Wheeler, 2001b) generated by POY in TNT (Goloboff et al., 2008, 2008-2018) using its new technology searches (Goloboff, 1999; Nixon, 1999) with 100 replications, 50 iterations of ratchet, 50 iterations of tree fusing and saving no more than 20 trees per replication (e.g., xmult: rep 100 ratchet 50 fuse 50 hold 20).

9 Results

Host Identiﬁcation

Molecular data

Twenty six specimens of H. cf. guttatus were sequenced for MT-CYB, 28 for MT-ATP6 and 25 for MT-ND2 (Appendix A.8). Fourteen specimens of F. geijskesi were sequenced for MT- CYB, 15 for MT-ATP6 and 14 for MT-ND2 (Appendix A.8). Unaligned sequences of MT-CYB varied from 565 to 1238 base pairs (bp) in length, MT-ATP6 varied from 854 to 1161 bp, and MT-ND2 varied from 569 to 1331 bp. Once aligned and trimmed to remove regions with leading and trailing gaps due to differential sequencing, the dataset included 565 bp of MT-CYB, 853 bp of MT-ATP6 and 990 bp of MT-ND2. Hence, the concatenated molecular matrix included 54 terminal and 2408 nucleotide characters. As presented in the tree resulted from the phylogenetic analysis of the concatenated sequences of MT-CYB, MT-ATP6 and MT-ND2 genes (Fig.1), H. cf. guttatus and F. geijskesi resulted as monophyletic groups with 30 and 35 Goodmand-Bremmer support (GBS) values, respectively. The clade H. cf guttatus+F. geijskesi are nested with GBS support of 17. Single genes trees (Appendices A.16, A.17, A.18) replicated the patterns presented in Fig.1. The area of study is the type locality of Fontitrygon colarensis. However, it was not possible to identify this species in the ﬁeld given the ambiguous nature of the diagnostic features of this species in comparison to Hypanus guttatus. The phylogenetic pattern found within the clade comprised by specimens that were initially identiﬁed as H. cf guttatus suggested two internal clades (A and B, Fig.1). We used those to clades to verify whether it was possible to distinguish them morfologically, which would allow us to recognize individuals of Fontitrygon colarensis in the samples (see bellow).

Morphological data

Fifty nine specimens, initially identiﬁed as H. cf guttatus, were measured with the purpose of identifying members of F. colarensis. The results of morphometric measurements of hosts OUTGROUPS

Hypanus guttatus (PA16-81) Hypanus guttatus (PA16-32) 30 Hypanus guttatus (PA16-50) Hypanus guttatus (PA16-01) Hypanus guttatus (PA16-58) 1 Hypanus guttatus (PA16-82) Hypanus guttatus (PA16-25) Hypanus guttatus (PA16-57) Hypanus guttatus (PA16-40) Hypanus guttatus (PA16-44) Hypanus guttatus (PA16-87) Hypanus guttatus (PA16-77) B Hypanus guttatus (PA16-62) Hypanus guttatus (PA16-02) Hypanus guttatus (PA16-80) Hypanus guttatus (PA16-53) 17 Hypanus guttatus (PA16-75) Hypanus guttatus (PA16-88) Hypanus guttatus (PA16-83) 1 Hypanus guttatus (PA16-37) Hypanus guttatus (PA16-11) Hypanus guttatus (PA16-91) Hypanus guttatus (PA16-07) A Hypanus guttatus (PA16-68) Hypanus guttatus (PA16-78) Hypanus guttatus (PA16-34) Hypanus guttatus (PA16-67) Hypanus guttatus (PA16-74) Fontitrygon geijskesi (PA16-72) Fontitrygon geijskesi (PA16-42) 35 Fontitrygon geijskesi (PA16-45) Fontitrygon geijskesi (PA16-52) Fontitrygon geijskesi (PA16-26) Fontitrygon geijskesi (PA16-60) Fontitrygon geijskesi (PA16-61) Fontitrygon geijskesi (PA16-48) 20.0 Fontitrygon geijskesi (PA16-28) Fontitrygon geijskesi (PA16-66) Fontitrygon geijskesi (PA16-41) Fontitrygon geijskesi (PA16-79) Fontitrygon geijskesi (PA16-49) Fontitrygon geijskesi (PA16-59) Fontitrygon geijskesi (PA16-76)

Figure 1: Phylogenetic relationships among host individuals based on the phylogenetic analysis under parsimony for the concatenated nucleotide data (MT-ND2, MT-CYB,and MT-ATP6; outgroup terminals omitted). Contents between parentheses refer to host accession code (Cestode Database). Numbers above branches refer to Goodman-Bremer support values for selected nodes. Scale bar represent number of transformations. 11 Golfetti, Y. Dissertação de Mestrado, 2018 initially identified as H. cf. guttatus, based on differential attributes to F. colarensis are presented in Table1. Only one specimen (PA16-007, Table1) exhibited right eye diameter/preoral length and eye average diameter/preoral length values in the range attributed to F. colarensis according to Santos et al. (2004). All other specimens possessed measurements within the range of H. guttatus. For preoral length/disk width, five male specimens (PA16-012, PA16-022, PA16- 033, PA16- 054 and PA16-082) were within the range proposed for H. guttatus by Santos et al. (2004), while 6 females specimens (PA16-007, PA16-027, PA16-029, PA16-030, PA16- 048, and PA16-050) showed measurements for POL/DW (%) dimensions described to for females of F. colarensis (Santos & Charvet-Almeida, 2007). All other specimens, males and females, are out of range of both species (n=57) for preoral length/disk width dimensions, Table 1). Finally, no specimens presented consistent morphometric values to either species for all variables considered to be of taxonomic importance by Santos et al. (2004). In an attempt to find morphological information that might be used to segregate these two nominal species, it was performed a Principal Component Analysis (PCA) of morphometric variables collected from host images. In this analysis, we color coded each individual reflecting the phylogenetic pattern found in Fig.1 for clade A (blue) and B (red). The rational was to seek congruence between phylogenetic signal (Fig.1) and morphometrical data (Table1). All remaining specimens, that is, those excluded from molecular analysis and those included but not nesting in any particular clade within H. cf. guttatus were coded in black (Fig.2). The Principal component analysis (PCA) dataset was based on four ratios, which were considered to be useful to distinguish these two species (Table1) by Santos et al. (2004) and Santos & Charvet-Almeida (2007). The results indicate that there is no congruence between cladistic structure and morphometric data. Specimens that nested in clade A (blue) and B (red) did not form any recognizable cluster as other individuals for which there were no molecular data. Based on these results, specimens of Clade A (blue), Clade B (red) and the remaining specimens sampled for this study (black) can not be distinguished morphologically. Hence, molecular and morphological data favors the hypotheses that only a H. guttatus was collected in the present study.

12 Table 1: Morphometric data obtained from specimens of Hypanus guttatus from Estuary of Bay of Marajó, Colares, Pará. Brazil. Measurements are presented in cm, except for POL/DW (%), RED/POL (%), LED/POL (%) and EAD/POL (%), which are percentages. Numbers in black indicate values congruent with ranges attributed to Hypanus guttatus; numbers in blue indicate values congruent with ranges attributed to females of Fontitrygon colarensis; and in red, for males of this species according to Santos et al. (2004) and Santos & Charvet-Almeida (2007). Legend: POL: Preoral length, DW: Disk width, POL/DW (%): Preoral length for Disk width, RED: Rigth eye diameter, LED: Left eye diameter, EAD: Eyes average diameter, RED/POL (%): Rigth eye diameter for preoral length, LED/POL (%): Left eye diameter for preoral length, and EAD/POL (%): Eye average diameter for preoral length.

Host code Sex POL DW POL/DW (%) RED LED EAD RED/POL (%) LED/POL (%) EAD/POL(%)

PA16-001 M 13.30 51.66 25.74 2.22 2.30 2.26 16.71 17.32 17.01 PA16-002 M 15.10 55.71 27.10 2.32 2.48 2.40 15.37 16.40 15.89 PA16-003 F 17.99 NA NA 2.41 2.83 2.62 13.41 15.75 14.58 PA16-004 F 13.35 49.82 26.79 2.14 2.08 2.11 16.03 15.55 15.79 PA16-005 F 14.47 56.33 25.69 2.58 2.43 2.51 17.86 16.78 17.32 PA16-007 F 29.17 95.97 30.39 2.47 3.35 2.91 8.47 11.48 9.98 PA16-008 M 14.77 49.71 29.72 2.38 2.51 2.45 16.13 17.00 16.56 PA16-011 F 14.97 55.20 27.11 2.31 2.12 2.22 15.47 14.15 14.81 PA16-012 M 11.09 43.85 25.30 1.88 2.01 1.95 16.99 18.09 17.54 PA16-013 M 12.43 42.70 29.10 2.19 2.11 2.15 17.61 17.00 17.31 PA16-014 M 13.28 46.14 28.78 2.29 2.03 2.16 17.26 15.32 16.29 PA16-015 M 12.78 47.86 26.70 2.12 1.98 2.05 16.63 15.47 16.05 PA16-022 F 13.74 54.03 25.43 2.30 2.13 2.21 16.74 15.47 16.10 PA16-023 F 13.45 52.36 25.70 2.52 2.67 2.59 18.74 19.81 19.28 PA16-024 M 14.91 51.53 28.94 2.11 2.25 2.18 14.15 15.08 14.61 PA16-025 M 12.88 48.63 26.49 2.15 2.11 2.13 16.72 16.39 16.55 PA16-030 F 20.48 75.55 27.11 3.03 2.51 2.77 14.80 12.26 13.53

Continued on next page Table 1 – Continued from previous page Host code Sex POL DW POL/DW (%) RED LED EAD RED/POL (%) LED/POL (%) EAD/POL(%)

PA16-031 F 15.22 54.96 27.70 2.49 2.28 2.39 16.37 14.98 15.67 PA16-032 M 15.19 54.40 27.92 2.08 1.99 2.03 13.69 13.09 13.39 PA16-033 M 11.23 46.36 24.22 2.31 2.33 2.32 20.57 20.72 20.64 PA16-034 F 14.06 53.39 26.34 1.93 2.40 2.16 13.69 17.05 15.37 PA16-035 M 17.35 60.03 28.90 2.49 2.17 2.33 14.38 12.50 13.44 PA16-036 M 12.69 48.28 26.28 2.49 1.84 2.17 19.67 14.50 17.08 PA16-037 M 12.39 44.03 28.15 1.85 1.89 1.87 14.91 15.27 15.09 PA16-038 F 20.77 74.27 27.96 3.22 2.75 2.99 15.53 13.23 14.38 PA16-039 F 18.80 63.87 29.43 2.87 2.30 2.58 15.25 12.24 13.75 PA16-040 F 17.96 62.35 28.80 3.02 2.61 2.81 16.84 14.51 15.67 PA16-043 F 23.36 75.58 30.91 3.13 2.61 2.87 13.39 11.18 12.28 PA16-044 F 19.67 63.75 30.85 2.54 2.39 2.47 12.92 12.15 12.54 PA16-050 F 16.30 59.91 27.20 2.97 2.73 2.85 18.22 16.76 17.49 PA16-053 F 21.51 79.75 26.98 2.36 2.96 2.66 10.97 13.74 12.36 PA16-054 M 17.16 68.76 24.96 2.98 2.66 2.82 17.39 15.52 16.45 PA16-055 F 17.73 67.01 26.45 2.56 2.50 2.53 14.43 14.12 14.28 PA16-056 M 13.64 52.02 26.22 2.28 2.29 2.28 16.68 16.81 16.75 PA16-057 M 14.15 51.51 27.47 2.39 2.51 2.45 16.92 17.71 17.32 PA16-058 M 12.99 47.92 27.10 1.96 2.26 2.11 15.07 17.44 16.25 PA16-062 F 17.16 62.87 27.29 2.72 2.88 2.80 15.88 16.79 16.33 PA16-063 M 12.68 46.68 27.15 2.51 2.15 2.33 19.80 16.96 18.38 PA16-064 M 14.54 52.67 27.62 2.27 1.90 2.08 15.58 13.08 14.33 PA16-065 M 15.22 52.06 29.23 2.41 2.16 2.29 15.84 14.19 15.02

Continued on next page Table 1 – Continued from previous page Host code Sex POL DW POL/DW (%) RED LED EAD RED/POL (%) LED/POL (%) EAD/POL(%)

PA16-067 F 17.23 60.31 28.57 2.59 2.61 2.60 15.01 15.18 15.09 PA16-068 M 16.46 58.06 28.35 2.11 2.30 2.20 12.84 13.94 13.39 PA16-069 M 13.32 45.41 29.33 2.32 2.12 2.22 17.45 15.91 16.68 PA16-070 M 14.21 46.13 30.81 1.76 2.08 1.92 12.36 14.62 13.49 PA16-071 F 22.25 78.07 28.49 3.56 3.21 3.39 16.02 14.44 15.23 PA16-074 F 20.59 72.84 28.27 3.21 2.27 2.74 15.59 11.02 13.30 PA16-075 F 26.82 89.31 30.03 3.52 3.59 3.55 13.13 13.38 13.25 PA16-077 F 17.60 62.89 27.98 2.34 2.37 2.35 13.31 13.45 13.38 PA16-078 F 21.08 69.75 30.22 2.55 2.33 2.44 12.11 11.04 11.58 PA16-080 M 15.94 57.91 27.53 2.44 2.44 2.44 15.33 15.31 15.32 PA16-081 M 16.70 56.09 29.77 2.20 2.41 2.30 13.18 14.41 13.80 PA16-082 M 15.32 60.22 25.43 2.73 2.42 2.58 17.82 15.81 16.81 PA16-083 M 17.02 57.38 29.67 2.45 2.61 2.53 14.42 15.36 14.89 PA16-084 M 15.92 57.40 27.74 2.31 2.54 2.43 14.51 15.96 15.24 PA16-087 M 14.56 47.45 30.69 2.47 2.40 2.43 16.93 16.47 16.70 PA16-088 M 14.10 53.07 26.56 2.36 2.21 2.29 16.74 15.68 16.21 PA16-089 M 14.98 51.81 28.92 2.21 2.09 2.15 14.73 13.96 14.34 PA16-090 M 12.34 46.23 26.69 2.43 2.29 2.36 19.73 18.52 19.13 PA16-091 M 12.62 45.32 27.85 2.08 2.72 2.40 16.50 21.58 19.04 Golfetti, Y. Dissertação de Mestrado, 2018

PA16−087

PA16−008 PA16−091

PA16−013

PA16−069 POLDW

PA16−070

PA16−083

PA16−014

PA16−044

− PA16 090 PA16−065 PA16−081 PA16−043 − PA16−063 PA16 040 PA16−075 PA16−057 LEDPOL EADPOL PA16−024 PA16−050 PA16−067

PA16−071 REDPOL PA16−089 PA16−039 PA16−023

PA16−037 PA16−062

PA16−078 PA16−058 PA16−031 PA16−084

PC 2 (13% explained var.) PC 2 (13% explained PA16−035

− PA16−080 PA16−003 PA16−033 PA16 002

PA16−007 PA16−025 PA16−038 PA16−068

− PA16 056 − PA16 015 − PA16−036 PA16−088 PA16 004 0.5 0.0 0.5 1.0 1.5 2.0

− PA16−005 PA16−064 PA16−001 PA16−077 − − PA16 012 − PA16 074 PA16 011 PA16−032

PA16−034

PA16−082 1.0 −

− PA16−022 PA16 030 PA16−055

PA16−054

PA16−053 1.5 −

−4 −2 0 2 4

PC 1 (77% explained var.)

Figure 2: General Principal Component Analysis (PCA) for morphometic data of selected hosts. Plotted individuals: red, clade A from Fig.1; blue, clade B from Fig.1; and black, specimens not sequenced and those not forming clades. Axes of the variables POL/DW (%): Preoral length for Disk width (in percentage), RED/POL (%): Rigth eye diameter for preoral length (in percentage), LED/POL (%): Left eye diameter for preoral length (in percentage), and EAD/POL (%): Eye average diameter for preoral length (in percentage) show dots displacement.

Phylogenetic Analysis of Acanthobothrium

Unaligned sequences of MT-RNR2 ranged from 283 to 561 bp in length, and sequences of LSU ranged from 424 to 950 bp. Aligned and trimmed sequences of MT-RNR2 and LSU resulted in datasets with 610 and 1076 bp, respectively. These regions were sequenced for 86 specimens of Acanthobothrium (Appendix A.9), which once added to Trevisan’s (2016) dataset resulted in a matrix with 236 terminals. The initial tree search by direct optimization completed 1075 random addition sequence (RAS), 2228 Tree Fusing and 407 Ratchets after 10 iterations. This analysis compiled 160 unique candidate trees raging from 6529 to 6614 steps in length. The re-diagnose of these candidate trees under IP found 3 equally parsimonious trees (MPTs) with 6500 steps for which the implied alignment consisted of sequences with 2019 bp. The reanalysis of the implied alignment in TNT (New technologies) resulted in 636 MPTs with 6496 steps, which strict

16 consensus is presented in Figure3. The phylogenetic analyses indicated the presence of six new different lineages (Fig.3). Of those new lineages, six are present in H. cf. guttatus, while four are hosted by F. geijskesi (except Acanthobothrium sp. 10 and Acanthobothrium sp. 11). Only the new taxon Acanthobothrium n. sp. 4 (blue in Fig.3) was recognized based on morphological data alone. Acanthobothrium n. sp. 1 and Acanthobothrium n. sp. 2 (purple and green in Fig. 3, respectively). Finally, Acanthobothrium n. sp. 3 is presented in red in Fig.3. All new lineages discovered in present study, except for Acanthobothrium sp. 10, are exclusively associated with marine clades of the Caribbean sea and the Brazilian coast. Acanthobothrium n. sp. 1 was recovered as sister group of the clade that includes the polyphyletyic Acanthobothrium sp. 5 sensu Trevisan (2016) ([b033/PN15-56.4] / [b058/TT14-06.5]) and Acanthobothrium sp. ex H. guttatus from Belize ([A164/BE-12-42]). Acanthobothrium n. sp. 2 is sister to an undescribed new lineage Acanthobothrium sp. 11. Acanthobothrium n. sp. 4 resulted as sister to the undescribed Acanthobothrium sp. ex Hypanus guttatus [A064/AL10-004.01] from Alagoas (Fig.3). The new taxon Acanthobothrium n. sp. 3 is sister to a big Northwest clade composed of the undescribed and newly discovered lineages of Acanthobothrium. Acanthobothrium n. sp. 3 is sister group of a clade that includes Acanthobothrium n. sp. 1 and nested species (described above), Acanthobothrium n. sp. 2, Acanthobothrium sp. 11, Acanthobothrium n. sp. 4 and Acanthobothrium sp. ex Hypanus guttatus [A064/AL10-004.01]. The new lineage Acanthobothrium sp. 10 is nested to a clade composed by marine Acanthobothrium sp. 9 sensu Trevisan (2016) and freshwater Acanthobothrium sp. 2 sensu Cardoso Jr. (2010). Despite the recognition of 6 lineages of Acanthobothrium, for only four lineages there was enough morphological material to provide taxonomic descriptions. This is because, there was only one specimen of Acanthobothrium sp. 10 found and included in the molecular study (Y078, Appendix A.9) and only two specimens of Acanthobothrium sp. 11 that were found and used likewise (Y050 and Y077, Appendix A.9), both immature worms. Based on these results, four new species of Acanthobothrium are described below. Of the six new lineages detected in present study, four are found in both hosts. Just Acanthobothrium sp. 10 and Acanthobothrium sp. 11 were found exclusively to H. guttatus. In addition, the prevalence of each putative species varied greatly. For instance, some lineages presented low prevalence, such as Acanthobothrium n. sp. 2 with 10.71% in Hypanus guttatus and 7.69% in Fontitrygon geijskesi; and Acanthobothrium n. sp. 4 showed 3.57 in Hypanus guttatus.

17 Golfetti, Y. Dissertação de Mestrado, 2018 M M M FW FW [AC07/BE-3] [b084/PN15-12.01] (Panama) spp. CLADE I sensu Cardoso Jr., 2010 [b033/PN15-56.4] [b058/TT14-06.5] Styracura schmardae Styracura pacifica ex ex sensu Trevisan, 2016 spp. ex Potamotrynidae Acanthobothrium sensu Trevisan, 2016 sensu Trevisan, 2016 sensu Trevisan, 2016 sensu Trevisan, 2016 sensu Trevisan, 2016 Potamotrygon schroederi [A064/AL10-004.01] [A164/BE-12-42] sensu Trevisan, 2016 Acanthobothrium sp. 2 ex [AC09/TM-9] Styracura pacifica Styracura schmardae CLADE II (ETP) Acanthobothroides thorsoni Hypanus guttatus Hypanus Fontitrygon geijskesi Fontitrygon Styracura pacifica Acanthobothroides pacificus Hypanus guttatus Hypanus Hypanus guttatus Hypanus Hypanus guttatus Hypanus Hypanus sabinus Hypanus guttatus Hypanus sp. 6 ex sp. 9 ex Styracura schmardae Styracura schmardae Hypanus guttatus Hypanus guttatus ex Styracura schmardae sp. 10 ex Styracura schmardae sp. ex n. sp. 4 ex 4 ex sp. n. sp. n. 8 ex CLADE III (ETP) n. sp. 1 ex Styracura schmardae Acanthobothrium n. sp. 3 ex 3 ex sp. n. sp. 11 ex n. sp. 2 ex 2 ex sp. n. sp. ex sp. ex Hypanus sabinus sp. 5 ex sp. 5 ex Acanthobothrium sp. ex sp. 1 ex himanturi sp. 3 ex based on the simultaneous analysis of MT-RNR2 and LSU, by direct Acanthobothrium CLADE IV (ETP) Acanthobothrium CLADE VI (ETP) Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium Onchobothrium? ex Acanthobothrium Acanthobothrium Styracura schmardae CLADE VII (ETP) Acanthobothrium Raja miraletus Acanthobothrium CLADE VIII (ETP) Acanthobothrium CLADE IX (ETP) CLADE X (ETP) sp. ex Acanthobothrium Acanthobothrium 60.0 Acanthobothrium Acanthobothrium Acanthobothrium Acanthobothrium [AC05/AF-72] [EU660532/-]* Dasyatis say ex Torpedo fuscomaculata OUTGROUP sp. ex . Phylogenetic relationships among lineages of Acanthobothrium brevissime Acanthobothrium optimization, regions usingfor parsimony vouchers. as the BoldAcanthobothrium optimality names criteria. represent new Contents lineages between recognized brackets in represent present molecular study. codes Sidebar and/or indicating accession freshwater number taxa (FW) and marine taxa (M) of Figure 3:

18 Taxonomic Actions

Acanthobothrium n. sp. 1

(Figs.5 and6)

Description (Based on 18 complete worms, 19 hologenophores and two scoleces observed with SEM): Worms acraspedote (Fig.4 A, Table3), euapolytic, 1.42–3.03 mm (n=17) long, with of 5–10 (n=17) proglottids. Scolex consisting of scolex proper and cephalic peduncle (Fig.5A,6 A). Cephalic peduncle 128–431 (n=13) long by 50–173 (=13) wide, covered with gladiate spinitriches with approximately 1–2 microtriches/휇푚2 (Fig.6 B). Scolex proper 205–303 (n=18) long by 131–221 (n=17) in maximum width (at level of anterior loculus), with four bothridia, bothridia 156–236 (n=18) long by 65–108 (n=19) in maximum width. Proximal surface of bothridia covered by gladiate microtriches 1–2 microtriches/휇푚2 and acicular filitriches 0.08 in diameter, approximately 20–22 microtriches/휇푚2 (Fig.6 C). Each bothridia with anterior muscular pad and three loculi divided by two horizontal septa. Anterior loculus 77–140 (n=18) long, middle loculus 26–50 (n=18) long, and posterior loculus 25–45 (n=17) long; loculi length ratio (A:M:P) 1:0.26–0.58:0.21–0.39 (n=17). Distal surface of bothridia and inter-loculi septa covered with papiliform filitriches with 0.08 in diameter, approximately 103- 106 microtriches/휇푚2 (Fig.6 D). Muscular pad 39–68 (n=18) long by 52–94 (n=18) wide, covered by papiliform filitriches with 0.09 in diameter, triangular shape and approximately 176–189 microtriches/휇푚2 (Fig.6 E). Bearing an apical sucker 9–25 (n=15) long by 12–21 (n=14) wide, and one pair of triangular hooks below posterior margin (Fig.5 B). Velum not present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surface of axial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongs and abaxial prongs with different lengths; lateral and medial hooks of different sizes (Fig.5 B). Lateral hook measurements: A 2–33 (n=34), B 41–68 (n=32), C 40–65 (n=33), D 61–93 (n=32). Medial hook measurements: A’ 15–28 (n=34), B’ 42–72 (n=32), C’ 39–64 (n=32) and D’ 58–94 (n=32). Medial hook base wider than lateral hook base. Thin layer of tissue anteriorly covering each prong of both set of hooks. (Fig.6 F). Immature proglottids wider than long; 4–9 (n=18) in number (Fig.5 C). Terminal mature proglottids longer than wide; 1–2 (n=18) in number, 364–1278 (n=17) long by 149–373 (n=17); mature proglottid length to width ratio 0.14–0.34 (n=17) (Fig.5 D). Genital pores irregularly alternating, 31-50% (n=34) from anterior end of proglottid. Some terminal proglottids with sperm-filled vas deferens. Gravid proglottids and eggs not observed. Testes round to elliptical, 22–55 (n=24) long by 32–60 (n=24) wide, arranged in two regular columns, extending from anterior region of proglottid to ovarian isthmus, 23–38 (n=27) in total number, 5–13 (n=28) pre-poral, 4–7 (n=29) post-poral and 13–20 (n=28) aporal. Cirrus sac pyriform 81–193 (n=33) long by 47–217 (n=33) wide,

19 Golfetti, Y. Dissertação de Mestrado, 2018 containing eversible cirrus armed with spinitriches. Vagina thick-walled, sinuous, extending from ootype along medial line of proglottid to anterior margin of cirrus sac to common genital atrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetrical inverted A-shaped in frontal view, lobulated, reaching or almost reaching posterior margin of cirrus sac. Poral arm 81–697 (n=33) long, aporal arm 124–674 (n=33) long by 45–156 (n=33) wide at isthmus. Vitellarium follicular in narrow lateral bands, each with 1–2 visible rows of follicles, extending from the ﬁrst line of distal testes to the ovarian isthmus, interrupted by vagina and cirrus sac dorsally and ventrally, not interrupted by ovary. Uterus linear, median, extending from ovarian isthmus to vagina level.

Taxonomic summary Type-host: Hypanus guttatus (Bloch & Schneider) Additional hosts: Fontitrygon geijskesi (Boeseman). Type-locality: Bay of Marajó, Colares - PA - Brazil (ca. 0°55’53.8”S-48°17’39.5”W). Site of infection: Spiral intestine. Specimens deposited: ### Prevalence: 44.44% in Hypanus guttatus and 7.69% in Fontitrygon geijskesi.

Remarks: The results of the phylogenetic analyses (Fig.3) suggested that Acanthobothrium n. sp. 1 is closely related to three undescribed lineages of the genus found in the Caribbean: the polyphyletic Acanthobothrium sp. 5 sensu Trevisan (2016) ([b033/PN15-56.4] / [b058/TT14- 06.5]) and Acanthobothrium sp. found in H. guttatus from Belize ([A164/BE-12-42]). Acanthobothrium n. sp. 1 can be easily distinguished from Acanthobothrium sp. 5 by its smaller total length (1.4-3.0 mm vs. 3.4–12.4 mm, respectively) and ovarian morphology (asymmetric vs. symmetric ovarian anterior lobes). There are 15 valid species of Acanthobothrium described for adjacent waters from where Acanthobothrium n. sp. 1 was found (see Table2). Within this group, six species are restricted to freshwater species of Potamotrygonidae and there is no evidence that they occur in marine Myliobatiformes (Table2). Among those marine species, Acanthobothrium n. sp. 1 is most similar to A. brevissime (as described by Campbell (1969)) since they share similar total length (1.4–3.0 vs. 1.5–4.2 mm, respectively), number of segments (5–10 vs. 7–29, respectively), and number of testes (23–38 vs. 19–40, respectively). However, Acanthobothrium n. sp. 1 can be distinguished from A. brevissime by possessing shorter scolex (205–303 vs. 320–475) and shorter bothridia (161–236 vs. 260–360). Also, Acanthobothrium n. sp. 1 share many morphometric attributes with A. lineatum, such as total length (1.4–3.0 vs. 1.8–6.1 mm, respectively), number of segments (5–10 vs. 6–19, respectively), and number of testes (23–38 vs. 28–45, respectively). Yet, Acanthobothrium n. sp. 1 possesses shorter scolex compared to A.

20 lineatum (205–303 vs. 380–600), shorter bothridia (161–236 vs. 275–624) and smaller anterior loculus length (77–140 vs. 144–325). Acanthobothrium n. sp. 1 can be easily differentiated from A. americanum and A. cairae by being a smaller worm (1.4–3.0 vs. 94 mm and 45–154 mm, respectively) with fewer segments (5–10 vs. 453–615 and 268–491, respectively), by having shorter scolex (205–303 vs. 554 and 1,100–1440, respectively) and fewer testes (23–38 vs. 59–78 and 82–166, respectively). Acanthobothrium n. sp. 1 can be distinguished from A. cartagenensis and A. colombianum by possessing shorter total length (1.4–3.0 mm vs. 25 mm and >35 mm, respectively) and fewer segments (5–10 vs. 13 and 31–48, respectively). This new species differs from A. himanturi and A. tasajerasi by having fewer segments (5–10 vs. 17–26 and 11–18, respectively). Acanthobothrium n. sp. 1 further differs from A. tasajerasi by the absence of an expanded poral atrium. Finally, Acanthobothrium n. sp. 1 can be distinguished from A. urotrygoni by its shorter total length (1.4–3.0 mm vs. > 15 mm) and longer scolex (205–303 vs. 154–161).

21 Golfetti, Y. Dissertação de Mestrado, 2018 DasyatidaeDasyatidae Warm Temperate Northwest Atlantic Warm Temperate Northwest Atlantic Dasyatidae Warm Temperate Northwest Atlantic Dasyatidae Warm Temperate Northwest Atlantic Aetobatidae Tropical Northwestern Atlantic Urotrygonidae Tropical Northwestern Atlantic Urotrygonidae Tropical Northwestern Atlantic PotamotrygonidaePotamotrygonidaePotamotrygonidaePotamotrygonidae Neotropical Freshwater Neotropical Freshwater Neotropical Freshwater Neotropical Freshwater PotamotrygonidaePotamotrygonidae Neotropical Freshwater Neotropical Freshwater Potamotrygonidae Tropical NorthwesternPotamotrygonidae Atlantic Tropical Northwestern Atlantic found in Northwest Atlantic and Neotropical freshwater river systems. Potamotrygon circularis Potamotrygon magdalenae Potamotrygon hystrix Bathytoshia centroura Urobatis jamaicensis Aetobatus narinari Urotrygon venezuelae Acanthobothrium Hypanus americanus Potamotrygon motoro Dasyatis americana Hypanus say Potamotrygon motoro Styracura schmardae Styracura schmardae Potamotrygon motoro Valid species of Brooks & Mayes, 1980 ex Brooks & Mayes, 1980 ex Mayes, Brooks & Thorson, 1978 ex Campbell, 1969 ex Table 2: Reyda, 2008 ex Brooks & Mayes, 1980 ex Linton, 1908 ex Brooks, 1977 ex Brooks, 1977 ex Mayes, Brooks & Thorson, 1978 ex Campbell, 1969 ex Ivanov, 2005 ex Rego & Dias, 1976 ex Vardo-Zalik & Campbell, 2011 ex Brooks, Mayes & Thorson, 1981 ex A. ramiroi A. regoi A. terezae A. amazonensis A. peruviense A. quinonesi A. cairae A. cartagenensis A. colombianum A. himanturi A. lineatum A. tasajerasi A. urotrygoni A. brevissime Species and HostA. americanum Host Family Biogeographical Realm

22 Figure 4: Light micrographs of new Acanthobothrium spp. described in the present study. A. Acanthobothrium n. sp. 1 (PA16-83-3); B. Acanthobothrium n. sp. 2 (PA16-87-8); C. Acanthobothrium n. sp. 3 (PA16-01-2); D. Acanthobothrium n. sp. 4. (PA16-48-1) 23 Golfetti, Y. Dissertação de Mestrado, 2018

Figure 5: Light micrographs of Acanthobothrium n. sp. 1 from Hypanus guttatus. A. Scolex (PA16- 83-3); B. hooks; C. subterminal proglottid; D. Terminal mature proglottid. 24 Figure 6: Scanning electron micrographs of Acanthobothrium n. sp. 1. A. Scolex (MY07); B. Distal surface of bothridia loculi; C. Proximal surface of bothridia; D. Cephalic peduncle surface; E. Distal surface of muscular pad; F. Detail of hooks.

25 Table 3: Morphometric data for new species of Acanthobothrium described in present study. Measurements are given as the range, followed by median and number of specimens in parenthesis. All measurements presented in 휇m, unless indicated, followed by median and number of specimens measured, in parenthesis.

Acanthobothrium sp. 1 Acanthobothrium sp. 2 Acanthobothrium sp. 3 Acanthobothrium sp. 4 Total length 1.42–3.03 mm (2.33, 17) 2.1–2.51 mm (2.17, 3) 3.34–12.11 mm (6.53, 50) 7.78–28.77 mm (18.37, 16) No. of proglottids 5–10 (8, 17) 7–8 (7, 3) 12–32 (18, 50) 49–133 (81.5, 16) Scolex length 205–303 (236.74, 18) 237–261 (251.8, 3) 350–609 (467.26, 50) 737–1127 (897.16, 14) Scolex width 131–221 (174.5, 17) 165–192 (176.48, 3) 241–435 (567.26, 50) 601–869 (707.14, 15) Bothridia length 161–236 (199.41, 18) 206–226 (208.75, 3) 273–461 (349.37, 50) 601–802 (712.44, 16) Bothridia width 65–108 (84.20, 18) 80–95 (87.5, 3) 120–234 (168.69, 50) 299–402 (343.91, 16) Muscular pad length 39–68 (52.98, 18) 55–60 (57.75, 2) 84–154 (111.12, 50) 178–251 (208.79, 12) Muscular pad width 52–94 (72.47, 18) 72–80 (76.25, 2) 127–209(161.05, 50) 259–343 (316.34, 12) Apical sucker length 9–25 (15.67, 15) 14–16 (15.42, 2) 13–54 (36.16, 50) 24–48 (36.32, 6) Apical sucker width 12–21 (15.20, 14) 21–28 (24.42, 2) 18–54 (34.7, 50) 32–122 (75.48, 10) Anterior loculus length 77–140 (111.53, 18) 106–126 (118.47, 3) 166–303 (217.43, 50) 341–546 (411.32, 16) Middle loculus length 26–50 (39.35, 18) 31–42 (39.14, 3) 53–100 (76.36, 50) 103–179 (125.91, 16) Posterior loculus length 25–45 (29.11, 17) 27–33 (30.15, 3) 29–75 (52.57, 50) 40–146 (91.73, 16) Lateral Hook A 21–33 (26.40, 34) 23–34 (28.16, 6) 39–74 (58.76, 50) 98–140 (123.18, 16) B 41–68 (54.02, 32) 55–69 (59.43, 6) 83–149 (116.76, 50) 154–210 (191.22, 15) C 40–65 (51.73, 33) 56–66 (60.21, 6) 80–143 (112.62, 50) 132–193 (165.29, 16) D 61–93 (75.40, 32) 73–98 (86.77, 6) 108–208 (160.78, 50) 219–316 (292.53, 15) Medial Hook A’ 15–28 (23.84, 34) 19–31 (27.46, 6) 41–66 (51.36, 50) 100–139 (118.79, 16)

Continued on next page Table 3 – Continued from previous page Specie Acanthobothrium sp. 1 Acanthobothrium sp. 2 Acanthobothrium sp. 3 Acanthobothrium sp. 4 B’ 42–72 (59.74, 32) 63–75 (71.12, 6) 73–140 (108.85, 50) 184–253 (228.2, 15) C’ 39–64 (53.78, 32) 54–65 (60.03, 5) 97–169 (127.83, 50) 122–179 (160.26, 15) D’ 59–94 (78.17, 32) 79–103 (91.15, 6) 117–226 (173.06, 50) 252–370 (339.5, 15) Cephalic peduncle length 128–431 (13) 220–340 (240, 3) 420–2090 (815, 50) 660–1470 (1050, 16) Cephalic peduncle width 50–173 (13) 80–90 (85, 3) 0.1–257 (51) 151–415 (16) No. immature proglottids 4–9 (6.5, 18) 6 (6, 3) 11–29 (16, 50) 46–114 (73.5, 16) No. mature proglottids 1–2 (1, 18) 1–2 (1, 3) 1–4 (2, 50) 3–19 (12, 15) Mature segment length 364–1278 (924.79, 17) 793–1200 (1139.27, 3) 757–2021 (1413.21, 50) 816–1172 (925.62, 16) Mature segment width 149–373 (214.69, 35) 187–296 (216.74, 6) 184–412 (283.74, 50) 373–546 (434.57, 16) Genital pore alternation irregular irregular irregular irregular Number of testes 23–38 (31, 27) 26–34 (32, 5) 24–41 (21, 49) 47–66 (56, 14) pre–poral testes 5–13 (9, 28) 8–9 (8, 5) 5–10 (7, 48) 9–19 (14.5, 14) post–poral testes 4–7 (5, 29) 5–7 (5.5, 6) 5–10 (7, 48) 9–12 (11, 14) aporal testes 13–20 (16.5, 28) 13–19 (16.5, 6) 14–21 (17, 49) 25–41 (30, 14) Testes length 22–55 (39.55, 24) 30–42 (36.09, 5) 30–65 (47.05, 39) 23–49 (41.06, 6) Testes width 32–60 (44.02, 24) 36–45 (40.61, 5) 31–63 (46.44, 39) 29–73 (35.39, 5) Testes shape round to eliptical round round round to eliptical Cirrus sac length 81–193 (134.28, 33) 104–182 (148.21, 6) 108–257 (177.96, 48) 60–206 (144.51, 16) Cirrus sac width 47–217 (115.97, 33) 82–157 (122.73, 6) 66–206 (116.71, 48) 107–198 (142.57, 16) Ovary width 45–156 (83.72, 33) 69–127 (83.37, 5) 63–189 (106.5, 49) 156–258 (193.31, 12) Ovary length (poral lobe) 81–697 (404.81, 33) 333–501 (427.87, 6) 305–885 (567.51, 49) 340–479 (426.23, 16) Ovary length (aporal lobe) 124–674 (3425.76, 33) 332–563 (444.60, 6) 311–946 (595.93, 49) 318–675 (498.39, 16) Genital pore position (% 31–50% (41.29, 34) 34–45 (38.04, 6) 26–46% (37.63, 49) 30–49% (41.06, 16) from posterior) 27 Golfetti, Y. Dissertação de Mestrado, 2018

Acanthobothrium n. sp. 2

(Figs.7)

Description (Based on 3 complete worms and 3 hologenophores): Worms acraspedote (Fig.4 B, Table3), euapolytic, 2,1–2,51 mm (n=3) long, with 7–8 (n=3) proglottids. Scolex consisting of scolex proper and cephalic peduncle (Fig.5 B). Cephalic peduncle 220–340 (n=3) long by 80–90 (=3) wide. Scolex proper 237–261 (n=3) long by 165–192 (n=3) in maximum width (at level of anterior loculus), composed by a proper and cephalic peduncle (Fig.7 A). Scolex proper with four bothridia, bothridia 206–226 (n=3) long by 80–95 (n=3) in maximum width, each with three loculi divided by two horizontal septa. Anterior loculus 106–126 (n=3) long, middle loculus 31–42 (n=3) long, and posterior loculus 27–33 (n=5) long; loculus length ratio 1:0.26–0.39:0.16–0.27 (n=3). Muscular pad 50–60 (n=2) long by 72–80 (n=2) wide, triangular shape. Bearing an apical sucker 14–16 (n=2) long by 21–28 (n=2) wide, and one pair of triangular hooks below posterior margin (Fig.7 B). Velum not- present. Hooks bipronged, hollow, with insconspicuous tubercle on proximal surface of axial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongs and abaxial prongs with different lengths; lateral and medial hooks of different shape and sizes (Fig.7 B). Lateral hook measurements: A 23–34 (n=6), B 55–69 (n=6), C 56–66 (n=6), D 73–98 (n=6). Medial hook measurements: A’ 19–31 (n=6), B’ 63–75 (n=6), C’ 54–65 (n=6) and D’ 79–103 (n=6). Medial hook base wider than lateral hook base. Thin layer of tissue anteriorly covering each prong of both set of hooks. Immature proglottids wider than long; 6 (n=3) in number (Fig.7 C). Terminal mature proglottids longer than wide; 1–2 (n=3) in number, 793–1200 (n=3) long by 187–296 (n=3); mature proglottid length to width ratio 3.7–6.4 (n=3) (Fig.7 D). Genital pores irregularly alternating, 34–45% (n=6) from anterior end of proglottid. Gravid proglottids and eggs not observed. Testes round, 30–42 (n=6) long by 36–45 (n=6) wide, arranged in two regular columns, extending from anterior region of proglottid to ovarian isthmus, 26–34 (n=5) in total number, 8–9 (n=5) pre-poral, 5–7 (n=6) post-poral and 13–19 (n=6) aporal. Cirrus sac round, 104–182 (n=6) long by 82–157 (n=6) wide, containing eversible cirrus armed with spinitriches. Vagina thick-walled, sinuous, extending from ootype along medial line of proglottid to anterior margin of cirrus sac to common genital atrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetrical (ovarian lobes with different lengths) inverted A-shaped in frontal view, lobulated, reaching or almost reaching posterior margin of cirrus sac. Poral lobe 333–501 (n=6) long, aporal lobe 332–563 (n=6) long by 69–127 (n=6) wide at isthmus. Vitellarium follicular form narrow lateral bands, each with 1–2 visible rows of follicles, extending from the ﬁrst line of distal testes to the ovarian isthmus, interrupted by vagina and cirrus sac dorsally and ventrally, not interrupted by ovary. Uterus linear, median, extending from ovarian isthmus to vagina level.

28 Taxonomic summary Type-host: Hypanus guttatus (Bloch & Schneider). Additional hosts: Fontitrygon geijskesi (Boeseman). Type-locality: Bay of Marajó, Colares - PA - Brazil (ca. 0°55’53.8”S-48°17’39.5”W). Site of infection: Spiral intestine. Specimens deposited: ### Prevalence: 11.11% in Hypanus guttatus and 7.69% in Fontitrygon geijskesi.

Remarks: Despite their relative phylogenetic distance, Acanthobothrium n. sp. 2 shares many morphometric attributes with Acanthobothrium n. sp. 1. All morphometric ranges between these species overlap. Although Acanthobothrium n. sp. 2 showed higher medians than Acanthobothrium. n. sp. 1 (see Table3), the new species is well supported by the molecular analyses, providing justiﬁcation to recognize it as a putative new species different from Acanthobothrium n. sp. 1. Compared to the 9 marine species found in adjacent waters to Bay of Marajó (see Table2), Acanthobothrium n. sp. 2 is also very similar to A. brevissime (as described by Campbell (1969)) since they share similar total length (2.1–2.5 vs. 1.5–4.2 mm, respectively), number of segments (7–8 vs. 7–29, respectively), and number of testes (26–34 vs. 19–40, respectively). However, Acanthobothrium n. sp. 2 can be distinguished from A. brevissime by possessing shorter scolex (237–261 vs. 320–475) and shorter bothridia (206–226 vs. 260–360). Acanthobothrium n. sp. 2 share some morphometric ranges with A. lineatum, such as total length (2.1–2.51 mm vs. 1.8–6.1 mm, respectively), number of segments (7–8 vs. 6–19, respectively), and number of testes (26–34 vs. 28–45, respectively). However, Acanthobothrium n. sp. 2 possesses shorter scolex compared to A. lineatum (237–261 vs. 380–600), shorter bothridia (206–226 vs. 275–624) and shorter anterior loculus (106–126 vs. 144–325). Also, Acanthobothrium n. sp. 2 can be easily differentiated from A. americanum and A. cairae by being a smaller worm (2.1–2.51 vs. 94 mm and 45–154 mm, respectively) with fewer segments (7–8 vs. 453–615 and 268–491, respectively), by having shorter scolex (237–261 vs. 554 and 1,100–1440, respectively) and fewer testes (26–34 vs. 59–78 and 82–166, respectively). Acanthobothrium n. sp. 2 can be distinguished from A. cartagenensis and A. colombianum by being a smaller worm (2.1–2.51 mm vs. 25 mm and > 35 mm, respectively) and having fewer segments (7–8 vs. 13 and 31–48, respectively). This new species differs from A. himanturi and A. tasajerasi by having fewer segments (7–8 vs. 17–26 and 11–18, respectively). Acanthobothrium n. sp. 2 further differs from A. tasajerasi by the absence of an expanded poral atrium. Finally, Acanthobothrium n. sp. 2 can be distinguished from A.

29 Golfetti, Y. Dissertação de Mestrado, 2018

urotrygoni by its shorter total length (2.1–2.51 mm vs. > 15 mm, respectively) and longer scolex length (231–261 vs. 154–161).

30 Figure 7: Light micrographs of Acanthobothrium n. sp. 2 from Hypanus guttatus. A. Scolex (PA16- 87-8); B. hooks; C. subterminal proglottid; D. Terminal mature proglottid. 31 Golfetti, Y. Dissertação de Mestrado, 2018

Acanthobothrium n. sp. 3

(Figs.8- 12)

Description (Based on 50 complete worms, three scoleces observed with SEM and two mature proglottids used for cross sections): Worms acraspedote (Fig.4 C, Table3), euapolytic, 3.01–12.11 mm (n=50) long, with 12–32 (n=50) proglottids. Scolex consisting of scolex proper and cephalic peduncle (Fig.8A,9 A). Cephalic peduncle 420–2090 (n=50) long by 0,1–257 (n=50) wide, covered with gladiate spinitriches whit approximately 2–4 microtriches/휇푚2(Fig.9 B). Scolex proper 350–609 (n=50) long by 241–435 (n=50) in maximum width (at level of anterior loculus), with for bothridia, bothridia 273–461 (n=50) long by 120–234 (n=50) in maximum width. Proximal surface of bothridia covered by gladiate microtriches whit approximately 1–2 microtriches/휇푚2 and acicular filitriches with 0.09 in diameter and approximately 46–53 microtriches/휇푚2 (Fig.9 C).Each bothridia with anterior muscular pad and three loculi divided by two horizontal septa. Anterior loculus 166–303 um (n=50) long, middle loculus 53–100 (n=50) long, and posterior loculus 29–75 (n=50) long; loculi length ratio (A:M:P) 1:0.23–0.52:0.11–0.34 (n=50). Distal surface of bothridia and inter- loculi septa covered by papiliform filitriches with 0.07 in diameter and approximately 54–56 microtriches/휇푚2 (Fig.9 D). Muscular pad 84–154 (n=50) long by 127–209 (n=50) wide, covered by papiliform filitriches with 0.09 in diameter, triangular shape and approximately 116–125 microtriches/휇푚2 (Fig.9 E). Bearing an apical sucker 13–54 (n=50) long by 18–54 (n=50) wide, and one pair of triangular hooks below posterior margin (Fig.8 B). Velum not–present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surface of axial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongs and abaxial prongs with different lengths; lateral and medial hooks of different sizes (Fig. 8 B). Lateral hook measurements: Lateral hook measurements: A 39–74 (n=50), B 83–149 (n=50), C 80–143 (n=50), D 108–208 (n=50). Medial hook measurements: A’ 41–66 (n=50), B’ 97–169 (n=50), C’ 73–140 (n=50), D 117–226 (n=50). Medial hook base wider than lateral hook base. Thin layer of tissue anteriorly covering each prong of both set of hooks. (Fig.9 F). Immature proglottids wider than long; 11–29 (n=50) in number (Fig.8 C). Terminal mature proglottids longer than wide; 1–4 (n=50) in number, 757–2021 (n=50) long by 184–412 (n=50); mature proglottid length to width ratio 4.1–4.9 (n=50) (Fig.8 D, E). Genital pores irregularly alternating, 26–46% (n=49) from anterior end of proglottid. Some terminal proglottids with sperm-filled vas deferens (Fig. 10 A). Gravid proglottids and eggs not observed. Testes round, 30–65 (n=39) long by 31–63 (n=39) wide, arranged in two regular columns, extending from anterior region of proglottid to ovarian isthmus, 24–41 (n=4) in total number, 5–10 (n=48) pre- poral, 5–10 (n=48) post-poral and 14–21 (n=49) aporal. Cirrus sac pyriform, 108–257 (n=48)

32 long by 66–206 (n=48) wide, containing eversible cirrus armed with spinitriches (Fig. 10 B). Vagina thick-walled, sinuous, extending from ootype along medial line of proglottid to anterior margin of cirrus sac to common genital atrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetrical (ovarian lobes with different lengths) inverted A- shaped in frontal view, lobulated, and tetra-lobed in cross section, reaching or almost reaching posterior margin of cirrus sac. Poral lobe 305–885 (n=49) long, aporal lobe 311–946 (n=49) long by 63–189 (n=4) wide at isthmus. Vitellarium follicular form narrow lateral bands, each with 1–2 visible rows of follicles, extending from the ﬁrst or second line of distal testes to after Mehlis’ gland level, being able of some follicles to reach the end of the ovarian lobes, interrupted by vagina and cirrus sac dorsally and ventrally, not interrupted by ovary. Uterus linear, median, extending from ovarian isthmus to vagina level.

Taxonomic summary Type-host: Hypanus guttatus (Bloch & Schneider). Additional hosts: Fontitrygon geijskesi (Boeseman). Type-locality: Bay of Marajó, Colares - PA - Brazil (ca. 0°55’53.8”S-48°17’39.5”W). Site of infection: Spiral intestine. Specimens deposited: ### Prevalence: 100% in Hypanus guttatus and 38.46% in Fontitrygon geijskesi.

Remarks: Of all species described here, Acanthobothrium n. sp. 3 was the most prevalent species and displayed great variability in scolex and proglottid morphology. Some specimens of Acanthobothrium n. sp. 3 possess thin and long terminal proglottids while others present more rounded and short ones (Fig.8 D, E). The scolex show some variations as well, mainly in middle and posterior loculi, where they can range in shape from more round to more pointed (Fig 12). However, this plasticity can not be associated with presence in different hosts, as the same morphological variation are found in parasite specimens collected for H. guttatus and F. geijskesi (Fig. 11). Acanthobothrium n. sp. 3 is morphometrically similar to A. brevissime, since all morphometric attributed overlap between them. However, Acanthobothrium n. sp. 3 is distinguishable from A. brevissime by molecular evidence (Fig.3). Acanthobothrium n. sp. 3 differs from A. lineatum by possessing longer (273–461 vs. 161–236) and wider bothridia (120–234 vs. 65–108), and by having a wider ovary (63–189 vs. 20–50). Acanthobothrium n. sp. 3 differs from A. cairae by its smaller total length (3.34–12.11 mm vs. 45–154 mm), fewer proglottids (12–32 vs. 268–491) and by possessing shorter (305–609 vs. 1.1–1.44 mm) and narrower scolex (241–435 vs. 975–1,225). The new species differs from A. americanum by

33 Golfetti, Y. Dissertação de Mestrado, 2018

having smaller total length (3.34–12.11 mm vs. 63–115mm) and by having fewer proglottids (12–32 vs. 453–615). Acanthobothrium n. sp. 3 is distinguishable from A. colombianum by possessing smaller total length (3.34–12.11 mm vs. 35 mm) and shorter mature proglottid (667–690 vs. 757–2021). Acanthobothrium n. sp. 3 differs from A. urotrygoni by having smaller total length (2.1–2.51 mm vs. > 15 mm), more proglottids (12–32 vs. 4–6) and longer scolex (250–609 vs. 154–161). Acanthobothrium n. sp. 3 is distinguishable from A. tasajerasi by having longer (39–68 vs. 84–154) and wider muscular pad (72–80 vs. 127–209), and for the absence of an expanded genital atrium. Acanthobothrium n. sp. 3 have all morphometric ranges overlapping with A. himanturi. However, the phylogenetic analyses supports that this new species differs from all other species (Clade IV (ETP), Fig.3). Acanthobothrium n. sp. 3 is distinguishable from Acanthobothrium n. sp. 1 by having longer total length (3.34–12.11 mm vs. 1.4–3.0 mm), more proglottids (12–32 vs. 5–10), longer (305–609 vs. 205–303) and wider scolex (241–435 vs. 131–221), and by having larger hook total lengths (D 108–208, D 117–226 vs. D 61–93, D’ 58–94). Finally, Acanthobothrium n. sp. 3 differs from Acanthobothrium n. sp. 2 by possessing a longer total length (3.34–12.11 mm vs.2.1–2.51 mm, respectively), more proglottids (12–32 vs. 7–8), longer (305–609 vs. 237–261) and wider scolex (241–435 vs. 165–192), and by having longer hooks total length (D 108–208, D 117–226 vs. D 73–98, D’ 79–103).

34 . A B

200

Figure 8: Light micrographs of Acanthobothrium n. sp. 3 from Hypanus guttatus. A. Scolex (PA16- 40-12); B. hooks; C. subterminal proglottid (PA16-72-2); D. Terminal mature proglottid; E. Terminal35 mature proglottid (PA16-02-3) Golfetti, Y. Dissertação de Mestrado, 2018

Figure 9: Scanning electron micrographs of Acanthobothrium n. sp. 3. A. Scolex (MY02); B. Distal surface of bothridia loculi; C. Proximal surface of bothridia; D. Cephalic peduncle surface; E. Distal surface of muscular pad (MY03); F. Detail of hooks.

36 Figure 10: Micrographs of transversal cross sections of Acanthobothrium n. sp. 3. A. Section at level of testes (PA16-77-3-1H). B. section at level of ovary (PA16-53-2-1H). Abbreviations: Ed. Excretory duct; Mg. Mehlis’ gland; O. Ovary; T. Testes; V. Vitelline follicles; Vd. Vas deferens.

37 Golfetti, Y. Dissertação de Mestrado, 2018

A Width 250 300 350 400 450 500

350 400 450 500 550 600

Length

B Width 200 250 300 350 400

800 1000 1200 1400 1600 1800 2000

Length

Figure 11: Scatter plot of attributes variation of Acanthobothrium n. sp. 3 based on type series. A. Scolex length vs. width; B. Terminal proglottids length vs. width. Black dots: specimens hosted by H. 38guttatus, red dots: specimens hosted by F. geijskesi. . A

Figure 12: Variation on bothridial morphology for Acanthobothrium n. sp. 3. A. PA16-40-12; B. PA16-34-3; C. PA16-83-4 39 Golfetti, Y. Dissertação de Mestrado, 2018

Acanthobothrium n. sp. 4

(Figs. 13- 15)

Description (Based on 16 mature complete worms, one scolex observed with SEM and one terminal proglottid used for cross sections): Worms acraspedote (Fig.4 D, Table3), euapolytic, 7.78–28.77 mm (n=16) long, with 49–119 (n=16) proglottids. Scolex consisting of scolex proper and cephalic peduncle (Fig. 13A, 14 A). Cephalic peduncle 660–1470 (n=16) long by 151–415 (=16) wide, covered with gladiate spinitriches with approximately 2–4 microtriches/휇푚2 (Fig. 14 B). Scolex proper 778–1127 (n=14) long by 601–869 (n=15) in maximum width (at level of anterior loculus), with four bothridia, bothridia 601–802 (n=16) long by 299–402 (n=16) in maximum width. Proximal surface of bothridia covered by gladiate spinitriches with approximately 1–2 microtriches/휇푚2 and acicular filitriches with 0.14 in diameter and approximately 24–32 microtriches/휇푚2 (Fig. 14 C). Each bothridia with anterior muscular pad and three loculi divided by two horizontal septa. Anterior loculus 341–546 um (n=16) long, middle loculus 103–179 (n=16) long, and posterior loculus 40–146 (n=16) long; loculus length ratio (A:M:P) 1:0.23–0.43:0.08–0.37 (n=16). Distal surface of bothridia and inter-loculi septa covered by papiliform filitriches with 0.12 in diameter and approximately 49–52 microtriches/휇푚2 (Fig. 14 D). Muscular pad 178–251 (n=12) long by 259–343 (n=12) wide, covered by papiliform filitriches with 0.12 in diameter, triangular shape and approximately 88–40 microtriches/휇푚2 (Fig. 14 E). Bearing an apical sucker 24–48 (n=6) long by 32–122 (n=10) wide, and one pair of triangular hooks below posterior margin (Fig. 13 B). Velum present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surface of axial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongs and abaxial prongs with different lengths; lateral and medial hooks of different sizes (Fig. 13 B). Lateral hook measurements: A 98–140 (n=16), B 154–209 (n=15), C 132–193 (n=16), D 219–316 (n=15). Medial hook measurements: A’ 100–139 (n=16), B’ 184–253 (n=15), C’ 122–179 (n=15) and D’ 252–370 (n=15). Medial hook base wider than lateral hook base. Thin layer of tissue anteriorly covering each prong of both set of hooks. Immature proglottids wider than long; 46–114 (n=16) in number (Fig. 13 C). Terminal mature proglottids longer than wide; 3–19 (n=15) in number, 816–1172 (n=16) long by 373–546 (n=16); mature proglottid length to width ratio 2.14–2.19 (Fig. 13 D). Genital pores irregularly alternating, 30–49% (n=16) from anterior end of proglottid. Some terminal proglottids with sperm-filled vas deferens (Fig. 15 A).Gravid proglottids and eggs not observed. Testes mostly round (eliptical in pre-poral and aporal testes anterior to cirrus sac at sperm-filled proglottids), 23–49 (n=6) long by 29–73 (n=5) wide, arranged in two regular columns, extending from anterior region of proglottid to ovarian isthmus, 47–66 (n=14) in total number, 9–19 (n=14) pre-poral, 9–12 (n=14) post-poral

40 and 25–41 (n=14) aporal. Cirrus sac pyriform, 60–206 (n=16) long by 107–198 (n=16) wide, containing eversible cirrus armed with spinitriches. Vagina thick-walled, sinuous, extending from ootype along medial line of proglottid to anterior margin of cirrus sac to common genital atrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetrical (ovarian lobes with different lengths) inverted A-shaped or in frontal view, lobulated, reaching or almost reaching posterior margin of cirrus sac, and tetra-lobed in cross section (Fig. 15 B). Poral lobe 340–479 (n=16) long, aporal lobe 318–675 (n=16) long by 156–258 (n=12) wide at isthmus. Vitellarium follicular form narrow lateral bands, each with 1–2 rows of follicles, extending from the ﬁrst line of distal testes to after Mehlis’ gland level not reaching the end of the ovarian lobes, interrupted by vagina and cirrus sac dorsally and ventrally, not interrupted by ovary. Uterus linear, median, extending from ovarian isthmus to vagina level.

Taxonomic summary Type-host: Fontitrygon geijskesi (Boeseman). Additional hosts: Hypanus guttatus (Bloch & Schneider). Type-locality: Bay of Marajó, Colares - PA - Brazil (ca. 0°55’53.8”S-48°17’39.5”W). Site of infection: Spiral intestine. Specimens deposited: ### Prevalence: 84.62% in Fontitrygon geijskesi and 3.07% in Hypanus guttatus

Remarks: The phylogenetic analyses suggested that Acanthobothrium n. sp. 4 is sister to Acanthobothrium sp. ex Hypanus guttatus [A064/AL10-004.01]. Acanthobothrium n. sp. 4 differs from the hologenophore of that sequence by having longer hooks handle (A 98–140 and A’ 100–139, vs. A 33.89 and A’ 35.38), larger total hook lengths (D 219–316 and D’ 252–370 vs. D 111.96 and D’113.50) and longer medial outer prong than inner prong (B’184–253 and C’ 122–179 vs. B’ 79.89 and C’ 89.96). Acanthobothrium n. sp. 4 is distinguishable from A. americanum and A. cairae by having smaller total length (7.78–28.77 mm vs. 63–115 mm and 45–154 mm, respectively), fewer proglottids (46–114 vs. 453–615 and 268–491, respectively) and fewer post-poral testes (9–12 vs. 18–25 and 42–85, respectively). Acanthobothrium n. sp. 4 is distinguishable from A. colombianum by having smaller total length (7.78–28.77 mm vs. 35 mm, respectively), more proglottids (49–133 vs. 31–48, respectively), and longer (737–1127 vs. 230, respectively) and wider scolex (601–869 vs. 230–246). This new species differs from A. himanturi by possessing more proglottids (49–133 vs. 17–26) and longer scolex (737–1127 vs. 240–350). Acanthobothrium n. sp. 4 differs from A. brevissime by having larger total length (7.78–28–77 mm vs. 1.5–4.2 mm), more proglottids (49–133 vs 7–29) and longer scolex

41 Golfetti, Y. Dissertação de Mestrado, 2018

(737–1127 vs. 240–350). Also, Acanthobothrium n. sp. 4 is distinguishable from A. lineatum by having larger total length (7.78–28.77 mm vs. 1.8–6.1 mm), more proglottids (49–133 vs. 11–18) and longer scolex (737–1127 vs. 380–600). Further, Acanthobothrium n. sp. 4 differs from A. tasajerasi by having larger total length (7.78–28,77 mm vs. 2.5–5.5 mm), longer scolex (737–1127 vs. 309–384) and more segments (49–113 v.s 5–10). Acanthobothrium n. sp. 4 can be distinguished from A. cartagenensis and A. urotrygoni, by possessing longer scolex (737–1127 vs. 320–475 and 154–161) and more segments (49–113 vs. 13 and 4–6). Acanthobothrium n. sp. 4 differs from Acanthobothrium n. sp. 1 by having larger total length (7.78–28.77 mm vs. 1.42–3.03 mm), more proglottids (49–133 vs. 5–10), longer total length (737–1,127 vs. 205–303), wider scolex (601–869 vs. 131–221) and by bearing more testes (47–66 vs. 23–28). Acanthobothrium n. sp. 4 differs from Acanthobothrium n. sp. 2 by its total length (7.78–28.77 mm vs. 2.1–2.51 mm, respectively), number of proglottids (49–133 vs. 7–8, respectively), longer (0.737–1.127 mm vs. 237–261), wider scolex (601–869 vs. 165–192) and for presenting more testes (47–66 vs. 26–34). Finally, Acanthobothrium n. sp. 4 is distinguishable from Acanthobothrium n. sp. 3 by possessing more proglottids (49–133 vs. 12–32), longer (0.737–1.127mm vs. 350–609), wider scolex (601–869 vs. 241–435), by presenting more testes (47–66 vs. 24–41) and longer hooks total length (D 219–316, D’ 252–370 vs. D 108–208, D 117–226).

42 Figure 13: Light micrographs of Acanthobothrium n. sp. 4 from Fontitrygon geijskesi. A. Scolex (PA16-41-1); B. hooks; C. subterminal proglottid; D. Terminal mature proglottid. 43 Golfetti, Y. Dissertação de Mestrado, 2018

Figure 14: Scanning electron micrographs of Acanthobothrium n. sp. 4. A. Scolex (MY04); B. Distal surface of bothridia loculi; C. Proximal surface of bothridia; D. Cephalic peduncle surface; E. Distal surface of muscular pad.

44 Figure 15: Micrographs of transversal cross sections of Acanthobothrium n. sp. 4. A. Section at level of testes (PA16-48-1-1H). B. section at level of ovary. Abbreviations: Ed. Excretory duct; Mg. Mehlis’ gland; O. Ovary; Ov. Oviduct; T. Testes; V. Vitelline follicles; Vd. Vas deferens.

45 Discussion

Hosts identity

The Bay of Marajó is inhabited by a unique fauna of batoids, as it includes lineages restricted to the Neotropical freshwater systems that can tolerate low salinity habitats, such as potamotrtygonines, as well as marine lineages that support euryhaline species, such as some dasyatids. Three dasyatids have been reported for this estuary, H. guttatus, F. geijskesi and F. colarensis (Santos et al., 2004). Among those, F. colarensis was described most recently by Santos et al. (2004) and, as all others, was considered to be a member of Dasyatis Rafinesque, 1810. However, Last et al. (2016) considered F. colarensis as members of the newly erected genus Fontitrygon Last, Naylor Manjaji-Matsumoto, 2016 together with F. geijskesi. Among these three dasyatids, H. guttatus and F. colarensis are most similar, despite the fact that Last et al. (2016) considered these taxa in different genera. It should be noticed, however, that the authors did not include any representative of F. colarensis in the phylogenetic study upon which they based generic assignment, nor did they examine the type material of either species (see Last et al., 2016). Morphologically, F. geijskesi is easily distinguishable from H. guttatus and F. colarensis by having sharp snout and pelvic fins laterally expanded and short, with acute tips (Santos et al., 2004). However, differentiating H. guttatus and F. colarensis revealed not to be an easy task. According to the description given by Santos et al. (2004), H. guttatus differs from F. colarensis by the ratio of horizontal eye diameter to preoral length (5.6 - 6.9% vs. 10 - 39%, respectively) and the ratio of preoral length to disk width (34.9-36.7% vs. 21.5 to 25.5%, respectively). In addition, the posterior margins of the pectoral fins were considered to be rounded and the posterior margins of the pelvic fins to be pointed in F. colarensis, whereas in H. guttatus the pectoral fins were considered to be angular and pelvic fins to be rounded (Santos et al., 2004). However, it should be noticed that all the morphometric attributes used to distinguish these species were based on a very small sample size two juvenile specimens and an adult, and our specimens displayed values that could not be assigned to any group unequivocally (Table ). Finally, we were unable to recognize the two morphologies described for the pelvic and pectoral fins in our samples. Because the morphological distinction between F. colarensis and H. guttatus presented itself to be difficult, we used molecular data to guide species assignments for hosts. The topologies recovered by the phylogenetic analyses of each partition (MT-CYB, MT-ATP6, MT-ND2) and of the concatenated dataset (Fig.1, Appendices A.16, A.18 and A.17) recovered consistently two clades attributed to F. geijskesi and H. guttatus. However, we observed two clades among terminals assigned to H. guttatus with low support that could indicate the existence of two species within. The addition of morphometric data did not support that these clades can be differentiated morphologically (Table1 and Fig.2). Therefore, we concluded that we collected two dasyatids, one clearly congruent with F. geijskesi and the other we considered to be H. guttatus. Our results may challenge the taxonomic status of F. colarensis. Our samples were collected in the type locality of this species. We collected 59 individuals that could be considered either species. We found no evidence indicating that any of the host specimens analyzed in this study were specimens of F. colarensis. Given the fact of a small number of specimens have been used in the type series and the morphological data presented by Santos et al. (2004) may not be representative of the full morphological range of this species. The absence of any specimen of F. colarensis and the incongruences between the information provided in the original description and the observed morphometric ranges found in the present study are surprising. Finally, the lack of molecular divergence among individuals that potentially could members of F. colarensis casts doubt in the validity of this nominal species species.

Patterns of host speciﬁcity

The degree of host speciﬁcity of cestodes of elasmobranchs observed in South America freshwater (SAFW) and marine environments are remarkably different. In marine stingrays, the infection by Acanthobothrium is characterized by a pattern of one parasite species in a single or few hosts - with few exceptions (Campbell & Beveridge, 2002; Fyler, 2009). Whereas in SAFW, the same Acanthobothrium species can be distributed in more than 15 different hosts (Cardoso Jr., 2010). Until now, there is no study addressing the patterns of infection for cestodes in estuarine waters. The prevalence levels for four species of Acanthobothrium parasites of dasyatids in Bay of Marajó revealed some interesting patterns (see Table4). First, all four species can be found in both dasyatid species, which seems to deviate from the general pattern of strict host speciﬁcity reported for marine species (Caira & Jensen, 2017; Fyler, 2009). Second, all species seem to have a primary host in which prevalence levels are higher in comparison to the other species – a

47 Golfetti, Y. Dissertação de Mestrado, 2018

secondary host. The difference in prevalence levels between primary and secondary hosts is also evident for Acanthobothrium sp. n. 1 (48.86 vs. 3.07, respectively), but it is less pronounced for Acanthobothrium sp. n. 2 (11.11 vs.7.69%, respectively; see Table4). Finally, the levels of prevalence in primary hosts varied from 100 to ∼11%, whereas for secondary hosts it varied from ∼38 to ∼3% in Acanthobothrium sp. n. 3.

Table 4: Prevalence for species of Acanthobothrium in two dasyatids from Bay of Marajó. Number of hosts surveyed: 27 for H. guttatus and 13 for F. geijskesi. Numbers between parentheses indicate sample size and those between square brackets indicate expected minimum number of host necessary (95% conﬁdence limit) to detect infection according to prevalence levels following Dohoo et al. (2012).

Acanthobothrium spp. H. guttatus (27) F. geijskesi (13) Acanthobothrium sp. n. 1 44.44% [5] 7.69% [37] Acanthobothrium sp. n. 2 11.11% [26] 7.69% [37] Acanthobothrium sp. n. 3 100.00% [1] 38.46% [8] Acanthobothrium sp. n. 4 3.07% [82] 84.62% [2]

The prevalence levels documented in this study stress the relevance of sample size for understanding patterns of parasite and host association. We can approximate the expected sample size required for the detection of a parasite species given its prevalence by the following equation:

푛 = ln(훼)/ ln(푞);

where, 훼 is the confidence limit – usually set to 0.05 or 0.01, and 푞 is 1 - minimum expected prevalence (see Dohoo et al., 2012:56-57). Accordingly, it would be expected the examination of 26 specimens of hosts to detect the presence of Acanthobothrium sp. n. 2 in its primary host H. guttatus using a confidence interval of 95%. Our study, however, also detected species of Acanthobothrium in cases for which the estimated sample size would be much larger. For instance, according to the model we used from Dohoo et al. (2012), it would be expected the examination of 84 hosts to detect Acanthobothrium sp. n. 4 in H. guttatus. Nonetheless, we were able to detect this species after examining 27 specimens. To a lesser extend, a similar case can be observed for Acanthobothrium sp. n. 1 and 2 in F. geijskesi (Table4). There are many reasonable explanations to explain these results, including violations of some assumptions of this model (i.e., infinite population, even distributions, among others) to errors associated with prevalence levels estimate, which are beyond the scope of this discussion. However, our data supports the contention that sample size has important roles in our understanding of host specificity. Generally, the number of host specimens examined with the objective of documenting

48 cestode diversity is low. For example, a quick survey of the number of hosts examined in taxonomic descriptions of Acanthobothrium species during a period of 10 years (2006-2016, Cestode Database – accessed on October, 2018), indicated that the number of hosts examined can vary from 1 to 341, but with a median of 4 hosts. Most studies focus on the fauna of a particular host species, and those that included multiple ones, the number of specimens examined is low. All indicate that our understanding of host speciﬁcity for Acanthobothrium might change as we increase the sample size of hosts examined.

Phylogeny of Acanthobothrium: implications on taxonomy and biogeography

Even with the inclusion of new terminals in this study, the general phylogenetic status of Acanthobothrium recovered mirrors the results of Trevisan (2016). Two terminals belonging to different genera nested within Acanthobothrium. The first was Onchobothrium ? ex Hypanus sabinus [AC09/TM-9], which we believe is a result of misidentification. Onchobothrium resembles to Acanthobothrium by possessing four bothridia divided into three loculi and by presenting similar proglottid morphology. However, they differ in hook morphology, since Acanthobothrium bears a pair of bipronged hooks in each bothridium, whereas in Onchobothrium those hooks are single-pronged. Despite conspicuous differences in hook morphology, species misidentification of both genera are common in the literature (Baer & Euzet, 1962; Caira & Machida, 1986; Linton, 1916; Yamaguti, 1952). The second genus to undermine the monophyly of Acanthobothrium was Acanthobothroides Brooks, 1977. Members of this genus have a scolex composed of four bothridia, each with three loculi, a muscular pad with a apical sucker and a pair of bipronged hooks (Marques et al., 1996, but see Brooks, 1977) - as for Acanthobothrium. Brooks (1977) erected Acanthobothoides based on the morphology of the medial hook, then described as having just one prong and a “base”. Marques et al. (1996) questioned this diagnose when they described Acanthobothroides pacificus Marques, Brooks & Ureña, 1996. According to Marques et al. (1996), all specimens in the type series of Acanthobothroides thorsoni had the inner prong of the medial hook broken - which was overlooked in the original description. The phylogeny of molecular data corroborates the suggestion of Marques et al. (1996) that Acanthobothroides is a junior synonym of Acanthobothrium and the hook morphology found in the two species of the genus are within the variability of Acanthobothrium. Brooks et al. (1981) pioneered the co-evolutionary studies on freshwater Potamotrygonidae adresssing their origins and host relationships using parasitological data (for marine Potamotrynonidae see Carvalho et al., 2016). They postulated that freshwater potamotrygonids

49 Golfetti, Y. Dissertação de Mestrado, 2018

derived from the Pacific Ocean and colonized the fluvial systems of South America by an urolophid ancestor (= Urolophus, which was later considered a member of Urobatis in Urotrygonidae). This event would have occurred as a result of the orogenesis of the Andes, which isolated the marine ancestor in a body of continental water that later suffered desalination and was integrated into the South American freshwater river system. This was first the study to present a phylogeny for Acanthobothrium. However, the study was hardly criticized for the methods employed, assumptions and taxonomic representation (Caira, 1990; Caira, 1994; Lovejoy, 1997; Simberloff, 1987; Straney, 1982). According to Brooks et al. (1981) hypothesis, the postulated isolation resulted in a single derivation event of stingrays and their parasites. They also assumed that the parasite monophyly would provide evidence for the monophyly of their host considering that both lineages were under co-evolutionary process (Brooks et al., 1981; Lovejoy, 1997). This hypothesis was reiterated by Brooks & Amato (1992:589): "It would appear that the helminth fauna of potamotrygonids is an assemblage of monophyletic groups and single species, supporting the hypothesis that potamotrygonids arose from a single invasion of freshwater habitats in South America.". However, as (Lovejoy, 1997:220) pointed out, any evaluation of host evolutionary history based on parasitological data have also to include "(1) data indicating which parasite species and lineages occur within different potamotrygonid hosts and (2) estimates of factors (such as host switching) that might obscure the original revolutionary patterns". We should also consider that inference on the monophyly of potamotrygonids stingrays has to consider other historical events besides parasite lineage co-divergence. After a series of studies (Lovejoy, 1996, 1997), Lovejoy et al. (1998) proposed the most accepted hypothesis for the origin of freshwater Potamotrygonids. According to the authors, freshwater potamotrygonids share the most recent common ancestral with the clade of amphi- American species of Styracura, S. schmardae and S. pacifica (then considered members of Himantura, Carvalho et al., 2016). The colonization of the freshwater river systen, argued Lovejoy et al. (1998), took place in the early Miocene during the marine incursions that occurred in the northern region of South America Lovejoy et al. (1998). This hypothesis is well supported by morphological, molecular, geological and parasitological data (Carvalho et al., 2016; Hoberg et al., 1988; Lovejoy, 1996, 1997; Lovejoy et al., 1998; Marques, 2000; Naylor et al., 2012; Trevisan & Marques, 2017). Since the publication of Lovejoy et al. (1998), parasitologists have tried to understand better the coevolutionary history between freshwater stingrays and their parasites. These studies, however, were still providing the taxonomic foundations required for historical association studies for parasite taxa such as Monogenoidea (Platyhelminthes, Cercomeromorpha) (Domingues & Marques, 2010), Potamotrygonocestus (Luchetti, 2011; Marques, 2000),

50 Rhinebothroides (Bueno, 2010), Rhinebothrium (Marques & Reyda, 2015; Reyda & Marques, 2011) and Acanthobothrium (Cardoso Jr., 2010; Machado, 2012). Although the criticisms pointed by Lovejoy (1997) have merit, there is one assumption that has been historically neglected by those ins=terested in the evoluvtion of this system (Brooks et al., 1981; Brooks, 1977; Lovejoy, 1997; Lovejoy et al., 1998). This is particularly true when the main goal is to use parasites to infer monophyly of freshwater potamotrygonids. All these studies assumed that the ancestor of freshwater potamotrygonids was infected by monophyletic groups of parasites. However, empirical data, including this, refute this assumption. The first molecular evidence for the non-monophyly of freshwater lineages of Acanthobothrium was provided by Machado (2012). This was later corroborated by Trevisan (2016) and now for this study. Freshwater lineages of Acanthobothrium are nested in two unrelated clades, one associated with Pacific species and other related to Atlantic species. The Atlantic group is closely related to Acanthobothrium species inhabiting rays from Northwest Atlantic and to a clade from Senegal (Clade III (ETP)) (Fig.3) (Machado, 2012). The phylogeny of Acanthobothrium (Fig.1) also reveal this interesting pattern - that is, host species not necessarily are infected by monophyletic assemblages of Acanthobothrium species. For instance, S. schmardae is inhabited by Acanthobothrium sp. 5 and Acanthobothrium sp. 3 sensu Trevisan, 2016; whereas H. guttatus is parasitized by Acanthobothrium n. sp. 4 and Acanthobothrium sp. 10. In none of these cases, the parasite lineages are phylogenetically related. Both Acanthobothrium lineages, nested within marine and freshwater lineages, sharing the same host, is an evidence of that to evaluate host history based in parasites data have to be looked wisely and be supported by other studies. In addition, we also observed phylogenetic relationships between marine and freshwater parasites lineages, far related, inhabiting the same host species. For instance, S. schmardae harbours the close marine lineages Acanthobothrium sp. 1 and Acanthobothrium sp. 3 sensu Trevisan, 2016, whereas the marine lineage Acanthobothrium sp. 9 is nested to the freshwater lineage Acanthobothrium sp. 2. For H. guttatus parasites, it is possible to nest the marine lineages Acanthobothrium n. sp. 3, Acanthobothrium n. sp. 4 and Acanthobothrium sp. 10 to freshwater groups. Our results also indicated that Acanthobothrium sp. 10 is the sister group of a clade composed by Acanthobothrium sp. 9 sensu Trevisan, 2016 and freshwater Acanthobothrium sp. 2 Cardoso Jr, 2010. This clade is sister group of Clade III (ETP) composed by marine species of Acanthobothrium from Senegal (Cardoso Jr., 2010; Machado, 2012; Trevisan, 2016) (Fig.3). The clade Acanthobothrium sp. 10 + Acanthobothrium sp. 9 + Acanthobothrium sp. 2 is closely related to Acanthobothrium lineages from the Atlantic and the Caribbean Sea, whereas the other lineages of freshwater Acanthobothrium are related to marine Pacific lineages (Machado, 2012). This close relationship of Acanthobothrium sp. 2 is one of the evidences of

51 Golfetti, Y. Dissertação de Mestrado, 2018

the non-monophyly of freshwater species of Acanthobothrium. The monophyly of South American freshwater lineages of cestodes, according to Brooks et al. (1981), considered all freshwater Acanthobothrium lineages closely nested to the Southeastern Pacific lineages. Looking at the phylogenetic pattern recovered in our analysis and previous analysis (Machado, 2012), there is no evidence to support the close relationship of Acanthobothrium sp. 2 with Pacific lineages. Our phylogenetic analyses of molecular data corroborated the previous studies (Machado, 2012; Trevisan, 2016) and suggested the non- monophyly of the freshwater lineages of Acanthobothrium. The present study increased our knowledge on estuarine species for Acanthobothrium of Bay of Marajó and on the phylogenetic relationships between the Acanthobothrium species of the Northwest Atlantic and South American freshwater systems. However, this is just the first step for the complete understanding of this relation. Except for the single specimen of Acanthobothrium sp. 10 found in H. guttatus, there is no evidence of parasite sharing between marine and freshwater stingrays. It can be associated with biotic and abiotic characteristics of the estuary, intermediary hosts availability, capacity to the contact of intermediary host and definitive host, capacity of establishment of the parasite in a host, in addition to sample size (Bush et al., 2001). Nonetheless, estuarine stingrays, their parasites, and estuarine environment should be observed with more attention in the future. In order to a better understand the relationships of, freshwater, estuarine and marine species of Acanthobothrium, the full appreciation of the Acanthobothrium fauna of Northwest Atlantic and their nested estuaries, is necessary. In addition, understand correctly the life cycle of marine cestodes parasites of stingrays, besides the increasing the number of hosts specimens analyzed in future studies (see section "Patterns of host specificity and parasite distribution"), is crucial it to interpret correctly the evolutionary relationships in brackish water Acanthobothrium.

52 Conclusions

The diagnose of Fontitrygon colarensis is not consistent and should be revised as well as its taxonomic status. Host sample size in previous studies have shaped our understanding on patters of host speciﬁcity (strict) and biogeographical distribution for Acanthobothrium. Acanthobothroides should be synonimyzed as Acanthobothrium. The monophyly of Acanthobothrium, was recovered as based on molecular data but with the inclusion of two species assigned to Acanthobothroides. The lineage Onchobothrium? ex Hypanus sabinus [AC09/TM-9] seems to be misidentiﬁed and should be considered as a member of Acanthobothrium. Six new lineages of Acanthobothrium were found in Bay of Marajó. Four new species of Acanthobothrium were described for Dasyatidae stingrays in Bay of Marajó. The four new species of Acanthobothrium are exclusively related to marine lineages of the Northwest Atlantic Acanthobothrium n. sp. 1 and Acanthobothrium n. sp. 2 presented full overlap of characters ranges and can only be distinguished on the basis of molecular data. Acanthobothrium n. sp. 3 and Acanthobothrium himanturi presented full overlap of characters ranges. The lineage Acanthobothrium sp. 10 are related to a clade that includes the marine Acanthobothrium sp. 9 sensu Trevisan, 2016 and freshwater Acanthobothrium sp. 2 Cardoso Jr. (sensu 2010) References

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60 Resumo

A documentação de organismos parasitas tem sido uma ferramenta importante para entender a história de seus hospedeiros e os processos coevolutivos implícitos nessas associações. Acanthobothrium é um gênero de cestóideos mundialmente distribuido, parasita de tubarões, "skates" e raias, com quase 200 espécies nominais. Estudos recentes vem apresentando novas hipóteses sobre a distribuição e especificidade dos cestóides. Devido à sua larga distribuição geográfica e em taxons hospedeiros, Acanthobothrium parece ser um bom modelo para avaliar estas hipóteses. A Baía de Marajó é uma área estuarina onde arraias de água doce da família Potamotrygonidae compartilham o mesmo ambiente com raias marinhas, especialmente dasiatídeos. Não há documentação sobre a fauna de Dasyatidae para Acanthobothrium a Baía de Marajó, nem sobre as relações destas com linhagens de água doce e seus hospedeiros. Com o objetivo de entender esses eventos evolutivos, nossos resultados mostraram seis novas linhagens de Acanthobothrium, parasitas de Hypanus guttatus e Fontitrygon geijskesi, das quais quatro são descritas. Cinco dessas novas linhagens estão incluídas em um clado exclusivamente do Noroeste Atlântico e do Mar do Caribe. Acanthobothrium sp. 10 foi recuperado como grupo irmão de um clado formado pela linhagem marinha Acanthobothrium sp. 9 sensu Trevisan e pela linhagem de água doce Acanthobothrium sp. 2 sensu Cardoso Jr. O padrão de especificidade de Acanthobothrium tem sido discutido e nossos resultados corroboram esta discussão quando observamos quatro espécies de Acanthobothrium compartilhando duas hospedeiras de diferentes gêneros. Além disso, tamanho amostral de hospedeiros pode estar relacionado com nosso entendimento sobre os padrões de especificidade estrita do parasita aos seus hospedeiros. A ausência de F. colarensis em nossas amostras e as incongruências em comparação com H. guttatus nos fazem questionar o status taxonômico de F. colarensis.

Palavras-chave: Coevolução, Neotropical, Especﬁcidade, Biogeograﬁa, Tamanho amostral.

Abstract

Parasite documentation has been an important tool to understand host history and co- evolutionary processes in these associations. Acanthobothrium is a worldwide genus of cestodes, and it is a parasite of sharks, skates and rays, with almost 200 nominal species. Recent studies are presenting new hypotheses on cestodes distribution and host specificity patterns. Due to their large distribution, geographical and in host taxa, Acanthobothrium seems to be a good model to evaluate these hypothesis. The Bay of Marajó is an estuarine area were freshwater stingrays of the family Potamotrygonidae share the same environment with marine dasyatid rays. There is no documentation about the dasyatid fauna of Acanthobothrium for Bay of Marajó, neither their relationships with freshwater lineages or their hosts. In our goal to understand those evolutionary events, our results revealed six new lineages of Acanthobothrium, parasites of Hypanus guttatus and Fontitrygon geijskesi, of which four are formally described. Five of those new lineages are included in a clade exclusive to Northwest Atlantic and Caribbean Sea. Acanthobothrium n. sp 10 was recovered as sister of clade formed by marine Acanthobothrium sp. 9 sensu Trevisan and freshwater Acanthobothrium sp. 2 sensu Cardoso Jr. The specificity pattern of Acanthobothrium has been discussed and our results corroborate this discussion when we observed four species of Acanthobothrium sharing two different hosts of different genera. Also, host sample size may be correlated with the specificity and strict specificity patterns of the parasite to their hosts. The absence of Fontitrygon colarensis in our samples and the incongruities in comparison to Hypanus guttatus make us question the taxonomic status of F. colarensis.

Keywords: Co-evolution, Neotropical, Host specﬁcity, Biogeography, Sample size

Appendices Appendix 66

A.1 RScript used for Primary Component Analyses (PCA)

#!/usr/bin/R library("RSvgDevice") library(MASS) raw.data <- read.table(file = "File", header = T, sep = "t", na.strings="NA") data <- raw.data[, first column of data: last column of data]# all character available host=raw.data$Host rownames(data)=host colors = c("red", "blue", "black") [unclass(raw.data$clade)] #This look substitutes NA for mean of the variable by all sample for (i in 1:ncol(data)) m = mean(na.omit(data[,i])) x = data[,i] x[is.na(x)] = m data[,i] = x #PCA_analises pc.data <- princomp(data, cor=TRUE, scores=TRUE) summary(pc.data) plot(pc.data) pdf("file.pdf", width = 10, height = 20) par(mfrow=c(2,1)) biplot(pc.data) plot(pc.data$scores[,1:2], pch = 15, col=colors, xlab="PC 1 (X% explained var.)", ylab="PC 2 (Y% explained var.)") text(pc.data$scores[,1:2], rownames(data),cex=0.3,pos=4) dev.off() 67 Appendix

A.2 DNA extraction protocol with Ammonium Acetate

Tissues in ethanol: remove the alcohol, press the paper into absorbent paper or put in a vacuum pump for 5 minutes, or in the oven (37°C.) for 15 minutes.

1. Cut the tissue into very small pieces and add 300 ml of lysis solution (Cell lysis solution (pH 8) - 1.21g of Tris base (10 mM), 37.4 g EDTA (100 mM), 20 g SDS (2%) 1 liter of H2O miliQ). 2. Add 5 휇푙 (at most 10 휇푙) of Proteinase-K (20 mg / ml). Mix the vortex and incubate at 65°C for one hour or until the glass is completely diluted (leave overnight at 5°C).

3. Let the sample cool and add 300 휇푙 of ammonium acetate (500 g of 푁퐻4퐶퐻3퐶푂2/ 865 ml of H2O). Vortex and incubate in ice (or freezer) for 30 minutes. 4. Centrifuge for 10 minutes (13,000 rpm at 4°C). 5. Transfer the supernatant to the new tubes. 6. Add 600 l of absolute isopropanol and inverter the tube gently. For parts with low extraction yield, sample as -20°C overnight to aid in precipitation. 7. Centrifuge for 10 minutes (13,000 rpm at 4°C) and discard the supernatant. 8. Add 600 휇푙 of 70% ethanol. 9. Centrifuge for 10 minutes (13,000 rpm at 4°C). Discard the ethanol. Invert the tube onto absorbent paper and dry the pellet in the vacuum pump for 30 minutes or at room temperature overnight. 10. Re-suspend the DNA in TE (50 or 100 휇푙) overnight at room temperature or at 37°C. Store in a refrigerator (2 to 8°C). Appendix 68

A.3 DNAdvance Extraction ProtocolTM

1. Remove the alcohol from the tube. Allow to dry to all alcohol. 2. Add 80 휇푙 of the following mix for each sample: lysis buffer 73 휇푙, 1M DTT 3 휇푙, Proteinase- K 4 휇푙. 3. Incubate at 55°C until the tissue is completely diluted (overnight if necessary). 4. Centrifuge to ensure that there is no condensation solution on the cap. 5. Add 50 휇푙 of the BIND 1 solution. 6. Vortex the BIND solution to resuspend the iron until it is homogeneous. 7. Add 80 휇푙 of the BIND 2 solution by mixing with the pipette. 8. Incubate at room temperature for 2 minutes. 9. Place on the magnetic plate and incubate for 5 minutes. 10. With the tube still on the plate, remove the supernatant taking care that the tip does not touch the walls of the tube. Discard the supernatant. 11. Add 150 휇푙 of 70% cooled Ethanol. 12. Repeat step 10 and 11. 13. Remove the alcohol again, taking care with the iron and making sure that nothing is left in the bottom of the tube. 14. Dry at room temperature. 15. Resuspend the iron in 50 휇푙 of elution Buffer. 16. Place on magnetic board for 5 minutes. 17. Separate the supernatant into another tube. 69 Appendix

A.4 Puriﬁcation Protocol with AMPURE

1. Vortex the AMPURE iron solution until it is homogeneous. 2. For each 1 휇푙 of PCR reaction, add 1.8 휇푙 of the AMPURE solution (usually 41.4). 3. Pipette to mix the AMPURE with the PCR (if using the vortex, give a quick spin). 4. Incubate for 5 minutes at room temperature. 5. Place ﬁrmly on the magnetic plate. 6. Incubate for 2 minutes. 7. With the tube still on the plate, remove the supernatant taking care that the tip does not touch the walls of the tube. Discard the supernatant. 8. Add 150 휇푙 of 70% cooled Ethanol 9. Repeat step 7 and 8 (total of 2 washes with alcohol). 10. Remove the alcohol again, taking care of the iron and making sure there is nothing left in the bottom of the tube. 11. Dry at 50°C for 3 minutes (dry bath or thermocycler if any are available). 12. Resuspend the iron in 15 휇푙 of miliQ water. 13. Centrifuge for 2 minutes at 13000 rpm. 14. Dose in the NanoDrop or agarose gel with Low Mass. A.5 Dasyatidae nucleotide sequences for MT-ATP6, MT-CYB and MT-ND2 for terminals used as outgroups

Dasyatidae nucleotide sequences, used to constitute hosts out group, with sequences of mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 2 (MT-ND2), mitochondrially encoded cytochrome b (MT-CYB) and mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6), obtained from NCBI GenBank.

Species MT-ATP6 MT-CYB MT-ND2

Neotrygon kuhlii NC_02176 NC_02176 — Dasyatis brevis JN184058 JN184058 — Hemitrygon akajei NC_021132 NC_021132 — Hypanus dipterurus — DQ082911 JQ518782 Hypanus americanus — — JN184288/ JQ518789 Hypanus sabinus — — JQ518787 Fontitrygon margaritella — — JQ518786 Fontitrygon margarita — — JQ518784 Bathytoshia brevicaudata — — JQ519028 Bathyoshia centroura — — JQ518781/ KY909637/ KY909636/ KY909635/ KY909634/ KY909633/ KY909632 A.6 Cestode nucleotide sequences acquired from previous studies

Cestode nucleotide sequences acquired from Fyler (2009), Machado (2012), Caira et al. (in prep) and Trevisan (2016) used to phylogenetic analyses with molecular codes, host codes, parasite specie, Hosts and Collection location, Coordinates and sequences obtained from mitochondrially encoded 16S RNA (MT-RNR2) and 28S cytoplasmatic ribosomal RNA (LSU) genes for each sample.

Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

A038 PR09-006.06 Acanthobothrium Potamotrygon Itaipu Dam, Brazil 25°24’45.96”S, Machado (2012) • • quinonesi motoro 54°35’39.24”W A055 MS10-043.01 Acanthobothrium Potamotrygon Rio Salobra, Miranda, MS, 20°12’59.34”S, Machado (2012) • • quinonesi falkneri Brazil 56°29”42.95” W A064 AL10-004.01 Acanthobothrium Hypanus guttatus Costa de Alagoas, Maceió, 9°40’25.32"S, Machado (2012) • • sp. Brazil 35°44’07.08"W A065 AL10-004.02 Acanthobothrium Hypanus guttatus Costa de Alagoas, Maceió, 9°40’25.32"S, 35° 44’ Machado (2012) • • sp. Brazil 07.08"W A096 VZ11-025.17 Acanthobothrium Paratrygon Rio Apure/Guaritico, 7°53’32.50"N, Machado (2012) • • sp. 1 sensu aiereba Venezuela 68°52’49.80"W Cardoso, 2010 A100 VZ11-018.04 Acanthobothrium Potamotrygon Laguna Sinamaica (Rio 11°2’39.00"N, Machado (2012) • • cf. quinonesi yepezi Limón), Venezuela 71°51’42.50"W A106 VZ11-025 Acanthobothrium Paratrygon Rio Apure/Guaritico, 7°53’32.50"N, Machado (2012) • - terezae aiereba Venezuela 68°52’49.80"W A112 VZ11-031.01 Acanthobothrium Paratrygon Rio Apure/Guaritico, 7°53’32.50"N, Machado (2012) • - terezae aiereba Venezuela 68°52’49.80"W

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A114 VZ11-026.04 Acanthobothrium Potamotrygon Rio Apure/Guaritico, 7°53’32.50"N, Machado (2012) • • sp. 1 sensu orbignyi Venezuela 68°52’49.80"W Cardoso, 2010 A117 RN11-043.03 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • amazonensis schroederi Barcelos, AM, Brazil 62°46’37.92”W A118 RN11-058.04 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • sp. 2 sensu schroederi Barcelos, AM, Brazil 62°46’37.92”W Cardoso, 2010 A121 RN11-058.03 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • cf. sp. 2 sensu schroederi Barcelos, AM, Brazil 62°46’37.92”W Cardoso, 2010 A122 RN11-058.06 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • sp. 1 sensu schroederi Barcelos, AM, Brazil 62°46’37.92”W Cardoso, 2010 A131 RN11-039.01 Acanthobothrium Paratrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • sp. 1 sensu aiereba Barcelos, AM, Brazil 62°46’37.92”W Cardoso, 2010 A150 RN11-050.01 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • quinonesi motoro Barcelos, AM, Brazil 62°46’37.92”W A154 RN11-056.01 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • amazonansis motoro Barcelos, AM, Brazil 62°46’37.92”W

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A160 RN11-063.02 Acanthobothrium Potamotrygon Rio Solimões/Negro/Branco, 0°52’11.28”S, Machado (2012) • • cf. amazonensis orbignyi Barcelos, AM, Brazil 62°46’37.92”W A162 BE-3-13 Acanthobothrium Styracura Gulf of Honduras, Head 16°13’20.8"N, Machado (2012) • • sp. schmardae Caye, Toledo, Belize 88°35’38.3"W A163 BE-3-14 Acanthobothrium Styracura Gulf of Honduras, Head 16°13’20.8"N, Machado (2012) • • sp. schmardae Caye, Toledo, Belize 88°35’38.3"W A164 BE-3-42 Acanthobothrium Hypanus guttatus Gales Point Manatee. Belize 17°13’29.9"N, Machado (2012) • • sp. 88°20’16.2"W AC01 AF-89 Acanthobothrium Dasyatis Indian Ocean, South Africa 34°1.5’S, 25°46.61’E Caira et al. (in •• sp. chrysonota prep) AC02 AF-63 Acanthobothrium Gymnura Indian Ocean, South Africa 34°27.36’S, Caira et al. (in •• sp. natalensis 21°33.20’E prep) AC03 AF-74 Acanthobothrium Myliobatis Indian Ocean, South Africa 34°14.51’S, Caira et al. (in •• sp. aquila 22°14.65’E prep) AC04 AF-139 Acanthobothrium Narke capensis Indian Ocean, South Africa 33°51.67’S, Caira et al. (in •• sp. 26°14.82’E prep) AC05 AF-72 Acanthobothrium Torpedo Indian Ocean, South Africa 34°23.33’S, Caira et al. (in •• sp. fuscomaculata 21°55.06’E prep) AC06 BJ-338 Acanthobothrium Aetobatus Gulf of California, 25°49’52"N, Caira et al. (in •• sp. laticeps Baja California Sur, Loreto, 111°19’38"W prep) Paciﬁc Ocean, Mexico

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AC07 BE-3 Acanthobothroides Styracura Gulf of Honduras, Head 16°13’20.8"N, Caira et al. (in •• thorsoni schmardae Caye, Toledo, Belize 88°35’38.3"W prep) AC08 AF-73 Acanthobothrium Rostroraja alba Indian Ocean, South Africa 34°19.86’S, 22°3.72’E Caira et al. (in •• sp. 1 sensu prep) Cardoso, 2010 AC09 TM-9 Onchobothrium ? Hypanus sabinus — — Caira et al. (in •• prep) AC10 AF-141 Acanthobothrium Acroteriobatus Indian Ocean, South Africa 33°51.67’S, Caira et al. (in •• sp. 1 sensu annulatus 26°14.82’E prep) Cardoso, 2010 AC12 AF-73 Acanthobothrium Rostroraja alba Indian Ocean, South Africa 34°19.86’S, 22°3.72’E Caira et al. (in •• sp. 2 sensu prep) Cardoso, 2010 AC13 BJ-637 Acanthobothrium Gymnura Gulf of California, Bahia de 28°59’9"N, Caira et al. (in •• sp. crebripunctata Los Angeles, Pacific Ocean 113°32’53"W prep) AC14 BJ-175 Acanthobothrium Gymnura Gulf of California, Bahia de 28°59’9"N, Caira et al. (in •• sp. marmorata Los Angeles, Pacific Ocean 113°32’53"W prep) AC15 SO-3 Acanthobothrium Pastinachus ater Western Province, Solomon 8°12’27.5"S, Caira et al. (in •• sp. Sea, Solomon Islands, Pacific 156°59’26.1"E prep) Ocean

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AC16 CM03-61 Acanthobothrium Aetomylaeus Gulf of Carpentaria, 12°35’11"S, Caira et al. (in •• sp. vespertilio Queensland, Weipa, Indian 141°42’34"E prep) Ocean, Australia AC17 KJG-17 Acanthobothrium Parascyllium Pacific Ocean, Australia — Caira et al. (in •• sp. collare prep) AC18 KA-162 Acanthobothrium Pastinachus Java Sea, Borneo, 01°03’30.60"N, Caira et al. (in •• sp. gracilicaudus West Kalimantan, Selakau, 108°58’24.60"E prep) Indonesia, Pacific Ocean AC19 AF-4 Acanthobothrium Holohalaelurus Indian Ocean, South Africa 36°17.60’S, 20°6.6’E Caira et al. (in •• sp. regani prep) AC20 AF-65 Acanthobothrium Mustelus Indian Ocean, South Africa 34°31.71’S, Caira et al. (in •• sp. mustelus 21°18.99’E prep) AC21 KJG-38 Acanthobothrium Trygonoptera sp. Pacific Ocean, Australia — Caira et al. (in •• sp. prep) AC23 BO-431 Acanthobothrium Chiloscyllium South China Sea, Borneo, 02°53’52.16"N, Caira et al. (in •• sp. punctatum Sarawak, Mukah, Malaysia, 112°05’44.12"E prep) Pacific Ocean AC24 KJG-40 Acanthobothrium Aptychotrema Pacific Ocean, Australia — Caira et al. (in •• sp. rostrata prep) AC25 BE-8 Acanthobothrium Aetobatus Tobacco Caye, Stann Creek, 16°54’3.5"N, Caira et al. (in •• tortum narinari Dangriga, Belize 88°03’40.3"W prep)

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AC26 CRP-55 Acanthobothrium Narcine Costa Rica, Pacific Ocean 10°42’29.592"N, Caira et al. (in •• inibiorum entemedor 85°40’9.264"W prep) AC27 CRP-71 Acanthobothrium Zapteryx xyster off Herradura, Pacific Ocean 9°30’02.0"N, Caira et al. (in •• sp. 84°41’32.0"W prep) AC28 CRP-51 Acanthobothrium Urotrygon sp. Costa Rica, Pacific Ocean 10°42’29.592"N, Caira et al. (in •• sp. 85°40’9.264"W prep) AC29 CRP-64 Acanthobothrium Diplobatis off Herradura, Pacific Ocean 09°25’5"N, Caira et al. (in •• sp. ommata 84°38’34"W prep) AC30 CRP-56 Acanthobothrium Hypanus longus Costa Rica, Pacific Ocean 10°42’29.592"N, Caira et al. (in •• sp. 85°40’9.264"W prep) AC31 SO-32 Acanthobothrium Urogymnus Western Province, 8°2’15.1"S, Caira et al. (in •• sp. asperrimus near Sagheraghi, Solomon 156°45’57.1"E prep) Sea, Solomon Islands, Pacific Ocean b033 PN15-56.4 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 5 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b034 PN15-56.5 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 9 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code b035 PN15-56.6 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 9 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b039 PN15-09.05 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 8 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b040 PN15-09.6 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 8 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b044 PN15-14.4 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 7 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b045 PN15-25.2 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 7 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b047 PN15-51.1 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 9 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b048 PN15-52.5 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi schmardae Bocas Del Toro, Panama b049 PN15-52.6 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi schmardae Bocas Del Toro, Panama

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code b050 PN15-52.7 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi schmardae Bocas Del Toro, Panama b052 PN15-53.4 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi schmardae Bocas Del Toro, Panama b053 PN15-53.5 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi schmardae Bocas Del Toro, Panama b054 PN15-53.6 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • himanturi sp. 3 schmardae Bocas Del Toro, Panama sensu Trevisan, 2016 b055 PN15-54.4 Acanthobothrium Styracura Caribbean Sea, Tobacco 16°54’N, 88°03’W Trevisan (2016) • • sp. 3 sensu schmardae Caye, Dangriga, Stann Trevisan, 2016 Creek, Belize b056 BE-09-15 Acanthobothrium Styracura Caribbean Sea, Tobacco 16°54’N, 88°03’W Trevisan (2016) • • sp. 5 sensu schmardae Caye, Dangriga, Stann Trevisan, 2016 Creek, Belize b058 TT14-06.5 Acanthobothrium Styracura Maracas bay, Maracas, San 10°45’N, 61°26’W Trevisan (2016) • • sp. 6 sensu schmardae Juan-Laventille, Trinidad & Trevisan, 2016 Tobago b076 PN15-09.9 Acanthobothrium Styracura eastern Paciﬁc Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 6 sensu paciﬁca Caleta, Montijo, Veráguas, Trevisan, 2016 Panama

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code b077 PN15-09.10 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 6 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b078 PN15-09.11 Acanthobothrium Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • sp. 6 sensu pacifica Caleta, Montijo, Veráguas, Trevisan, 2016 Panama b079 PN15-53.7 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 1 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b080 PN15-53.8 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 1 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b081 PN15-53.9 Acanthobothrium Styracura Caribbean Sea, Almirante, 09°17’N, 82°20’W Trevisan (2016) • • sp. 1 sensu schmardae Bocas Del Toro, Panama Trevisan, 2016 b084 PN15-12.01 Acanthobothroides Styracura eastern Pacific Ocean, Playa 07°29’N, 81°13’W Trevisan (2016) • • pacificus pacifica Caleta, Montijo, Veráguas, Panama CF01 NT-37 Acanthobothrium Himantura Northen Territory, east of 11°17’44"S, Fyler (2009) • • sp. leoparda Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF02 BJ-107 Acanthobothrium Narcine Bahia de Los Angeles, Baja 28°59’9"N, Fyler (2009) • • sp. entemedor California, Pacific Ocean, 113°32’53"W Mexico CF03 SE-205 Acanthobothrium Rhinobatos Atlantic Ocean, Senegal 14°40’42"N, Fyler (2009) • • sp. rhinobatos 17°27’42"W CF04 BJ-243 Acanthobothrium Raja velezi Santa 27°19’51"N, Fyler (2009) • • sp. Rosalia, Baja California Sur, 112°15’30"W Pacific Ocean, Mexico CF05 BO-496 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • etini polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia CF06 SE-222 Acanthobothrium Dasyatis sp. Mbour, Atlantic Ocean, 14°24’22"N, Fyler (2009) • • cf. woodsholei Senegal 16°58’6"W CF07 SE-56 Acanthobothrium Raja miraletus Atlantic Ocean, Senegal 14°40’42"N, Fyler (2009) • • sp. 17°27’42"W CF09 NT-105 Acanthobothrium Pastinachus ater Northen Territory, east of 11°17’44"S, Fyler (2009) • • sp. Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF24 KJG-1 Acanthobothrium Brachaelurus Coast of Sydney, Pacific — Fyler (2009) • • sp. waddi Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF25 KJG-7 Acanthobothrium Hypnos Coast of Sydney, Pacific — Fyler (2009) • • cf. angelae monopterygius Ocean, Australia CF26 KJG-7 Acanthobothrium Hypnos Coast of Sydney, Pacific — Fyler (2009) • • cf. angelae monopterygius Ocean, Australia CF31 NT-7 Acanthobothrium Rhynchobatus Nhulunbuy (Gove), Northern 12°11’38"S, Fyler (2009) • • mattaylori laevis Territory, Gove 136°41’11"E Harbor, Gulf of Carpentaria, Pacific Ocean, Australia CF32 NT-7 Acanthobothrium Rhynchobatus Nhulunbuy (Gove), Northern 12°11’38"S, Fyler (2009) • • jaenneae laevis Territory, Gove 136°41’11"E Harbor, Gulf of Carpentaria, Pacific Ocean, Australia CF33 NT-7 Acanthobothrium Rhynchobatus Nhulunbuy (Gove), Northern 12°11’38"S, Fyler (2009) • • hypermekkolpos laevis Territory, Gove 136°41’11"E Harbor, Gulf of Carpentaria, Pacific Ocean, Australia CF34 NT-7 Acanthobothrium Rhynchobatus Nhulunbuy (Gove), Northern 12°11’38"S, Fyler (2009) • • hypermekkolpos laevis Territory, Gove 136°41’11"E Harbor, Gulf of Carpentaria, Pacific Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF35 NT-66 Acanthobothrium Rhynchobatus cf. Northen Territory, east of 11°17’44"S, Fyler (2009) • • herronorum laevis Wessel Islands, Arafura Sea, 136°59’48"E Paciﬁc Ocean, Australia CF37 AZ-10 Acanthobothrium Dipturus batis Horta, 38°31’N, 28°37’W Fyler (2009) • • sp. Azores Islands, Faial Island, Atlantic Ocean, Portugal CF40 SE-134 Acanthobothrium Glaucostegus Atlantic Ocean, Senegal 14°40’42"N, Fyler (2009) • • sp. cemiculus 17°27’42"W CF41 SE-134 Acanthobothrium Glaucostegus Atlantic Ocean, Senegal 14°40’42"N, Fyler (2009) • • sp. cemiculus 17°27’42"W CF45 SE-143 Acanthobothrium Aetomylaeus St. Louis, Atlantic Ocean, 16° 1’28"N, Fyler (2009) • • sp. bovinus Senegal 16°30’33"W CF46 BO-409 Acanthobothrium Neotrygon cf. Sarawak, 02°49’01.20"N, Fyler (2009) • • sp. kuhlii Borneo, South China Sean, 110°52’47.16"E Pacifc Ocean, Malaysia CF47 MS05-402 Acanthobothrium Hypanus say Florida, Crooked Island Bay, 29°59’37"N, Fyler (2009) • • cf. brevissime Gulf of Mexico, Atlantic 85°31’48"W Ocean, U.S.A. CF50 MS05-431 Acanthobothrium Hypanus sabinus Florida, Indian Pass, Gulf 29°40’8"N, Fyler (2009) • • sp. of Mexico, Atlantic Ocean, 85°13’30"W U.S.A.

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF51 MS05-421 Acanthobothrium Raja eglanteria Florida, Indian Pass, Gulf 29°40’8"N, Fyler (2009) • • sp. of Mexico, Atlantic Ocean, 85°13’30"W U.S.A. CF52 MS05-421 Acanthobothrium Raja eglanteria Florida, Indian Pass, Gulf 29°40’8"N, Fyler (2009) • • sp. of Mexico, Atlantic Ocean, 85°13’30"W U.S.A. CF53 BO-268 Acanthobothrium Urogymnus Mukah, Sarawak, Borneo, 02°53’52.16"N, Fyler (2009) • • sp. lobistoma South China Sean, Pacifc 112°05’44.12"E Ocean, Malaysia CF61 SE-222 Acanthobothrium Dasyatis sp. Mbour, Atlantic Ocean, 14°24’22"N, Fyler (2009) • • sp. Senegal 16°58’6"W CF65 SE-129 Acanthobothrium Paragaleus Ouakam, Atlantic Ocean, 14°42’54"N, Fyler (2009) • • sp. pectoralis Senegal 17°29’28"W CF68 CM03-3 Uncibilocularis Himantura cf. Weipa, Queensland, Gulf of 12°35’11"S, Fyler (2009) • • squireorum urarnak Carpentaria, Indian Ocean, 141°42’34"E Australia CF69 KJG-1 Acanthobothrium Brachaelurus Coast of Sydney, Paciﬁc — Fyler (2009) • • sp. waddi Ocean, Australia CF70 NT-117 Acanthobothrium Himantura Northen Territory, east of 11°17’44"S, Fyler (2009) • • cf. lasti leoparda Wessel Islands, Arafura Sea, 136°59’48"E Paciﬁc Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF75 SE-275 Acanthobothrium Gymnura Kafountine, Casamance, 12°55’41"N, Fyler (2009) • • sp. altavela Atlantic Ocean, Senegal 16°45’10"W CF77 SE-305 Acanthobothrium Gymnura Joal, Atlantic Ocean, Senegal 14°10’30"N, Fyler (2009) • • sp. altavela 16°51’12"W CF78 SE-199 Acanthobothrium Zanobatus Atlantic Ocean, Senegal 14°40’42"N, Fyler (2009) • • sp. schoenleinii 17°27’42"W CF79 SE-224 Acanthobothrium Taeniura grabata Mbour, Atlantic Ocean, 14°24’22"N, Fyler (2009) • • sp. Senegal 16°58’6"W CF80 SE-234 Acanthobothrium Fontitrygon Djifere, Atlantic Ocean, 13°55’50”N, Fyler (2009) • • sp. margaritella Senegal 16°45’42”W CF85 PU-4 Acanthobothrium Potomotrygon cf. Boca Manu, Madre de Dios, 12°16’S 70°55’W Fyler (2009) • • cf. terezae motoro Madre de Dios River, Peru CF87 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • rodmani Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF89 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • romanowi Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF93 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • oceanharvestae Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF100 TW-19 Acanthobothrium Orectolobus Magong, Penghu, Taiwan 23°33’49"N, Fyler (2009) • • margieae japonicus Strait, Pacific Ocean, Taiwan 119°34’31"E CF103 KA-43 Acanthobothrium Himantura Muara Pasir, 01°45’58.92"S, Fyler (2009) • • sp. undulata East Kalimantan, Borneo, 116°23’36.09"E Makassar Strait, Pacific Ocean, Indonesia CF105 BJ-300 Acanthobothrium Heterodontus Santa 27°19’51"N, Fyler (2009) • • santarosaliense mexicanus Rosalia, Baja California Sur, 112°15’30"W Pacific Ocean, Mexico CF108 BJ-626 Acanthobothrium Myliobatis Bahia de Los Angeles, Baja 28°59’9"N, Fyler (2009) • • cf. microcephalum californica California, Pacific Ocean, 113°32’53"W Mexico CF116 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • romanowi Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF120 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • popi Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF121 NT-96 Acanthobothrium. Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • zimmeri Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF122 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • zimmeri Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF124 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • rodmani Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF126 NT-96 Acanthobothrium Himantura sp. Northen Territory, east of 11°17’44"S, Fyler (2009) • • oceanharvestae Wessel Islands, Arafura Sea, 136°59’48"E Pacific Ocean, Australia CF128 TW-19 Acanthobothrium Orectolobus Magong, Penghu, Taiwan 23°33’49"N, Fyler (2009) • • margieae japonicus Strait, Pacific Ocean, Taiwan 119°34’31"E CF130 KA-211 Acanthobothrium Maculabatis cf. Sidu, West Kalimantan, 01°21’45.20"S, Fyler (2009) • • sp. gerrardi Borneo, Java Sea, Pacific 110°04’10.30"E Ocean, Indonesia CF134 BO480 Acanthobothrium Urogymnus Mukah, Sarawak, Borneo, 02°53’52.16"N, Fyler (2009) • • sp. lobistoma South China Sean, Pacifc 112°05’44.12"E Ocean, Malaysia CF136 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • etini polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF138 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • masnihae polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia CF139 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • saliki polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia CF140 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • zainili polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia CF142 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • masnihae polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia CF145 BO-108 Acanthobothrium Urogymnus Kampung Abai, 05°41’10.81"N, Fyler (2009) • • saliki polylepis Kinebatangan River, Sabah, 118°23’08.35"E Borneo, Kinabatangan River, Malaysia

Continued on next page Table A.6 – Continued from previous page Molecular Host code† Parasite species Host Collection location Coordinates Sequence author RNR2 LSU code

CF147 TM-1 Acanthobothrium Raja eglanteria Cape Point, North Carolina, 35°15’N, 75°31’W Fyler (2009) • • sp. Western Atlantic Ocean, U.S.A. CF151 VI-1 Acanthobothrium Potamotrygon Colastiné River, Santa Fé, 31°40’S, 60°46’W Fyler (2009) • • ramiroi motoro Argentina CF152 VI-2 Acanthobothrium Potamotrygon Colastiné River, Santa Fé, 31°40’S, 60°46’W Fyler (2009) • • ramiroi motoro Argentina CF153 KA-57 Acanthobothrium Maculabatis cf. Kota Baru, 03°14’44.80"S, Fyler (2009) • • sp. gerrardi South Kalimantan, Borneo, 116°13’23.80"E Makassar Strait, Paciﬁc Ocean, Indonesia CF154 KA-436 Acanthobothrium Pateobatis fai Manggar, East Kalimantan, 01°12’55.20"S, Fyler (2009) • • sp. Borneo, Makassar Strait, 116°58’27.50"E Paciﬁc Ocean, Indonesia A.7 GenBank nucleotide sequences of cestodes for MT-RNR2 and LSU regions

GenBank nucleotide sequences of Proteocephalidea and Acanthobothrium obtained from mitochondrially encoded 16S RNA (MT-RNR2) and 28S cytoplasmatic ribosomal RNA (LSU) genes and used in present study to compose the MT-RNR2+LSU phylogenetic analyses.

Species Host Marine water body Gen Bank freshwater river system MT-RNR2 LSU

Acanthobothrium spp. Acanthobothrium brevissime Hypanus say Gulf of Mexico, Florida, U.S.A. EU660532 A. parviuncinatum Urobatis maculatus Baja California, Mexico EF095264 Acanthobothrium sp. 1 Hypanus logus Baja California, Mexico AF286953 Proteocephalidea specimens Acanthotaenia sp. Varanus exanthematicus Ghana AJ389480 AJ388593 Gangesia parasiluri Silurus asotus Nagano prefecture, Lake Suwa, Japan AJ389477 AJ388590 Silurotaenia siluri Silurus glanis Orlik water reserve, Czech Republic AJ389479 AJ388592 Scholzia emarginata Phractocephalus hemioliopterus Amazonas, Itacoatiara, Brazil AJ388616 Monticellia piracatinga Calophysus macropterus Amazonas, Itacoatiara, Brazil AJ388627 Ophiotaenia jarara Bothrops jararaca Domingos Martins, Espirito Santo, Brazil AJ388607 Proteocephalus percae Perca ﬂuviatilis Neuchatel Lake, Switzerland AJ388594 Proteocephalus tetrastomus Hypomesus nipponensis Nagano prefecture, Lake Suwa, Japan AJ388635 Spasskyellina lenha Sorubimichthys planiceps Amazonas, Itacoatiara, Brazil AJ388611 Zygobothrium megacephalum Phractocephalus hemioliopterus Amazonas, Itacoatiara, Brazil AJ388621 A.8 Original hosts nucleotide sequences

Original hosts nucleotide sequences collected from hosts of Estuary of Bay of Marajó, Colares, Pará. Brazil. Sequences obtained from mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 2 (MT-ND2), mitochondrially encoded cytochrome b (MT-CYB) and mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) genes.

Host code Specie Collection Location Coordinates MT-ATP6 MT-CYB MT-ND2

PA16-001 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-002 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-007 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-011 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-025 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-026 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-028 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-032 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • - • PA16-034 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-037 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-040 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-041 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-042 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-044 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-045 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-048 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-049 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-050 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • - • PA16-052 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • - •

Continued on next page Table A.8 – Continued from previous page Host code Specie Collection Location Coordinates MT-ATP6 MT-CYB MT-ND2

PA16-053 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-057 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-058 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-059 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-060 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-061 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-062 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-066 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-067 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-068 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-072 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • - • PA16-074 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • - PA16-075 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-076 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-077 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-078 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-079 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-080 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-081 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-082 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • - PA16-083 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-087 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • • PA16-088 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • •

Continued on next page Table A.8 – Continued from previous page Host code Specie Collection Location Coordinates MT-ATP6 MT-CYB MT-ND2

PA16-091 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S-48°17’39.5”W • • - 93 Appendix

2 Hypanus americanus 46 Hypanus sabinus 11 34 Hypanus dipterurus 12 20 Bathyoshia brevicaudata 27 26 19 Bathyoshia centroura 25 76 Fontitrygon margarita 31 Fontitrygon margaritella Hypanus guttatus (PA16-01) Hypanus guttatus (PA16-02) Hypanus guttatus (PA16-07) Hypanus guttatus (PA16-11) Hypanus guttatus (PA16-25) Hypanus guttatus (PA16-32) 5 Hypanus guttatus (PA16-34) 3 Hypanus guttatus (PA16-37) Hypanus guttatus (PA16-40) 23 Hypanus guttatus (PA16-44) Hypanus guttatus (PA16-50) 1 Hypanus guttatus (PA16-53) 16 Hypanus guttatus (PA16-57) Hypanus guttatus (PA16-58) Hypanus guttatus (PA16-62) 4 Hypanus guttatus (PA16-67) 3 Hypanus guttatus (PA16-68) 1 Hypanus guttatus (PA16-75) Hypanus guttatus (PA16-77) Hypanus guttatus (PA16-78) Hypanus guttatus (PA16-80) 17 Hypanus guttatus (PA16-81) 3 Hypanus guttatus (PA16-83) 1 Hypanus guttatus (PA16-87) Hypanus guttatus (PA16-88) 2 Fontitrygon geijskesi (PA16-26) Fontitrygon geijskesi (PA16-42) 2 Fontitrygon geijskesi (PA16-45) Fontitrygon geijskesi (PA16-48) 1 Fontitrygon geijskesi (PA16-49) 2 21 Fontitrygon geijskesi (PA16-52) 1 Fontitrygon geijskesi (PA16-59) 2 Fontitrygon geijskesi (PA16-60) 1 Fontitrygon geijskesi (PA16-61) 1 Fontitrygon geijskesi (PA16-76) Fontitrygon geijskesi (PA16-28) Fontitrygon geijskesi (PA16-66) 1 2 Fontitrygon geijskesi (PA16-72) 3 Fontitrygon geijskesi (PA16-41) 1 Fontitrygon geijskesi (PA16-79)

20.0 Figure A.16: Phylogenetic relationship between hosts, based in mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 2 (MT-ND2) gene, of present work and GenBank sequences under parsimony as the optimality criteria. Contents between parentheses refer to host accesion code. Goodman-Bremer values implemented. Branchs without Goodman-Bremer values are zero. Appendix 94

Hypanus dipterurus

Dasyatis brevis 14 Hemitrygon akajei 39 Neotrygon kuhlii 1 Hypanus guttatus (PA16-81) 1 Hypanus guttatus (PA16-01) 1 Hypanus guttatus (PA16-83) 16 Hypanus guttatus (PA16-02) 1 Hypanus guttatus (PA16-25)

Hypanus guttatus (PA16-40)

Hypanus guttatus (PA16-44)

Hypanus guttatus (PA16-53) 13 2 Hypanus guttatus (PA16-57)

3 Hypanus guttatus (PA16-58) Hypanus guttatus (PA16-62)

Hypanus guttatus (PA16-75)

Hypanus guttatus (PA16-77)

Hypanus guttatus (PA16-80)

Hypanus guttatus (PA16-82)

Hypanus guttatus (PA16-87)

25 Hypanus guttatus (PA16-88) 1 Hypanus guttatus (PA16-11)

Hypanus guttatus (PA16-34)

Hypanus guttatus (PA16-37) 1 3 Hypanus guttatus (PA16-67) 1 Hypanus guttatus (PA16-74)

Hypanus guttatus (PA16-78) 1 Hypanus guttatus (PA16-07) 2 Hypanus guttatus (PA16-68)

Hypanus guttatus (PA16-91)

Fontitrygon geijskesi (PA16-42)

Fontitrygon geijskesi (PA16-79)

Fontitrygon geijskesi (PA16-26) 2 Fontitrygon geijskesi (PA16-28)

Fontitrygon geijskesi (PA16-41)

Fontitrygon geijskesi (PA16-45)

Fontitrygon geijskesi (PA16-48) 1 Fontitrygon geijskesi (PA16-49)

Fontitrygon geijskesi (PA16-59) 1 Fontitrygon geijskesi (PA16-60)

Fontitrygon geijskesi (PA16-61)

Fontitrygon geijskesi (PA16-66)

Fontitrygon geijskesi (PA16-76)

7.0 Figure A.17: Phylogenetic relationship between hosts, based in mitochondrially encoded cytochrome b (MT-CYB) gene, of present work and GenBank sequences under parsimony as the optimality criteria. Contents between parentheses refer to host accesion code. Goodman-Bremer values implemented. Branchs without Goodman-Bremer values are zero. 95 Appendix

Neotrygon kuhlii 21 Dasyatis brevis 18 Hemitrygon akajei Hypanus guttatus (PA16-01) Hypanus guttatus (PA16-02) Hypanus guttatus (PA16-07) Hypanus guttatus (PA16-11) Hypanus guttatus (PA16-25) Hypanus guttatus (PA16-32) 4 Hypanus guttatus (PA16-34) Hypanus guttatus (PA16-37) Hypanus guttatus (PA16-40) Hypanus guttatus (PA16-44) Hypanus guttatus (PA16-50) 1 19 Hypanus guttatus (PA16-53) Hypanus guttatus (PA16-57) 17 Hypanus guttatus (PA16-58) Hypanus guttatus (PA16-62) 5 Hypanus guttatus (PA16-67) Hypanus guttatus (PA16-68) 3 Hypanus guttatus (PA16-74) 1 Hypanus guttatus (PA16-75) Hypanus guttatus (PA16-77) 1 Hypanus guttatus (PA16-78) Hypanus guttatus (PA16-80) Hypanus guttatus (PA16-81) 20 Hypanus guttatus (PA16-82) Hypanus guttatus (PA16-83) Hypanus guttatus (PA16-87) Hypanus guttatus (PA16-88) Hypanus guttatus (PA16-91) Fontitrygon geijskesi (PA16-26) 2 Fontitrygon geijskesi (PA16-42) Fontitrygon geijskesi (PA16-45) 1 Fontitrygon geijskesi (PA16-48) Fontitrygon geijskesi (PA16-49) 14 Fontitrygon geijskesi (PA16-52) Fontitrygon geijskesi (PA16-59) Fontitrygon geijskesi (PA16-60) Fontitrygon geijskesi (PA16-61) Fontitrygon geijskesi (PA16-76) Fontitrygon geijskesi (PA16-28) Fontitrygon geijskesi (PA16-41) 1 Fontitrygon geijskesi (PA16-66) Fontitrygon geijskesi (PA16-72) Fontitrygon geijskesi (PA16-79)

7.0 Figure A.18: Phylogenetic relationship between hosts, based in mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) gene, of present work and GenBank sequences under parsimony as the optimality criteria. Contents between parentheses refer to host accesion code. Goodman-Bremer values implemented. Branchs without Goodman-Bremer values are zero. A.9 Original Acanthobothrium nucleotide sequences

Original Acanthobothrium nucleotide sequences collected in Estuary of Bay of Marajó, Colares, Pará. Brazil

Molecular Hologenophores Acanthobothrium species Host species Collection location Coordinates code code

Y001 PA16-68-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y002 PA16-68-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y003 PA16-61-1v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y004 PA16-81-1v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y005 PA16-81-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y006 PA16-62-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y007 PA16-62-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y008 PA16-07-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y009 PA16-40-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y010 PA16-40-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y011 PA16-40-3v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y012 PA16-40-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y013 PA16-40-5v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y014 PA16-40-6v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y015 PA16-25-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y016 PA16-44-1v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y017 PA16-44-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y018 PA16-44-3v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y019 PA16-44-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y020 PA16-79-1v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W

Continued on next page Table A.9 – Continued from previous page Molecular Hologenophores Acanthobothrium species Host species Collection location Coordinates code code

Y021 PA16-66-1v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y022 PA16-87-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y023 PA16-87-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y024 PA16-87-3v Acanthobothrium n. sp. 2 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y025 PA16-50-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y026 PA16-50-2v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y027 PA16-82-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y028 PA16-82-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y029 PA16-57-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y030 PA16-60-1v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y031 PA16-26-1v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y032 PA16-45-1v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y033 PA16-76-1v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y034 PA16-76-2v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y035 PA16-88-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y036 PA16-88-2v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y037 PA16-33-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y038 PA16-77-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y039 PA16-77-2v Acanthobothrium n. sp. 2 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y040 PA16-83-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y041 PA16-83-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y042 PA16-72-1v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W

Continued on next page Table A.9 – Continued from previous page Molecular Hologenophores Acanthobothrium species Host species Collection location Coordinates code code

Y043 PA16-72-2v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y044 PA16-72-3v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y045 PA16-74-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y046 PA16-74-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y047 PA16-74-3v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y048 PA16-74-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y049 PA16-80-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y050 PA16-80-2v Acanthobothrium sp. 11 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y051 PA16-80-3v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y052 PA16-80-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y053 PA16-80-5v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y054 PA16-67-1v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y055 PA16-67-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y056 PA16-49-1v Acanthobothrium n. sp. 4 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y057 PA16-49-2v Acanthobothrium n. sp. 3 Fontitrygon geijskesi Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y058 PA16-53-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y059 PA16-53-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y060 PA16-53-3v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y061 PA16-53-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y062 PA16-75-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y063 PA16-75-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y064 PA16-75-3v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W

Continued on next page Table A.9 – Continued from previous page Molecular Hologenophores Acanthobothrium species Host species Collection location Coordinates code code

Y065 PA16-75-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y066 PA16-75-5v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y067 PA16-78-1v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y068 PA16-78-2v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y069 PA16-78-3v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y070 PA16-78-4v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y071 PA16-78-5v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y072 PA16-81-3v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y073 PA16-81-4v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y074 PA16-81-5v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y075 PA16-81-6v Acanthobothrium n. sp. 2 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y076 PA16-67-3v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y077 PA16-68-3v Acanthobothrium n. sp. 11 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y078 PA16-80-6v Acanthobothrium n. sp. 10 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y079 PA16-74-5v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y080 PA16-74-6v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y081 PA16-74-7v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y082 PA16-74-8v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y083 PA16-74-9v Acanthobothrium n. sp. 3 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y084 PA16-74-10v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y085 PA16-74-11v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W Y086 PA16-77-3v Acanthobothrium n. sp. 1 Hypanus guttatus Estuary of Bay of Marajó, Colares, Pará, Brazil 0°55’53.8”S48°17’39.5”W