Acanthobothrium Blanchard, 1948 from the Northwest Atlantic … · 2019. 3. 1. · o escotismo...

Yu Golfetti

Acanthobothrium Blanchard, 1948 from theNorthwest Atlantic and their phylogenetic

relationships with freshwater lineages.

São Paulo2018

Yu Golfetti

Acanthobothrium Blanchard, 1948 from the NorthwestAtlantic and their phylogenetic relationships with

freshwater lineages.

Acanthobothrium Blanchard, 1948 do NoroesteAtlântico e seu relacionamento filogenético com

linhagens de água doce.

Dissertação apresentada ao Institutode Biociências da Universidade deSão Paulo, para a obtenção de Títulode Mestre em Ciências, na Área deZoologia.

Orientador: Fernando P. de LunaMarques

São Paulo2018

Golfetti, YuAcanthobothrium Blanchard, 1948 from the

Northwest Atlantic and their phylogenetic relationshipswith freshwater lineages.

99 páginasDissertação (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 financeiroCAPES e CNPQ (processo 130093/2018-1)

Apoio técnico e científicoLaboratório de Helmintologia Evolutiva, Dpto. de Zoologia, IB-USPLaboratório 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 desavençasque tenhamos, vem sendo meu pilar desde sempre. Mais específicamente eu queriaagradecer aos meus avós Elio e Sonia, além de minha Tia Carla, por me apoiaremincondicionalmente desde de meu início de vida até hoje. Família, amo vocês.

Não posso deixar de agradecer a família Tiradentes. Nunca imaginei, isso em 2003, queo escotismo seria algo de tanta importância dentro da minha vida. É no Tiradentes quetenho 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 tornandomelhores cidadãos é gratificante e emocionante.

Eu tenho muitos amigos, muitos desses eu conheci dentro da USP. Agradeço de coraçãoà Isabela Rodrigues (Tonks), Kléber Mathubara (Fanta), Giulia Magri, Ana Perticarrari,Thomas Creedy, Rachel Montesinos, Yu Oliveira, Ariadne Vilaça, Edgard Lopes, CarolTieko, 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, MariaAngélica (Keka), Jéssica Cardana, Ricardo Correia, Luli Brunelli e GeovannaKerkhoven, 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 luzquando 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 teremdividido o sentimento mais bonito comigo mesmo quando eu era apenas cacos. Obrigadapor 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 quedividiram 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 dividiré muito mais que dar, é um receber diário.

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

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

Eu amo vocês todos meus amigos.

Também não posso deixar de citar as pessoas que me proporcionaram a chance de chegara USP e estar apresentando hoje essa tese. Sabrina, Madalena, Kaká, Francis, Ana eAline, sinto muito a falta de vocês. Rogério, Renatinho, Amanda e Joaber... meus quatroorientadores. Os melhores orientadores que um pessoa pode ter nesse mundo. Euconsegui. Sem vocês eu não conseguiria. Vocês me ensinaram tanto. Eu agradeço a cadadia 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 lhesalcançar.

Não posso deixar de agradecer aos técnicos Enio, Phillip, Bia e Sabrina, sem vocêsmetade das minhas coisas não teriam sido realizadas e eu continuaria sem saber ondecolocar minhas amostras hahahah. Porém não posso deixar de agradecer ao Manu portodo apoio na parte de bancada molecular, fazendo mil e uma reanálises, testando primere 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, Liliane Luana (mesmo que só por um mês) por todas as conversas descompromissadas,risadas, cafés, recadinhos na lousa, preocupações comigo no whatsapp e por meescutarem. Obrigada ao Bjorn por financiar a coleta dos hospedeiros ao qual sem esseestudo não teria sido realizado, por me puxar a orelha pelo inglês mal falado e os papossobre NFL. Obrigada a Nati por toda a parte laboratorial ensinada, por todas as dicas devida, 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, portodos 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 seique 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 sobrea 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 suaorientanda e ter me dado a chance de conhecer tanta coisa e presenciar o que desde oinício da graduação eu ansiava. Eu sei que não foi das melhores orientandas, eu sei quedei trabalho, e quando eu estava melhorando, vieram os problemas. Sua compreensãocomigo foi crucial para esse dia ter chego. Desculpa não ter conseguido me dedicar tantonesse final, porém fui até onde meus limites de corpo e mente me autorizavam. Obrigadapelos ensinamentos, pelas chances de te questionar, pelas conversas e brigas (porquenão?)... Você me fez crescer demais.

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

Obrigada!

.

Table of Contents

Introduction 1General goals of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Material and Methods 4Collection Biological Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Morphological Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Molecular Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Results 10Host Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Molecular data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Morphological data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Phylogenetic Analysis of Acanthobothrium . . . . . . . . . . . . . . . . . . . . . . 16Taxonomic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Acanthobothrium n. sp. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Acanthobothrium n. sp. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Acanthobothrium n. sp. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Acanthobothrium n. sp. 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

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

Phylogeny of Acanthobothrium . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Conclusions 53

References 60

Resumo 61

Abstract 63

Appendices 65A.1 Script in R used for Primary Component Analyses (PCA) . . . . . . . . . . . . 66A.2 DNA extraction protocol with Ammonium Acetate . . . . . . . . . . . . . . . 67A.3 DNAdvance Extraction ProtocolTM . . . . . . . . . . . . . . . . . . . . . . . . 68A.4 Purification Protocol with AMPURE . . . . . . . . . . . . . . . . . . . . . . . 69

A.5 Dasyatidae nucleotide sequences for MT-ATP6, MT-CYB and MT-ND2 for ter-minals used as outgroups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

A.6 Cestode nucleotide sequences acquired from previous studies . . . . . . . . . . 71A.7 GenBank nucleotide sequences of cestodes for MT-RNR2 and LSU regions . . 89A.8 Original hosts nucleotide sequences . . . . . . . . . . . . . . . . . . . . . . . 90A.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 Neotropicalfreshwater river systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

3 Morphometric data for new species of Acanthobothrium described in presentstudy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

4 Prevalence for species of Acantobothrium . . . . . . . . . . . . . . . . . . . . 48

List of Figures

1 Phylogenetic relationships among host individuals - Concatenated nucleotide data 112 General PCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Phylogenetic hypothesis for Acanthobothrium . . . . . . . . . . . . . . . . . . 184 New Acanthobothrium spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Acanthobothrium n. sp. 1 morphology . . . . . . . . . . . . . . . . . . . . . . 246 Acanthobothrium n. sp. 1 SEM . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Acanthobothrium n. sp. 2 morphology . . . . . . . . . . . . . . . . . . . . . . 318 Acanthobothrium n. sp. 3 morphology . . . . . . . . . . . . . . . . . . . . . . 359 Acanthobothrium n. sp. 3 SEM . . . . . . . . . . . . . . . . . . . . . . . . . . 3610 Acanthobothrium n. sp. 3 cross sections . . . . . . . . . . . . . . . . . . . . . 3711 Scatter plot of attributes variation of Acanthobothrium n. sp. 3 . . . . . . . . . 3812 Variation on bothridial morphology for Acanthobothrium n. sp. 3 . . . . . . . . 3913 Acanthobothrium n. sp. 4 morphology . . . . . . . . . . . . . . . . . . . . . . 4314 Acanthobothrium n. sp. 4 SEM . . . . . . . . . . . . . . . . . . . . . . . . . . 4415 Acanthobothrium n. sp. 4 cross sections . . . . . . . . . . . . . . . . . . . . . 45A.16 Hosts phylogenetic relationship - MT-ND2 . . . . . . . . . . . . . . . . . . . . 93A.17 Hosts phylogenetic relationship - MT-CYB . . . . . . . . . . . . . . . . . . . 94A.18 Hosts phylogenetic relationship - MT-ATP6 . . . . . . . . . . . . . . . . . . . 95

Introduction

Studies on metazoan parasitic fauna have been historically neglected (Windsor, 1995), despiterecent estimates suggesting that the diversity of parasitic lineages may account for 1

3to 1

2of

the global biota (Poulin, 2014). The documentation of this diversity is important becauseit has the potential of revealing the historical and ecological events that could account forthe diversification of these neglected groups (Adamson & Caira, 1994; Bush et al., 2001).However, in order to study the evolutionary processes responsible for the diversification ofparasite lineages, the taxonomy and systematics of their hosts needs to be well developed. Inaddition, understanding the evolutionary history of associated parasites and their hosts makesco-evolutionary inference possible, which would allow us to elucidate and understand implicithistorical 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 freshwaterstingrays (Myliobatiformes: Potamotrygonidae) has challenged the current paradigm of hostspecificity attributed to most cestode genera hosted by elasmobranchs (Cardoso Jr., 2010;Marques & Reyda, 2015; Reyda & Marques, 2011; Trevisan et al., 2017). The paradigmof specific cestodes species to a single host species, and exclusively to their occurrencearea, has been questioned. Freshwater potamotrygonids host cestodes that vary widely intheir degree of specificity, ranging from taxa that are found in more than 19 hosts speciessuch 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 infectfreshwater stingrays in estuarine areas (Trevisan & Marques, 2017). It is unclear whether thepatterns observed in potamotrygonids are the result of the complex history of colonization offreshwater habitats and diversification - in this case unique or particular to this system - or if ourunderstanding 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 restrictedto Neotropical river systems. However, unpublished data (Marques, com. pess.) revealoccurrences 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 OrinocoDelta and Caulobothrium Baer, 1948, a phyllobothridean genus that is restricted to marinebatoids, in Potamotrygon yepezi Castex & Castello in Lake Maracaibo. Also, the marineAnindobothrium anacolum Marques, Brooks & Lasso, 2001 was found infecting the freshwaterpotamotrygonid Potamotrygon yepezi (Trevisan et al., 2017). These examples, provide someevidence that cross infections may occur in areas where the distribution of marine andfreshwater 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 makeit difficult to assess if the cestode distribution and specificity in marine/estuarine environmentsare in fact different from the patterns observed in freshwater or if they are simply the resultof poor sampling of estuarine regions. To date, the number of studies made of elasmobranchcestodes from estuarine regions are scarce.

Cestodes that parasitize elasmobranchs have been studied from the American continent andadjacent marine region since the middle of 19th’ century. These studies mainly focused ondocumenting the diversity of cestode fauna and did not provide any analyses of the observedinfection patterns. The majority of the studies in Brazil focused on cestodes infecting freshwaterstingrays (Potamotrygoninae).

The origin of Potamotrygoninae appears to have imposed a distinct pattern of diversificationin its parasitic fauna. This unique group of freshwater batoids, now represented by about30 species, distributed into four genera (Carvalho & Lovejoy, 2011; Carvalho & Ragno,2011; Fontenelle et al., 2014; Froese & Pauly, 2018; Loboda & Carvalho, 2013; Rosaet al., 2008) share a common ancestor with the clade represented by two amphi-Americanspecies of Styracura (Carvalho et al., 2016). The freshwater potamotrygonids colonized theriver systems of South America during marine incursions of the Paleogene period, moreprecisely between the lower Miocene and middle Eocene (22.5-46.0 mya; see Carvalho etal., 2004; Lovejoy et al., 1998; Marques, 2000). During this process of colonization, thereis 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 sametime 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 riversystems, tolerate low salinity (Thorson et al., 1983, 1978), and several species can be foundin the estuaries of the largest Neotropical river basins (e.g., the Orinoco Delta , Maracaibo Lakeand Bay of Marajó). In these environments, potamotrygonids and marine stingrays, mainly ofthe 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 acestode infection is acquired via food chain when the hosts consumes a food item that is infectedwith 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 theirgeographic distribution and how their parasitic fauna is related to one another has been poorlyexplored.

The estuarine region of the Bay of Marajó, State of Pará, Brazil, is an interesting area tostudy the cestode fauna of dasyatids. Bay of Marajó is characterized by having a large estuarywith great variation in salinity. The tides and salinity levels are controlled by the discharge of theAmazonian 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 thecestode fauna of the freshwater potamotrigonids species reported from this area are relativelywell 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). Inaddition, there are no records of cestodes for F. colarensis and F. geijskesi.

Only a fewcestode genera, including Acanthobothrium, Rhinebothroides and Rhinebothrium, have beenstudied 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 therelationships between marine and freshwater cestodes, and with their hosts. In addition, thereis no known species of Acanthobothrium from batoids that occur off the Brazilian coast.

Acanthobothrium is distributed worldwide and represent the most diverse genus of theOnchobotriidae, representing almost 76% of the species in the family (Caira et al., 2017;Campbell & Beveridge, 2002; Machado & Marques, 2012), with more than 195 nominalspecies described (Caira et al., 2012-2018). Members of this genus parasitize severalspecies of Elasmobranchii: rays, skates (Myliobatiformes, Rajiformes, Rhinopristiformes andTorpediniformres) and sharks (Charchaniformes, Orectolobiformes, Heterodontiformes andSqualiformes) (Caira et al., 2017). However, most of the diversity of Acanthobothrium isfound 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 oftheir scolex. All species posses a scolex that is divided into four bothridia (large sucker-likestructures), each of which is further subdivided by two horizontal septa resulting in three loculi.

3


Each bothridia exhibits a pair of bipronged hooks, and an apical muscular pad bearing an apicalsucker (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 studiedby Brooks (1977). Trevisan (2016) recovered Acanthobothrium sp. 9 as sister-taxon to thefreshwater lineage Acanthobothrium sp. 2 sensu Cardoso Jr., 2010. This clade is nested withina larger clade of marine species of Acanthobothrium from Senegal (Machado, 2012; Trevisan,2016). This phylogenetic pattern could be interpreted as evidence of an evolutionary processesthat resulted from multiple entries of marine cestodes taxa, such as Acanthobothrium, into theSouth American freshwater system. The Bay of Marajó, is one of the estuaries in the North ofSouth America where conditions of co-existence of marine and freshwater stingrays could makethis entries events possible to have occurred. Studies of the cestode fauna of batoid species thatoccur in estuaries have the potential to help elucidate the specific patterns of distribution anddiversification of these parasites and their implications on historical patterns of association inboth 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 Marajóprohibits any further studies on their relationships with freshwater cestode lineages, which couldpossibly reveal a secondary invasion of Acanthobothrium into the South American freshwatersystem were the motivation for this study. Within this context, the goals of the present studyare:

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 thedry season, when marine waters flows into estuary bringing in marine stingrays. Stingrayswere collected by fishermen using longlines. While still alive, the rays were anaesthetized,photographed with a scale, and then euthanized by cervical incision. Spiral intestines ofFontitrygon geijskesi (n=22) and Hypanus cf. guttatus (n=59) were collected (the specimensnot recognized as F. geijskesi collected were initially assigned to H. cf. guttatus, see Resultsand Discussion), removed from hosts after an abdominal longitudinal incision and washed witha saline solution and cut in half. One half and the sift were fixed in hot 10% formalin solutionand transferred to ETOH 70% for storage after 48 hours. The other half of the spiral intestinewas fixed in ETOH 100% and kept at -20°C temperature. Of those collected hosts, 27 specimensof 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 inthe laboratory. Specimens selected for morphological analysis were serially hydrated, stainedwith Delafield’s hematoxylin, destained in acid an ethanol solution (1% HCl), followed bybasic ethanol solution (1% NaOH), dehydrated by an ethanol series to 100%, cleared in methylsalicilate 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 ethanolseries, submerged in 1.5% osmium tetroxide for 1̃2 hours, completely dehydrated using


Hexamethyldisilazane (HMDS) and fixed on metallic supports (stubs). The strobila weremounted as SEM voucher, as described in the section above. Scoleces were examined witha Zeiss Sigma VP scanning electron microscope at the Instituto de Biociências – Universidadede São Paulo (IB-USP) for the identification of microtriches. The terminology employed indescribing 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 thesection above to serve as vouchers. Mature proglottids were prepared following conventionaltechniques: dehydrated by an ethanol series to 100%, cleared in xylene, included in paraffinmedium and sectioned to 7𝜇 thickness using Leica RM 2025 retracting rotary microtome, placedin glass slides, stained in eosin and hematoxylin, and permanently mounted with Canada’sbalsam.

Morphometric Data:

Morphological hook data and nomenclature follow Ghoshroy & Caira (2001). The hook handlebase was named A for lateral hooks and A’ for medial hooks, inner or abaxial prong (distancefrom the extremity of the most internal prong in bothridia middle line to the anteriormost pointof hook internal curve) B and B’, outer or abaxial prong (distance from the extremity of themost 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 outerprong) 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 microscopewith a standardized ocular ruler. All measurements provided in the descriptions are given inmicrometers (𝜇m), unless otherwise stated and followed by the number of specimens fromwhich the measurements were taken between parentheses. Loculi lengths ratio were calculatedin 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. Theseincluded preoral length (POL), disk width (DW), the ratio between preoral length and Diskwidth (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 dividedinto 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 PrincipalComponents 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 hostand fixed in ETOH 100% in the field. An aliquot of each muscle sample was was used forDNA extraction using the Ammonium Acetate (𝑁𝐻4𝐶𝐻3𝐶𝑂2) protocol (Appendix A.2). Theextractions were quantified using NanoDrop 2000 (Thermo Scientific) and amplified usingPolymerase Chain Reaction (PCR). Each PCR cycle had initial denaturation for 5 minutes at95°C, 35 cycles of denaturations for 30 seconds at 95°C, annealing for 30 seconds at specifictemperature depending on primers (see below), 1 minute to 1 minute 10 seconds extensionat 72°C, and a final extension for 7 minutes at 72°C. The amplification and sequencing wereperformed 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 GAARTA TCA TTC – 5’ at 57°C, ATP synthase membrane subunit 6 (MT-ATP6) with primersCOX2MODF 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: ubiquinoneoxidoreductase core subunit 2 (MT-ND2) with primers ILEMF 5 –AAG GAG CAG TTT GATAGA 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 middleportion. Scolex and posterior portion were mounted as hologenophores vouchers (sensu Pleijelet 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 usingNanoDrop 2000 (Thermo Scientific). Polymerase Chain Reaction was used to amplify theD1-D3 regions of the nuclear 28S cytoplasmatic ribosomal RNA (LSU) and mitochondriallyencoded 16S RNA (MT-RNR2).

7


Amplifications for cestode specimens were performed in a 25 𝜇l volume containing 1 𝜇l ofDNA, 200 mM Tris-HCl (pH 8.4), 50 mM Kcl, 200 1M dNTPs, 1.0-3.0 nM MGCl2, 0.4 1Mof each primer, 1U of Taq DNA polymerase recombinant (Fermantas, Thermo Scientific). Foreach PCR cycle included initial denaturation for 5 minutes at 95°C, 35 cycles of denaturationsfor 30 seconds at 95°C, annealing for 30 seconds at specific gene temperature (see below), 1minute to 1:10 minutes for extension at 72°C, and final extension for 7 minutes at 72°C. Theamplification and sequencing were performed with the follow primer sets: LSU with LSU-5F3’ – TAG GTC GAC CCG CTG AAY TTA AGC A – 5´ and LSA-1500R 3’– TGC CTT TTGCAT 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 werepurify 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 AMPuretXP DNA Purification and Cleanup kit (Beckman Coulter). Products were subsequentlycycle-sequenced directly from both forward and reverse directions using ABI Big-DyeSequence Terminator version 3.1, cleaned with ethanol precipitation, and sequenced on an ABIPrism 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 usingMAFFT with options –maxiterate 1000 –globalpair (v7.271; Katoh et al., 2002) and editedin BioEdit (version 7.1.3.0; Hall, 1999) to remove leading and trailing gaps resulted fromdifferential sequencing.

Phylogenetic inference:

The phylogenetic analyses of host sequences considered partial sequences of 18 terminalsas outgroups, 14 sequences for mitochondrially encoded NADH ubiquinone oxidoreductasecore 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 membranesubunit 6 (MT-ATP6) (N. kuhlii [1], D. brevis [1] and H. akajei [1]) A.5). As ingroup, it wasconsidered 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 phylogeneticanalysis was performed with TNT (Goloboff et al., 2008, 2008-2018) with 100 replicates ofTNT’s new technology searches (i.e., xmult). Nodal support of selected nodes was estimatedusing 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 thisstudy 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 fromall major body waters of the world. The outgroup was composed by Onchoproteochephalideasequences obtained from NCBI GenBank (n=10). Also, Acanthobothrium sequences wereacquired from NCBI GenBank to compose the ingroup (n=10) (Appendix A.7). Sequencesof 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 parsimonyas the optimality criteria. Direct optimization was selected because these regions requirealignment. 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 independentsearches for 1 h 30min using the command search [i.e., search(maxtime:0:01:30)] assumingequal weights for all character transformations and no gap opening cost (gap opening:0). Thissearch was conducted in a 10 X 2.83 GHz Intel ® CoreTM2 Quad Processor Q9550 computercluster. After compiling candidate trees by DO, re-diagnosis was performed using iterative passalignment (DO/IP; Wheeler, 2003a). Finally, the results of POY were verified by performinga phylogenetic analysis of the implied alignment (sensu Wheeler, 2001b) generated by POYin 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 andsaving 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 and25 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-CYBvaried from 565 to 1238 base pairs (bp) in length, MT-ATP6 varied from 854 to 1161 bp, andMT-ND2 varied from 569 to 1331 bp. Once aligned and trimmed to remove regions with leadingand trailing gaps due to differential sequencing, the dataset included 565 bp of MT-CYB, 853bp of MT-ATP6 and 990 bp of MT-ND2. Hence, the concatenated molecular matrix included54 terminal and 2408 nucleotide characters.

As presented in the tree resulted from the phylogenetic analysis of the concatenatedsequences 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. Singlegenes 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 possibleto identify this species in the field given the ambiguous nature of the diagnostic features of thisspecies in comparison to Hypanus guttatus. The phylogenetic pattern found within the cladecomprised by specimens that were initially identified as H. cf guttatus suggested two internalclades (A and B, Fig. 1). We used those to clades to verify whether it was possible to distinguishthem 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 ofidentifying members of F. colarensis. The results of morphometric measurements of hosts

20.0

Fontitrygon geijskesi (PA16-52)

Hypanus guttatus (PA16-07)










































OUTGROUPS

30

35

17

1

1

B

A

Figure 1: Phylogenetic relationships among host individuals based on the phylogenetic analysis underparsimony for the concatenated nucleotide data (MT-ND2, MT-CYB,and MT-ATP6; outgroup terminalsomitted). Contents between parentheses refer to host accession code (Cestode Database). Numbersabove branches refer to Goodman-Bremer support values for selected nodes. Scale bar represent numberof transformations.

11

http://tapewormdb.uconn.edu/index.php/hosts/specimen_search/elasmobranch


initially identified as H. cf. guttatus, based on differential attributes to F. colarensis arepresented in Table 1.

Only one specimen (PA16-007, Table 1) exhibited right eye diameter/preoral length andeye average diameter/preoral length values in the range attributed to F. colarensis accordingto 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 etal. (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 forfemales of F. colarensis (Santos & Charvet-Almeida, 2007). All other specimens, males andfemales, are out of range of both species (n=57) for preoral length/disk width dimensions, Table1). Finally, no specimens presented consistent morphometric values to either species for allvariables 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 twonominal species, it was performed a Principal Component Analysis (PCA) of morphometricvariables collected from host images. In this analysis, we color coded each individual reflectingthe phylogenetic pattern found in Fig. 1 for clade A (blue) and B (red). The rational was toseek congruence between phylogenetic signal (Fig. 1) and morphometrical data (Table 1). Allremaining specimens, that is, those excluded from molecular analysis and those included butnot 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 wereconsidered to be useful to distinguish these two species (Table 1) by Santos et al. (2004) andSantos & Charvet-Almeida (2007). The results indicate that there is no congruence betweencladistic 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 moleculardata. Based on these results, specimens of Clade A (blue), Clade B (red) and the remainingspecimens sampled for this study (black) can not be distinguished morphologically. Hence,molecular and morphological data favors the hypotheses that only a H. guttatus was collectedin the present study.

12

Table 1: Morphometric data obtained from specimens of Hypanus guttatus from Estuary of Bay of Marajó, Colares, Pará. Brazil. Measurementsare presented in cm, except for POL/DW (%), RED/POL (%), LED/POL (%) and EAD/POL (%), which are percentages. Numbers in black indicatevalues congruent with ranges attributed to Hypanus guttatus; numbers in blue indicate values congruent with ranges attributed to females of Fontitrygoncolarensis; 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 averagediameter 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


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




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


−4 −2 0 2 4

−1

.5−

1.0

−0

.50

.00

.51

.01

.52

.0

PC 1 (77% explained var.)

PC

2 (

13

% e

xp

lain

ed

va

r.)

PA16−001

PA16−002 PA16−003

PA16−004

PA16−005

PA16−007

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PA16−013

PA16−014

PA16−015

PA16−022

PA16−023

PA16−024

PA16−025

PA16−030

PA16−031

PA16−032

PA16−033

PA16−034

PA16−035

PA16−036

PA16−037

PA16−038

PA16−039

PA16−040PA16−043

PA16−044

PA16−050

PA16−053

PA16−054

PA16−055

PA16−056

PA16−057

PA16−058

PA16−062

PA16−063

PA16−064

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PA16−082

PA16−083

PA16−084

PA16−087

PA16−088

PA16−089

PA16−090

PA16−091

POLDW

REDPOL

LEDPOLEADPOL

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 notsequenced and those not forming clades. Axes of the variables POL/DW (%): Preoral length for Diskwidth (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 diameterfor 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 LSUranged from 424 to 950 bp. Aligned and trimmed sequences of MT-RNR2 and LSU resulted indatasets with 610 and 1076 bp, respectively. These regions were sequenced for 86 specimensof Acanthobothrium (Appendix A.9), which once added to Trevisan’s (2016) dataset resulted ina 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 160unique candidate trees raging from 6529 to 6614 steps in length. The re-diagnose of thesecandidate trees under IP found 3 equally parsimonious trees (MPTs) with 6500 steps for whichthe implied alignment consisted of sequences with 2019 bp. The reanalysis of the impliedalignment in TNT (New technologies) resulted in 636 MPTs with 6496 steps, which strict

16

consensus is presented in Figure 3.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 taxonAcanthobothrium n. sp. 4 (blue in Fig. 3) was recognized based on morphological dataalone. 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, areexclusively 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 includesthe 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 discoveredlineages of Acanthobothrium. Acanthobothrium n. sp. 3 is sister group of a clade thatincludes Acanthobothrium n. sp. 1 and nested species (described above), Acanthobothrium

n. sp. 2, Acanthobothrium sp. 11, Acanthobothrium n. sp. 4 and Acanthobothrium sp. exHypanus guttatus [A064/AL10-004.01]. The new lineage Acanthobothrium sp. 10 is nestedto a clade composed by marine Acanthobothrium sp. 9 sensu Trevisan (2016) and freshwaterAcanthobothrium sp. 2 sensu Cardoso Jr. (2010).

Despite the recognition of 6 lineages of Acanthobothrium, for only four lineages there wasenough morphological material to provide taxonomic descriptions. This is because, there wasonly 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 usedlikewise (Y050 and Y077, Appendix A.9), both immature worms. Based on these results, fournew species of Acanthobothrium are described below.

Of the six new lineages detected in present study, four are found in both hosts. JustAcanthobothrium 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 lineagespresented 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.D

issertaçãode

Mestrado,2018

60.0

Onchobothrium? ex Hypanus sabinus [AC09/TM-9]

Acanthobothrium sp. ex Hypanus guttatus [A164/BE-12-42]

Acanthobothrium sp. ex Raja miraletus

OUTGROUP

Acanthobothrium sp. 1 ex Styracura schmardae sensu Trevisan, 2016

Acanthobothrium sp. 11 ex Hypanus guttatus

Acanthobothrium sp. 5 ex Styracura schmardae sensu Trevisan, 2016 [b033/PN15-56.4]

Acanthobothrium sp. ex Hypanus sabinus

Acanthobothrium CLADE X (ETP)

Acanthobothroides thorsoni ex Styracura schmardae [AC07/BE-3]

Acanthobothrium CLADE II (ETP)

Acanthobothrium sp. 3 ex Styracura schmardae sensu Trevisan, 2016

Acanthobothrium CLADE VIII (ETP)

Acanthobothrium CLADE IV (ETP)

Acanthobothrium sp. 6 ex Styracura pacifica sensu Trevisan, 2016

Acanthobothrium sp. 5 ex Styracura schmardae sensu Trevisan, 2016 [b058/TT14-06.5]

Acanthobothrium sp. ex Hypanus guttatus [A064/AL10-004.01]

Acanthobothrium brevissime ex Dasyatis say [EU660532/-]*

Acanthobothrium CLADE IX (ETP)

Acanthobothrium n. sp. 3 ex Hypanus guttatus

Acanthobothrium sp. 9 exStyracura schmardae sensu Trevisan, 2016 (Panama)

Acanthobothrium CLADE VI (ETP)

Acanthobothrium sp. n. 8 ex Styracura pacifica sensu Trevisan, 2016

Acanthobothrium spp. ex Potamotrynidae

Acanthobothrium Styracura schmardae


Acanthobothrium sp. ex Torpedo fuscomaculata [AC05/AF-72]

Acanthobothrium CLADE III (ETP)

Acanthobothrium spp. CLADE I

Acanthobothrium sp. 2 ex Potamotrygon schroederi sensu Cardoso Jr., 2010

Acanthobothrium CLADE VII (ETP)

Acanthobothrium n. sp. 4 ex Fontitrygon geijskesi

Acanthobothroides pacificus ex Styracura pacifica [b084/PN15-12.01]

Acanthobothrium sp. ex Styracura schmardae


FW

M

Acanthobothrium sp. 10 exHypanus guttatus

FW

M

M

himanturi ex

Figure 3: Phylogenetic relationships among lineages of Acanthobothrium based on the simultaneous analysis of MT-RNR2 and LSU, by directoptimization, regions using parsimony as the optimality criteria. Contents between brackets represent molecular codes and/or accession numberfor vouchers. Bold names represent new lineages recognized in present study. Sidebar indicating freshwater taxa (FW) and marine taxa (M) ofAcanthobothrium.

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Taxonomic Actions

Acanthobothrium n. sp. 1

(Figs. 5 and 6)

Description (Based on 18 complete worms, 19 hologenophores and two scoleces observedwith SEM): Worms acraspedote (Fig. 4 A, Table 3), 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. 5 A, 6 A). Cephalic peduncle 128–431 (n=13) long by 50–173 (=13) wide, coveredwith gladiate spinitriches with approximately 1–2 microtriches/𝜇𝑚2 (Fig. 6 B). Scolex proper205–303 (n=18) long by 131–221 (n=17) in maximum width (at level of anterior loculus), withfour bothridia, bothridia 156–236 (n=18) long by 65–108 (n=19) in maximum width. Proximalsurface of bothridia covered by gladiate microtriches 1–2 microtriches/𝜇𝑚2 and acicularfilitriches 0.08 in diameter, approximately 20–22 microtriches/𝜇𝑚2 (Fig. 6 C). Each bothridiawith anterior muscular pad and three loculi divided by two horizontal septa. Anterior loculus77–140 (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 andinter-loculi septa covered with papiliform filitriches with 0.08 in diameter, approximately 103-106 microtriches/𝜇𝑚2 (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 approximately176–189 microtriches/𝜇𝑚2 (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). Velumnot present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surface ofaxial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongsand abaxial prongs with different lengths; lateral and medial hooks of different sizes (Fig. 5B). 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 tissueanteriorly covering each prong of both set of hooks. (Fig. 6 F). Immature proglottids widerthan 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 widthratio 0.14–0.34 (n=17) (Fig. 5 D). Genital pores irregularly alternating, 31-50% (n=34) fromanterior end of proglottid. Some terminal proglottids with sperm-filled vas deferens. Gravidproglottids 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 toovarian isthmus, 23–38 (n=27) in total number, 5–13 (n=28) pre-poral, 4–7 (n=29) post-poraland 13–20 (n=28) aporal. Cirrus sac pyriform 81–193 (n=33) long by 47–217 (n=33) wide,

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containing eversible cirrus armed with spinitriches. Vagina thick-walled, sinuous, extendingfrom ootype along medial line of proglottid to anterior margin of cirrus sac to common genitalatrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetricalinverted A-shaped in frontal view, lobulated, reaching or almost reaching posterior margin ofcirrus 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 offollicles, extending from the first line of distal testes to the ovarian isthmus, interrupted byvagina and cirrus sac dorsally and ventrally, not interrupted by ovary. Uterus linear, median,extending from ovarian isthmus to vagina level.

Taxonomic summaryType-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: thepolyphyletic 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 smallertotal length (1.4-3.0 mm vs. 3.4–12.4 mm, respectively) and ovarian morphology (asymmetricvs. symmetric ovarian anterior lobes).

There are 15 valid species of Acanthobothrium described for adjacent waters from whereAcanthobothrium n. sp. 1 was found (see Table 2). Within this group, six species are restrictedto freshwater species of Potamotrygonidae and there is no evidence that they occur in marineMyliobatiformes (Table 2). Among those marine species, Acanthobothrium n. sp. 1 is mostsimilar 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. 1can 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 manymorphometric 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 (23–38vs. 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 anteriorloculus length (77–140 vs. 144–325). Acanthobothrium n. sp. 1 can be easily differentiatedfrom A. americanum and A. cairae by being a smaller worm (1.4–3.0 vs. 94 mm and 45–154mm, respectively) with fewer segments (5–10 vs. 453–615 and 268–491, respectively), byhaving shorter scolex (205–303 vs. 554 and 1,100–1440, respectively) and fewer testes (23–38vs. 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 mmand >35 mm, respectively) and fewer segments (5–10 vs. 13 and 31–48, respectively). This newspecies differs from A. himanturi and A. tasajerasi by having fewer segments (5–10 vs. 17–26and 11–18, respectively). Acanthobothrium n. sp. 1 further differs from A. tasajerasi by theabsence of an expanded poral atrium. Finally, Acanthobothrium n. sp. 1 can be distinguishedfrom 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.D

issertaçãode

Mestrado,2018

Table 2: Valid species of Acanthobothrium found in Northwest Atlantic and Neotropical freshwater river systems.

Species and Host Host Family Biogeographical RealmA. americanum Campbell, 1969 ex Hypanus americanus Dasyatidae Warm Temperate Northwest AtlanticA. brevissime Linton, 1908 ex Hypanus say Dasyatidae Warm Temperate Northwest AtlanticA. cairae Vardo-Zalik & Campbell, 2011 ex Bathytoshia centroura Dasyatidae Warm Temperate Northwest AtlanticA. cartagenensis Brooks & Mayes, 1980 ex Urobatis jamaicensis Urotrygonidae Tropical Northwestern AtlanticA. colombianum Brooks & Mayes, 1980 ex Aetobatus narinari Aetobatidae Tropical Northwestern AtlanticA. himanturi Brooks, 1977 ex Styracura schmardae Potamotrygonidae Tropical Northwestern AtlanticA. lineatum Campbell, 1969 ex Dasyatis americana Dasyatidae Warm Temperate Northwest AtlanticA. tasajerasi Brooks, 1977 ex Styracura schmardae Potamotrygonidae Tropical Northwestern AtlanticA. urotrygoni Brooks & Mayes, 1980 ex Urotrygon venezuelae Urotrygonidae Tropical Northwestern AtlanticA. amazonensis Mayes, Brooks & Thorson, 1978 ex Potamotrygon circularis Potamotrygonidae Neotropical FreshwaterA. peruviense Reyda, 2008 ex Potamotrygon motoro Potamotrygonidae Neotropical FreshwaterA. quinonesi Mayes, Brooks & Thorson, 1978 ex Potamotrygon magdalenae Potamotrygonidae Neotropical FreshwaterA. ramiroi Ivanov, 2005 ex Potamotrygon motoro Potamotrygonidae Neotropical FreshwaterA. regoi Brooks, Mayes & Thorson, 1981 ex Potamotrygon hystrix Potamotrygonidae Neotropical FreshwaterA. terezae Rego & Dias, 1976 ex Potamotrygon motoro Potamotrygonidae Neotropical Freshwater

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. Acanthobothriumn. sp. 3 (PA16-01-2); D. Acanthobothrium n. sp. 4. (PA16-48-1) 23


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. Distalsurface of bothridia loculi; C. Proximal surface of bothridia; D. Cephalic peduncle surface; E. Distalsurface of muscular pad; F. Detail of hooks.

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

Acanthobothrium sp. 1 Acanthobothrium sp. 2 Acanthobothrium sp. 3 Acanthobothrium sp. 4Total 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 HookA 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 HookA’ 15–28 (23.84, 34) 19–31 (27.46, 6) 41–66 (51.36, 50) 100–139 (118.79, 16)



Specie Acanthobothrium sp. 1 Acanthobothrium sp. 2 Acanthobothrium sp. 3 Acanthobothrium sp. 4B’ 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 (%from posterior)

31–50% (41.29, 34) 34–45 (38.04, 6) 26–46% (37.63, 49) 30–49% (41.06, 16)

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(Figs. 7)

Description (Based on 3 complete worms and 3 hologenophores): Worms acraspedote(Fig. 4 B, Table 3), euapolytic, 2,1–2,51 mm (n=3) long, with 7–8 (n=3) proglottids. Scolexconsisting 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) inmaximum 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 loculus106–126 (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 by72–80 (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 axialprong; internal channels of axial and abaxial prongs continuous, smooth; axial prongs andabaxial 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), D73–98 (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 tissueanteriorly 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 ovarianisthmus, 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 eversiblecirrus armed with spinitriches. Vagina thick-walled, sinuous, extending from ootype alongmedial line of proglottid to anterior margin of cirrus sac to common genital atrium. Vaginalsphincter absent. Ovary located near posterior end of proglottid, asymmetrical (ovarian lobeswith different lengths) inverted A-shaped in frontal view, lobulated, reaching or almost reachingposterior margin of cirrus sac. Poral lobe 333–501 (n=6) long, aporal lobe 332–563 (n=6) longby 69–127 (n=6) wide at isthmus. Vitellarium follicular form narrow lateral bands, each with1–2 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. Uteruslinear, median, extending from ovarian isthmus to vagina level.

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Taxonomic summaryType-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 sharesmany morphometric attributes with Acanthobothrium n. sp. 1. All morphometric rangesbetween these species overlap. Although Acanthobothrium n. sp. 2 showed higher mediansthan Acanthobothrium. n. sp. 1 (see Table 3), the new species is well supported by themolecular analyses, providing justification to recognize it as a putative new species differentfrom Acanthobothrium n. sp. 1.

Compared to the 9 marine species found in adjacent waters to Bay of Marajó (seeTable 2), Acanthobothrium n. sp. 2 is also very similar to A. brevissime (as described byCampbell (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 (7–8vs. 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 differsfrom 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 ofan expanded poral atrium. Finally, Acanthobothrium n. sp. 2 can be distinguished from A.

29


urotrygoni by its shorter total length (2.1–2.51 mm vs. > 15 mm, respectively) and longerscolex 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



(Figs. 8 - 12)

Description (Based on 50 complete worms, three scoleces observed with SEM andtwo mature proglottids used for cross sections): Worms acraspedote (Fig. 4 C, Table 3),euapolytic, 3.01–12.11 mm (n=50) long, with 12–32 (n=50) proglottids. Scolex consisting ofscolex proper and cephalic peduncle (Fig. 8 A, 9 A). Cephalic peduncle 420–2090 (n=50)long by 0,1–257 (n=50) wide, covered with gladiate spinitriches whit approximately 2–4microtriches/𝜇𝑚2(Fig. 9 B). Scolex proper 350–609 (n=50) long by 241–435 (n=50) inmaximum 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 gladiatemicrotriches whit approximately 1–2 microtriches/𝜇𝑚2 and acicular filitriches with 0.09 indiameter and approximately 46–53 microtriches/𝜇𝑚2 (Fig. 9 C).Each bothridia with anteriormuscular 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 54–56microtriches/𝜇𝑚2 (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 approximately116–125 microtriches/𝜇𝑚2 (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). Velumnot–present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surface ofaxial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongsand 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 lateralhook base. Thin layer of tissue anteriorly covering each prong of both set of hooks. (Fig. 9F). Immature proglottids wider than long; 11–29 (n=50) in number (Fig. 8 C). Terminal matureproglottids 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 irregularlyalternating, 26–46% (n=49) from anterior end of proglottid. Some terminal proglottids withsperm-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 fromanterior 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)

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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 anteriormargin of cirrus sac to common genital atrium. Vaginal sphincter absent. Ovary located nearposterior 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 reachingposterior 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, eachwith 1–2 visible rows of follicles, extending from the first or second line of distal testes toafter 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. Uteruslinear, median, extending from ovarian isthmus to vagina level.

Taxonomic summaryType-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 prevalentspecies and displayed great variability in scolex and proglottid morphology. Some specimensof Acanthobothrium n. sp. 3 possess thin and long terminal proglottids while others presentmore rounded and short ones (Fig. 8 D, E). The scolex show some variations as well, mainlyin 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 thesame 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 allmorphometric attributed overlap between them. However, Acanthobothrium n. sp. 3 isdistinguishable 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


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 havingsmaller total length (2.1–2.51 mm vs. > 15 mm), more proglottids (12–32 vs. 4–6) and longerscolex (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 forthe absence of an expanded genital atrium. Acanthobothrium n. sp. 3 have all morphometricranges overlapping with A. himanturi. However, the phylogenetic analyses supports that thisnew species differs from all other species (Clade IV (ETP), Fig. 3). Acanthobothrium n. sp. 3is distinguishable from Acanthobothrium n. sp. 1 by having longer total length (3.34–12.11 mmvs. 1.4–3.0 mm), more proglottids (12–32 vs. 5–10), longer (305–609 vs. 205–303) and widerscolex (241–435 vs. 131–221), and by having larger hook total lengths (D 108–208, D 117–226vs. 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 (241–435vs. 165–192), and by having longer hooks total length (D 108–208, D 117–226 vs. D 73–98,D’ 79–103).

34

.

200

200

A B

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. Terminalmature proglottid (PA16-02-3)

35


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

36

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

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800 1000 1200 1400 1600 1800 2000

200

250

300

350

400

Length

Wid

th

350 400 450 500 550 600

250

300

350

400

450

500

Length

Wid

th

A

B

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.guttatus, red dots: specimens hosted by F. geijskesi.38

.

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



(Figs. 13 - 15)

Description (Based on 16 mature complete worms, one scolex observed with SEM andone terminal proglottid used for cross sections): Worms acraspedote (Fig. 4 D, Table 3),euapolytic, 7.78–28.77 mm (n=16) long, with 49–119 (n=16) proglottids. Scolex consistingof scolex proper and cephalic peduncle (Fig. 13 A, 14 A). Cephalic peduncle 660–1470(n=16) long by 151–415 (=16) wide, covered with gladiate spinitriches with approximately2–4 microtriches/𝜇𝑚2 (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 coveredby gladiate spinitriches with approximately 1–2 microtriches/𝜇𝑚2 and acicular filitriches with0.14 in diameter and approximately 24–32 microtriches/𝜇𝑚2 (Fig. 14 C). Each bothridiawith anterior muscular pad and three loculi divided by two horizontal septa. Anterior loculus341–546 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 ofbothridia and inter-loculi septa covered by papiliform filitriches with 0.12 in diameter andapproximately 49–52 microtriches/𝜇𝑚2 (Fig. 14 D). Muscular pad 178–251 (n=12) long by259–343 (n=12) wide, covered by papiliform filitriches with 0.12 in diameter, triangular shapeand approximately 88–40 microtriches/𝜇𝑚2 (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. 13B). Velum present. Hooks bipronged, hollow, with inconspicuous tubercle on proximal surfaceof axial prong; internal channels of axial and abaxial prongs continuous, smooth; axial prongsand abaxial prongs with different lengths; lateral and medial hooks of different sizes (Fig. 13B). Lateral hook measurements: A 98–140 (n=16), B 154–209 (n=15), C 132–193 (n=16), D219–316 (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. Thinlayer of tissue anteriorly covering each prong of both set of hooks. Immature proglottids widerthan 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 lengthto 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-poraland 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 toovarian 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, extendingfrom ootype along medial line of proglottid to anterior margin of cirrus sac to common genitalatrium. Vaginal sphincter absent. Ovary located near posterior end of proglottid, asymmetrical(ovarian lobes with different lengths) inverted A-shaped or in frontal view, lobulated, reachingor 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) wideat 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 ofthe ovarian lobes, interrupted by vagina and cirrus sac dorsally and ventrally, not interrupted byovary. Uterus linear, median, extending from ovarian isthmus to vagina level.

Taxonomic summaryType-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 toAcanthobothrium sp. ex Hypanus guttatus [A064/AL10-004.01]. Acanthobothrium n. sp. 4differs from the hologenophore of that sequence by having longer hooks handle (A 98–140 andA’ 100–139, vs. A 33.89 and A’ 35.38), larger total hook lengths (D 219–316 and D’ 252–370vs. D 111.96 and D’113.50) and longer medial outer prong than inner prong (B’184–253 andC’ 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 and45–154 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 (737–1127vs. 230, respectively) and wider scolex (601–869 vs. 230–246). This new species differs fromA. himanturi by possessing more proglottids (49–133 vs. 17–26) and longer scolex (737–1127vs. 240–350). Acanthobothrium n. sp. 4 differs from A. brevissime by having larger totallength (7.78–28–77 mm vs. 1.5–4.2 mm), more proglottids (49–133 vs 7–29) and longer scolex

41


(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 differsfrom A. tasajerasi by having larger total length (7.78–28,77 mm vs. 2.5–5.5 mm), longerscolex (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 longerscolex (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 itstotal length (7.78–28.77 mm vs. 2.1–2.51 mm, respectively), number of proglottids (49–133vs. 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 (49–133vs. 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).

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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


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.

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Figure 15: Micrographs of transversal cross sections of Acanthobothrium n. sp. 4. A. Section atlevel 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.

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Discussion

Hosts identity

The Bay of Marajó is inhabited by a unique fauna of batoids, as it includes lineagesrestricted to the Neotropical freshwater systems that can tolerate low salinity habitats, suchas potamotrtygonines, as well as marine lineages that support euryhaline species, such as somedasyatids. 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 bySantos 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 erectedgenus Fontitrygon Last, Naylor Manjaji-Matsumoto, 2016 together with F. geijskesi.

Among these three dasyatids, H. guttatus and F. colarensis are most similar, despite the factthat 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 studyupon which they based generic assignment, nor did they examine the type material of eitherspecies (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 berounded and the posterior margins of the pelvic fins to be pointed in F. colarensis, whereasin 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 todistinguish these species were based on a very small sample size two juvenile specimens and anadult, 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 itselfto be difficult, we used molecular data to guide species assignments for hosts. The topologiesrecovered by the phylogenetic analyses of each partition (MT-CYB, MT-ATP6, MT-ND2) andof the concatenated dataset (Fig. 1, Appendices A.16, A.18 and A.17) recovered consistentlytwo clades attributed to F. geijskesi and H. guttatus. However, we observed two clades amongterminals assigned to H. guttatus with low support that could indicate the existence of twospecies within. The addition of morphometric data did not support that these clades can bedifferentiated morphologically (Table 1 and Fig. 2). Therefore, we concluded that we collectedtwo 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 collectedin the type locality of this species. We collected 59 individuals that could be considered eitherspecies. We found no evidence indicating that any of the host specimens analyzed in this studywere specimens of F. colarensis. Given the fact of a small number of specimens have beenused in the type series and the morphological data presented by Santos et al. (2004) may not berepresentative 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 descriptionand the observed morphometric ranges found in the present study are surprising. Finally, thelack 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 Americafreshwater (SAFW) and marine environments are remarkably different. In marine stingrays,the infection by Acanthobothrium is characterized by a pattern of one parasite species in asingle or few hosts - with few exceptions (Campbell & Beveridge, 2002; Fyler, 2009). Whereasin 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 cestodesin estuarine waters.

The prevalence levels for four species of Acanthobothrium parasites of dasyatids in Bay ofMarajó revealed some interesting patterns (see Table 4). First, all four species can be found inboth dasyatid species, which seems to deviate from the general pattern of strict host specificityreported for marine species (Caira & Jensen, 2017; Fyler, 2009). Second, all species seem tohave a primary host in which prevalence levels are higher in comparison to the other species – a

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secondary host. The difference in prevalence levels between primary and secondary hosts is alsoevident for Acanthobothrium sp. n. 1 (48.86 vs. 3.07, respectively), but it is less pronouncedfor Acanthobothrium sp. n. 2 (11.11 vs.7.69%, respectively; see Table 4). Finally, the levelsof prevalence in primary hosts varied from 100 to ∼11%, whereas for secondary hosts it variedfrom ∼38 to ∼3% in Acanthobothrium sp. n. 3.

Table 4: Prevalence for species of Acanthobothrium in two dasyatids from Bay of Marajó. Numberof hosts surveyed: 27 for H. guttatus and 13 for F. geijskesi. Numbers between parentheses indicatesample 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 forunderstanding patterns of parasite and host association. We can approximate the expectedsample size required for the detection of a parasite species given its prevalence by the followingequation:

𝑛 = ln(𝛼)/ ln(𝑞);

where, 𝛼 is the confidence limit – usually set to 0.05 or 0.01, and 𝑞 is 1 - minimum expectedprevalence (see Dohoo et al., 2012:56-57). Accordingly, it would be expected the examinationof 26 specimens of hosts to detect the presence of Acanthobothrium sp. n. 2 in its primary hostH. guttatus using a confidence interval of 95%.

Our study, however, also detected species of Acanthobothrium in cases for which theestimated sample size would be much larger. For instance, according to the model we used fromDohoo 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 27specimens. To a lesser extend, a similar case can be observed for Acanthobothrium sp. n.1 and 2 in F. geijskesi (Table 4). There are many reasonable explanations to explain theseresults, including violations of some assumptions of this model (i.e., infinite population, evendistributions, among others) to errors associated with prevalence levels estimate, which arebeyond the scope of this discussion. However, our data supports the contention that sample sizehas important roles in our understanding of host specificity.

Generally, the number of host specimens examined with the objective of documenting

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cestode diversity is low. For example, a quick survey of the number of hosts examined intaxonomic descriptions of Acanthobothrium species during a period of 10 years (2006-2016,Cestode Database – accessed on October, 2018), indicated that the number of hosts examinedcan vary from 1 to 341, but with a median of 4 hosts. Most studies focus on the fauna ofa particular host species, and those that included multiple ones, the number of specimensexamined 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 andbiogeography

Even with the inclusion of new terminals in this study, the general phylogenetic status ofAcanthobothrium recovered mirrors the results of Trevisan (2016). Two terminals belongingto 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 andby presenting similar proglottid morphology. However, they differ in hook morphology,since Acanthobothrium bears a pair of bipronged hooks in each bothridium, whereas inOnchobothrium those hooks are single-pronged. Despite conspicuous differences in hookmorphology, species misidentification of both genera are common in the literature (Baer &Euzet, 1962; Caira & Machida, 1986; Linton, 1916; Yamaguti, 1952). The second genusto 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 specimensin the type series of Acanthobothroides thorsoni had the inner prong of the medial hookbroken - which was overlooked in the original description. The phylogeny of molecular datacorroborates the suggestion of Marques et al. (1996) that Acanthobothroides is a junior synonymof Acanthobothrium and the hook morphology found in the two species of the genus are withinthe variability of Acanthobothrium.

Brooks et al. (1981) pioneered the co-evolutionary studies on freshwater Potamotrygonidaeadresssing their origins and host relationships using parasitological data (for marinePotamotrynonidae see Carvalho et al., 2016). They postulated that freshwater potamotrygonids

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derived from the Pacific Ocean and colonized the fluvial systems of South America byan urolophid ancestor (= Urolophus, which was later considered a member of Urobatis inUrotrygonidae). 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 desalinationand was integrated into the South American freshwater river system. This was first the studyto present a phylogeny for Acanthobothrium. However, the study was hardly criticized forthe 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 singlederivation event of stingrays and their parasites. They also assumed that the parasite monophylywould provide evidence for the monophyly of their host considering that both lineages wereunder co-evolutionary process (Brooks et al., 1981; Lovejoy, 1997). This hypothesis wasreiterated 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 evolutionaryhistory 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 shouldalso consider that inference on the monophyly of potamotrygonids stingrays has to considerother historical events besides parasite lineage co-divergence.

After a series of studies (Lovejoy, 1996, 1997), Lovejoy et al. (1998) proposed the mostaccepted 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 ofHimantura, Carvalho et al., 2016). The colonization of the freshwater river systen, arguedLovejoy et al. (1998), took place in the early Miocene during the marine incursions that occurredin the northern region of South America Lovejoy et al. (1998). This hypothesis is well supportedby morphological, molecular, geological and parasitological data (Carvalho et al., 2016; Hoberget 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 betterthe coevolutionary history between freshwater stingrays and their parasites. These studies,however, were still providing the taxonomic foundations required for historical associationstudies for parasite taxa such as Monogenoidea (Platyhelminthes, Cercomeromorpha)(Domingues & Marques, 2010), Potamotrygonocestus (Luchetti, 2011; Marques, 2000),

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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 thathas been historically neglected by those ins=terested in the evoluvtion of this system (Brookset al., 1981; Brooks, 1977; Lovejoy, 1997; Lovejoy et al., 1998). This is particularly true whenthe main goal is to use parasites to infer monophyly of freshwater potamotrygonids. All thesestudies assumed that the ancestor of freshwater potamotrygonids was infected by monophyleticgroups of parasites. However, empirical data, including this, refute this assumption.

The first molecular evidence for the non-monophyly of freshwater lineages ofAcanthobothrium was provided by Machado (2012). This was later corroborated by Trevisan(2016) and now for this study. Freshwater lineages of Acanthobothrium are nested in twounrelated clades, one associated with Pacific species and other related to Atlantic species. TheAtlantic group is closely related to Acanthobothrium species inhabiting rays from NorthwestAtlantic 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, hostspecies 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 andAcanthobothrium sp. 10. In none of these cases, the parasite lineages are phylogeneticallyrelated. Both Acanthobothrium lineages, nested within marine and freshwater lineages, sharingthe same host, is an evidence of that to evaluate host history based in parasites data have to belooked wisely and be supported by other studies. In addition, we also observed phylogeneticrelationships between marine and freshwater parasites lineages, far related, inhabiting the samehost species. For instance, S. schmardae harbours the close marine lineages Acanthobothrium

sp. 1 and Acanthobothrium sp. 3 sensu Trevisan, 2016, whereas the marine lineageAcanthobothrium sp. 9 is nested to the freshwater lineage Acanthobothrium sp. 2. ForH. 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 cladecomposed 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 marinespecies 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


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 theSoutheastern Pacific lineages. Looking at the phylogenetic pattern recovered in our analysisand previous analysis (Machado, 2012), there is no evidence to support the close relationshipof Acanthobothrium sp. 2 with Pacific lineages. Our phylogenetic analyses of molecular datacorroborated 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 ofBay of Marajó and on the phylogenetic relationships between the Acanthobothrium speciesof the Northwest Atlantic and South American freshwater systems. However, this is just thefirst step for the complete understanding of this relation. Except for the single specimen ofAcanthobothrium sp. 10 found in H. guttatus, there is no evidence of parasite sharing betweenmarine and freshwater stingrays. It can be associated with biotic and abiotic characteristicsof the estuary, intermediary hosts availability, capacity to the contact of intermediary host anddefinitive 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 environmentshould be observed with more attention in the future. In order to a better understandthe relationships of, freshwater, estuarine and marine species of Acanthobothrium, the fullappreciation of the Acanthobothrium fauna of Northwest Atlantic and their nested estuaries,is necessary. In addition, understand correctly the life cycle of marine cestodes parasites ofstingrays, besides the increasing the number of hosts specimens analyzed in future studies (seesection "Patterns of host specificity and parasite distribution"), is crucial it to interpret correctlythe evolutionary relationships in brackish water Acanthobothrium.

52

Conclusions

The diagnose of Fontitrygon colarensis is not consistent and should be revised as well as itstaxonomic 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 theinclusion of two species assigned to Acanthobothroides.

The lineage Onchobothrium? ex Hypanus sabinus [AC09/TM-9] seems to be misidentified andshould 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 ofMarajó.

The four new species of Acanthobothrium are exclusively related to marine lineages of theNorthwest Atlantic

Acanthobothrium n. sp. 1 and Acanthobothrium n. sp. 2 presented full overlap of charactersranges and can only be distinguished on the basis of molecular data.

Acanthobothrium n. sp. 3 and Acanthobothrium himanturi presented full overlap of charactersranges.

The lineage Acanthobothrium sp. 10 are related to a clade that includes the marineAcanthobothrium sp. 9 sensu Trevisan, 2016 and freshwater Acanthobothrium sp. 2 CardosoJr. (sensu 2010)

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Alignment Method. Cladistics 19: 261–268.Wheeler, W. C. 1996. Optimization Alignment: the end of multiple sequence alignment in

phylogenetics? Cladistics 12: 1–9.Windsor, D. A. 1995. Equal Rights for Parasites. Conservation Biology 9(1): 1–2.Yamaguti, S. 1952. Studies on the Helminth Fauna of Japan. Part 49. Cestodes of Fishes, II.

Acta Medicinae Okayama 8(1): 1–76.

60

https://github.com/nelas/WormBox

Resumo

A documentação de organismos parasitas tem sido uma ferramenta importante para entendera 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 novashipóteses sobre a distribuição e especificidade dos cestóides. Devido à sua larga distribuiçãogeográfica e em taxons hospedeiros, Acanthobothrium parece ser um bom modelo para avaliarestas hipóteses. A Baía de Marajó é uma área estuarina onde arraias de água doce dafamília Potamotrygonidae compartilham o mesmo ambiente com raias marinhas, especialmentedasiatídeos. Não há documentação sobre a fauna de Dasyatidae para Acanthobothrium a Baíade 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 novaslinhagens de Acanthobothrium, parasitas de Hypanus guttatus e Fontitrygon geijskesi, das quaisquatro são descritas. Cinco dessas novas linhagens estão incluídas em um clado exclusivamentedo Noroeste Atlântico e do Mar do Caribe. Acanthobothrium sp. 10 foi recuperado comogrupo irmão de um clado formado pela linhagem marinha Acanthobothrium sp. 9 sensuTrevisan e pela linhagem de água doce Acanthobothrium sp. 2 sensu Cardoso Jr. O padrãode especificidade de Acanthobothrium tem sido discutido e nossos resultados corroboramesta discussão quando observamos quatro espécies de Acanthobothrium compartilhando duashospedeiras de diferentes gêneros. Além disso, tamanho amostral de hospedeiros pode estarrelacionado com nosso entendimento sobre os padrões de especificidade estrita do parasita aosseus hospedeiros. A ausência de F. colarensis em nossas amostras e as incongruências emcomparação com H. guttatus nos fazem questionar o status taxonômico de F. colarensis.

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

61

Abstract

Parasite documentation has been an important tool to understand host history and co-evolutionary processes in these associations. Acanthobothrium is a worldwide genus ofcestodes, 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 specificitypatterns. Due to their large distribution, geographical and in host taxa, Acanthobothrium seemsto be a good model to evaluate these hypothesis. The Bay of Marajó is an estuarine areawere freshwater stingrays of the family Potamotrygonidae share the same environment withmarine 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. Inour goal to understand those evolutionary events, our results revealed six new lineages ofAcanthobothrium, parasites of Hypanus guttatus and Fontitrygon geijskesi, of which four areformally described. Five of those new lineages are included in a clade exclusive to NorthwestAtlantic and Caribbean Sea. Acanthobothrium n. sp 10 was recovered as sister of clade formedby marine Acanthobothrium sp. 9 sensu Trevisan and freshwater Acanthobothrium sp. 2 sensuCardoso Jr. The specificity pattern of Acanthobothrium has been discussed and our resultscorroborate this discussion when we observed four species of Acanthobothrium sharing twodifferent hosts of different genera. Also, host sample size may be correlated with the specificityand 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 thetaxonomic status of F. colarensis.

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

63

Appendices

Appendix 66

A.1 RScript used for Primary Component Analyses (PCA)#!/usr/bin/Rlibrary("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 availablehost=raw.data$Hostrownames(data)=hostcolors = c("red", "blue", "black") [unclass(raw.data$clade)]#This look substitutes NA for mean of the variable by all samplefor (i in 1:ncol(data))

m = mean(na.omit(data[,i]))x = data[,i]x[is.na(x)] = mdata[,i] = x

#PCA_analisespc.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 vacuumpump 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 (pH8) - 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 𝜇𝑙 (at most 10 𝜇𝑙) of Proteinase-K (20 mg / ml). Mix the vortex and incubate at 65°Cfor one hour or until the glass is completely diluted (leave overnight at 5°C).

3. Let the sample cool and add 300 𝜇𝑙 of ammonium acetate (500 g of 𝑁𝐻4𝐶𝐻3𝐶𝑂2/ 865 mlof 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 extractionyield, 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 𝜇𝑙 of 70% ethanol.

9. Centrifuge for 10 minutes (13,000 rpm at 4°C). Discard the ethanol. Invert the tube ontoabsorbent paper and dry the pellet in the vacuum pump for 30 minutes or at room temperatureovernight.

10. Re-suspend the DNA in TE (50 or 100 𝜇𝑙) overnight at room temperature or at 37°C. Storein 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 𝜇𝑙 of the following mix for each sample: lysis buffer 73 𝜇𝑙, 1M DTT 3 𝜇𝑙, Proteinase-K 4 𝜇𝑙.

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 𝜇𝑙 of the BIND 1 solution.

6. Vortex the BIND solution to resuspend the iron until it is homogeneous.

7. Add 80 𝜇𝑙 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 nottouch the walls of the tube. Discard the supernatant.

11. Add 150 𝜇𝑙 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 inthe bottom of the tube.

14. Dry at room temperature.

15. Resuspend the iron in 50 𝜇𝑙 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 𝜇𝑙 of PCR reaction, add 1.8 𝜇𝑙 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 touchthe walls of the tube. Discard the supernatant.

8. Add 150 𝜇𝑙 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 inthe 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 𝜇𝑙 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 terminalsused as outgroups

Dasyatidae nucleotide sequences, used to constitute hosts out group, with sequences of mitochondrially encoded NADH: ubiquinoneoxidoreductase 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 tophylogenetic 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.

Molecularcode

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

A038 PR09-006.06 Acanthobothrium

quinonesi

Potamotrygon

motoro

Itaipu Dam, Brazil 25°24’45.96”S,

54°35’39.24”W

Machado (2012) • •

A055 MS10-043.01 Acanthobothrium

quinonesi

Potamotrygon

falkneri

Rio Salobra, Miranda, MS,

Brazil

20°12’59.34”S,

56°29”42.95” W

Machado (2012) • •

A064 AL10-004.01 Acanthobothrium

sp.

Hypanus guttatus Costa de Alagoas, Maceió,

Brazil

9°40’25.32"S,

35°44’07.08"W

Machado (2012) • •

A065 AL10-004.02 Acanthobothrium

sp.

Hypanus guttatus Costa de Alagoas, Maceió,

Brazil

9°40’25.32"S, 35° 44’

07.08"W

Machado (2012) • •

A096 VZ11-025.17 Acanthobothrium

sp. 1 sensu

Cardoso, 2010

Paratrygon

aiereba

Rio Apure/Guaritico,

Venezuela

7°53’32.50"N,

68°52’49.80"W

Machado (2012) • •


cf. quinonesi

Potamotrygon

yepezi

Laguna Sinamaica (Rio

Limón), Venezuela

11°2’39.00"N,

71°51’42.50"W

Machado (2012) • •

A106 VZ11-025 Acanthobothrium

terezae

Paratrygon

aiereba


Venezuela

7°53’32.50"N,

68°52’49.80"W

Machado (2012) • -


terezae

Paratrygon

aiereba


Venezuela

7°53’32.50"N,

68°52’49.80"W

Machado (2012) • -


Table A.6 – Continued from previous page

Molecularcode



sp. 1 sensu

Cardoso, 2010

Potamotrygon

orbignyi


Venezuela

7°53’32.50"N,

68°52’49.80"W

Machado (2012) • •

A117 RN11-043.03 Acanthobothrium

amazonensis

Potamotrygon

schroederi

Rio Solimões/Negro/Branco,

Barcelos, AM, Brazil

0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


sp. 2 sensu

Cardoso, 2010

Potamotrygon

schroederi



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


cf. sp. 2 sensu

Cardoso, 2010

Potamotrygon

schroederi



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


sp. 1 sensu

Cardoso, 2010

Potamotrygon

schroederi



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


sp. 1 sensu

Cardoso, 2010

Paratrygon

aiereba



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


quinonesi

Potamotrygon

motoro



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •


amazonansis

Potamotrygon

motoro



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •



Molecularcode



cf. amazonensis

Potamotrygon

orbignyi



0°52’11.28”S,

62°46’37.92”W

Machado (2012) • •

A162 BE-3-13 Acanthobothrium

sp.

Styracura

schmardae

Gulf of Honduras, Head

Caye, Toledo, Belize

16°13’20.8"N,

88°35’38.3"W

Machado (2012) • •


sp.

Styracura

schmardae



16°13’20.8"N,

88°35’38.3"W

Machado (2012) • •


sp.

Hypanus guttatus Gales Point Manatee. Belize 17°13’29.9"N,

88°20’16.2"W

Machado (2012) • •

AC01 AF-89 Acanthobothrium

sp.

Dasyatis

chrysonota

Indian Ocean, South Africa 34°1.5’S, 25°46.61’E Caira et al. (in

prep)

• •


sp.

Gymnura

natalensis

Indian Ocean, South Africa 34°27.36’S,

21°33.20’E

Caira et al. (in

prep)

• •


sp.

Myliobatis

aquila


22°14.65’E

Caira et al. (in

prep)

• •


sp.

Narke capensis Indian Ocean, South Africa 33°51.67’S,

26°14.82’E

Caira et al. (in

prep)

• •


sp.

Torpedo

fuscomaculata


21°55.06’E

Caira et al. (in

prep)

• •

AC06 BJ-338 Acanthobothrium

sp.

Aetobatus

laticeps

Gulf of California,

Baja California Sur, Loreto,

Pacific Ocean, Mexico

25°49’52"N,

111°19’38"W

Caira et al. (in

prep)

• •



Molecularcode


AC07 BE-3 Acanthobothroides

thorsoni

Styracura

schmardae



16°13’20.8"N,

88°35’38.3"W

Caira et al. (in

prep)

• •


sp. 1 sensu

Cardoso, 2010

Rostroraja alba Indian Ocean, South Africa 34°19.86’S, 22°3.72’E Caira et al. (in

prep)

• •

AC09 TM-9 Onchobothrium ? Hypanus sabinus — — Caira et al. (in

prep)

• •


sp. 1 sensu

Cardoso, 2010

Acroteriobatus

annulatus


26°14.82’E

Caira et al. (in

prep)

• •


sp. 2 sensu

Cardoso, 2010

Rostroraja alba Indian Ocean, South Africa 34°19.86’S, 22°3.72’E Caira et al. (in

prep)

• •


sp.

Gymnura

crebripunctata

Gulf of California, Bahia de

Los Angeles, Pacific Ocean

28°59’9"N,

113°32’53"W

Caira et al. (in

prep)

• •


sp.

Gymnura

marmorata

Gulf of California, Bahia de

Los Angeles, Pacific Ocean

28°59’9"N,

113°32’53"W

Caira et al. (in

prep)

• •

AC15 SO-3 Acanthobothrium

sp.

Pastinachus ater Western Province, Solomon

Sea, Solomon Islands, Pacific

Ocean

8°12’27.5"S,

156°59’26.1"E

Caira et al. (in

prep)

• •



Molecularcode


AC16 CM03-61 Acanthobothrium

sp.

Aetomylaeus

vespertilio

Gulf of Carpentaria,

Queensland, Weipa, Indian

Ocean, Australia

12°35’11"S,

141°42’34"E

Caira et al. (in

prep)

• •

AC17 KJG-17 Acanthobothrium

sp.

Parascyllium

collare

Pacific Ocean, Australia — Caira et al. (in

prep)

• •

AC18 KA-162 Acanthobothrium

sp.

Pastinachus

gracilicaudus

Java Sea, Borneo,

West Kalimantan, Selakau,

Indonesia, Pacific Ocean

01°03’30.60"N,

108°58’24.60"E

Caira et al. (in

prep)

• •


sp.

Holohalaelurus

regani

Indian Ocean, South Africa 36°17.60’S, 20°6.6’E Caira et al. (in

prep)

• •


sp.

Mustelus

mustelus


21°18.99’E

Caira et al. (in

prep)

• •


sp.

Trygonoptera sp. Pacific Ocean, Australia — Caira et al. (in

prep)

• •

AC23 BO-431 Acanthobothrium

sp.

Chiloscyllium

punctatum

South China Sea, Borneo,

Sarawak, Mukah, Malaysia,

Pacific Ocean

02°53’52.16"N,

112°05’44.12"E

Caira et al. (in

prep)

• •


sp.

Aptychotrema

rostrata

Pacific Ocean, Australia — Caira et al. (in

prep)

• •

AC25 BE-8 Acanthobothrium

tortum

Aetobatus

narinari

Tobacco Caye, Stann Creek,

Dangriga, Belize

16°54’3.5"N,

88°03’40.3"W

Caira et al. (in

prep)

• •



Molecularcode


AC26 CRP-55 Acanthobothrium

inibiorum

Narcine

entemedor

Costa Rica, Pacific Ocean 10°42’29.592"N,

85°40’9.264"W

Caira et al. (in

prep)

• •


sp.

Zapteryx xyster off Herradura, Pacific Ocean 9°30’02.0"N,

84°41’32.0"W

Caira et al. (in

prep)

• •


sp.

Urotrygon sp. Costa Rica, Pacific Ocean 10°42’29.592"N,

85°40’9.264"W

Caira et al. (in

prep)

• •


sp.

Diplobatis

ommata

off Herradura, Pacific Ocean 09°25’5"N,

84°38’34"W

Caira et al. (in

prep)

• •


sp.

Hypanus longus Costa Rica, Pacific Ocean 10°42’29.592"N,

85°40’9.264"W

Caira et al. (in

prep)

• •

AC31 SO-32 Acanthobothrium

sp.

Urogymnus

asperrimus

Western Province,

near Sagheraghi, Solomon

Sea, Solomon Islands, Pacific

Ocean

8°2’15.1"S,

156°45’57.1"E

Caira et al. (in

prep)

• •

b033 PN15-56.4 Acanthobothrium

sp. 5 sensu

Trevisan, 2016

Styracura

schmardae

Caribbean Sea, Almirante,

Bocas Del Toro, Panama

09°17’N, 82°20’W Trevisan (2016) • •


sp. 9 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •



Molecularcode



sp. 9 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


sp. 8 sensu

Trevisan, 2016

Styracura

pacifica

eastern Pacific Ocean, Playa

Caleta, Montijo, Veráguas,

Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 8 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 7 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 7 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 9 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


himanturi

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


himanturi

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •



Molecularcode



himanturi

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


himanturi

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


himanturi

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


himanturi sp. 3

sensu Trevisan,

2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


sp. 3 sensu

Trevisan, 2016

Styracura

schmardae

Caribbean Sea, Tobacco

Caye, Dangriga, Stann

Creek, Belize

16°54’N, 88°03’W Trevisan (2016) • •

b056 BE-09-15 Acanthobothrium

sp. 5 sensu

Trevisan, 2016

Styracura

schmardae

Caribbean Sea, Tobacco

Caye, Dangriga, Stann

Creek, Belize

16°54’N, 88°03’W Trevisan (2016) • •

b058 TT14-06.5 Acanthobothrium

sp. 6 sensu

Trevisan, 2016

Styracura

schmardae

Maracas bay, Maracas, San

Juan-Laventille, Trinidad &

Tobago

10°45’N, 61°26’W Trevisan (2016) • •


sp. 6 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •



Molecularcode



sp. 6 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 6 sensu

Trevisan, 2016

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •


sp. 1 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


sp. 1 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •


sp. 1 sensu

Trevisan, 2016

Styracura

schmardae



09°17’N, 82°20’W Trevisan (2016) • •

b084 PN15-12.01 Acanthobothroides

pacificus

Styracura

pacifica



Panama

07°29’N, 81°13’W Trevisan (2016) • •

CF01 NT-37 Acanthobothrium

sp.

Himantura

leoparda

Northen Territory, east of

Wessel Islands, Arafura Sea,

Pacific Ocean, Australia

11°17’44"S,

136°59’48"E

Fyler (2009) • •



Molecularcode


CF02 BJ-107 Acanthobothrium

sp.

Narcine

entemedor

Bahia de Los Angeles, Baja

California, Pacific Ocean,

Mexico

28°59’9"N,

113°32’53"W

Fyler (2009) • •

CF03 SE-205 Acanthobothrium

sp.

Rhinobatos

rhinobatos

Atlantic Ocean, Senegal 14°40’42"N,

17°27’42"W

Fyler (2009) • •


sp.

Raja velezi Santa

Rosalia, Baja California Sur,


27°19’51"N,

112°15’30"W

Fyler (2009) • •

CF05 BO-496 Acanthobothrium

etini

Urogymnus

polylepis

Kampung Abai,

Kinebatangan River, Sabah,

Borneo, Kinabatangan River,

Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •


cf. woodsholei

Dasyatis sp. Mbour, Atlantic Ocean,

Senegal

14°24’22"N,

16°58’6"W

Fyler (2009) • •


sp.

Raja miraletus Atlantic Ocean, Senegal 14°40’42"N,

17°27’42"W

Fyler (2009) • •


sp.

Pastinachus ater Northen Territory, east of



11°17’44"S,

136°59’48"E

Fyler (2009) • •

CF24 KJG-1 Acanthobothrium

sp.

Brachaelurus

waddi

Coast of Sydney, Pacific

Ocean, Australia

— Fyler (2009) • •



Molecularcode



cf. angelae

Hypnos

monopterygius


Ocean, Australia

— Fyler (2009) • •


cf. angelae

Hypnos

monopterygius


Ocean, Australia

— Fyler (2009) • •


mattaylori

Rhynchobatus

laevis

Nhulunbuy (Gove), Northern

Territory, Gove

Harbor, Gulf of Carpentaria,


12°11’38"S,

136°41’11"E

Fyler (2009) • •


jaenneae

Rhynchobatus

laevis


Territory, Gove



12°11’38"S,

136°41’11"E

Fyler (2009) • •


hypermekkolpos

Rhynchobatus

laevis


Territory, Gove



12°11’38"S,

136°41’11"E

Fyler (2009) • •


hypermekkolpos

Rhynchobatus

laevis


Territory, Gove



12°11’38"S,

136°41’11"E

Fyler (2009) • •



Molecularcode



herronorum

Rhynchobatus cf.

laevis




11°17’44"S,

136°59’48"E

Fyler (2009) • •

CF37 AZ-10 Acanthobothrium

sp.

Dipturus batis Horta,

Azores Islands, Faial Island,

Atlantic Ocean, Portugal

38°31’N, 28°37’W Fyler (2009) • •


sp.

Glaucostegus

cemiculus


17°27’42"W

Fyler (2009) • •


sp.

Glaucostegus

cemiculus


17°27’42"W

Fyler (2009) • •


sp.

Aetomylaeus

bovinus

St. Louis, Atlantic Ocean,

Senegal

16° 1’28"N,

16°30’33"W

Fyler (2009) • •


sp.

Neotrygon cf.

kuhlii

Sarawak,

Borneo, South China Sean,

Pacifc Ocean, Malaysia

02°49’01.20"N,

110°52’47.16"E

Fyler (2009) • •

CF47 MS05-402 Acanthobothrium

cf. brevissime

Hypanus say Florida, Crooked Island Bay,

Gulf of Mexico, Atlantic

Ocean, U.S.A.

29°59’37"N,

85°31’48"W

Fyler (2009) • •


sp.

Hypanus sabinus Florida, Indian Pass, Gulf

of Mexico, Atlantic Ocean,

U.S.A.

29°40’8"N,

85°13’30"W

Fyler (2009) • •



Molecularcode



sp.

Raja eglanteria Florida, Indian Pass, Gulf


U.S.A.

29°40’8"N,

85°13’30"W

Fyler (2009) • •


sp.

Raja eglanteria Florida, Indian Pass, Gulf


U.S.A.

29°40’8"N,

85°13’30"W

Fyler (2009) • •


sp.

Urogymnus

lobistoma

Mukah, Sarawak, Borneo,

South China Sean, Pacifc

Ocean, Malaysia

02°53’52.16"N,

112°05’44.12"E

Fyler (2009) • •


sp.

Dasyatis sp. Mbour, Atlantic Ocean,

Senegal

14°24’22"N,

16°58’6"W

Fyler (2009) • •


sp.

Paragaleus

pectoralis

Ouakam, Atlantic Ocean,

Senegal

14°42’54"N,

17°29’28"W

Fyler (2009) • •

CF68 CM03-3 Uncibilocularis

squireorum

Himantura cf.

urarnak

Weipa, Queensland, Gulf of

Carpentaria, Indian Ocean,

Australia

12°35’11"S,

141°42’34"E

Fyler (2009) • •


sp.

Brachaelurus

waddi


Ocean, Australia

— Fyler (2009) • •


cf. lasti

Himantura

leoparda




11°17’44"S,

136°59’48"E

Fyler (2009) • •



Molecularcode



sp.

Gymnura

altavela

Kafountine, Casamance,

Atlantic Ocean, Senegal

12°55’41"N,

16°45’10"W

Fyler (2009) • •


sp.

Gymnura

altavela

Joal, Atlantic Ocean, Senegal 14°10’30"N,

16°51’12"W

Fyler (2009) • •


sp.

Zanobatus

schoenleinii


17°27’42"W

Fyler (2009) • •


sp.

Taeniura grabata Mbour, Atlantic Ocean,

Senegal

14°24’22"N,

16°58’6"W

Fyler (2009) • •


sp.

Fontitrygon

margaritella

Djifere, Atlantic Ocean,

Senegal

13°55’50”N,

16°45’42”W

Fyler (2009) • •

CF85 PU-4 Acanthobothrium

cf. terezae

Potomotrygon cf.

motoro

Boca Manu, Madre de Dios,

Madre de Dios River, Peru

12°16’S 70°55’W Fyler (2009) • •


rodmani

Himantura sp. Northen Territory, east of



11°17’44"S,

136°59’48"E

Fyler (2009) • •


romanowi




11°17’44"S,

136°59’48"E

Fyler (2009) • •


oceanharvestae




11°17’44"S,

136°59’48"E

Fyler (2009) • •



Molecularcode


CF100 TW-19 Acanthobothrium

margieae

Orectolobus

japonicus

Magong, Penghu, Taiwan

Strait, Pacific Ocean, Taiwan

23°33’49"N,

119°34’31"E

Fyler (2009) • •

CF103 KA-43 Acanthobothrium

sp.

Himantura

undulata

Muara Pasir,

East Kalimantan, Borneo,

Makassar Strait, Pacific

Ocean, Indonesia

01°45’58.92"S,

116°23’36.09"E

Fyler (2009) • •


santarosaliense

Heterodontus

mexicanus

Santa

Rosalia, Baja California Sur,


27°19’51"N,

112°15’30"W

Fyler (2009) • •


cf. microcephalum

Myliobatis

californica

Bahia de Los Angeles, Baja

California, Pacific Ocean,

Mexico

28°59’9"N,

113°32’53"W

Fyler (2009) • •


romanowi




11°17’44"S,

136°59’48"E

Fyler (2009) • •


popi




11°17’44"S,

136°59’48"E

Fyler (2009) • •

CF121 NT-96 Acanthobothrium.

zimmeri




11°17’44"S,

136°59’48"E

Fyler (2009) • •



Molecularcode



zimmeri




11°17’44"S,

136°59’48"E

Fyler (2009) • •


rodmani




11°17’44"S,

136°59’48"E

Fyler (2009) • •


oceanharvestae




11°17’44"S,

136°59’48"E

Fyler (2009) • •

CF128 TW-19 Acanthobothrium

margieae

Orectolobus

japonicus

Magong, Penghu, Taiwan

Strait, Pacific Ocean, Taiwan

23°33’49"N,

119°34’31"E

Fyler (2009) • •


sp.

Maculabatis cf.

gerrardi

Sidu, West Kalimantan,

Borneo, Java Sea, Pacific

Ocean, Indonesia

01°21’45.20"S,

110°04’10.30"E

Fyler (2009) • •

CF134 BO480 Acanthobothrium

sp.

Urogymnus

lobistoma

Mukah, Sarawak, Borneo,

South China Sean, Pacifc

Ocean, Malaysia

02°53’52.16"N,

112°05’44.12"E

Fyler (2009) • •


etini

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •



Molecularcode



masnihae

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •


saliki

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •


zainili

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •


masnihae

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •


saliki

Urogymnus

polylepis

Kampung Abai,



Malaysia

05°41’10.81"N,

118°23’08.35"E

Fyler (2009) • •



Molecularcode


CF147 TM-1 Acanthobothrium

sp.

Raja eglanteria Cape Point, North Carolina,

Western Atlantic Ocean,

U.S.A.

35°15’N, 75°31’W Fyler (2009) • •

CF151 VI-1 Acanthobothrium

ramiroi

Potamotrygon

motoro

Colastiné River, Santa Fé,

Argentina

31°40’S, 60°46’W Fyler (2009) • •

CF152 VI-2 Acanthobothrium

ramiroi

Potamotrygon

motoro

Colastiné River, Santa Fé,

Argentina

31°40’S, 60°46’W Fyler (2009) • •


sp.

Maculabatis cf.

gerrardi

Kota Baru,

South Kalimantan, Borneo,

Makassar Strait, Pacific

Ocean, Indonesia

03°14’44.80"S,

116°13’23.80"E

Fyler (2009) • •


sp.

Pateobatis fai Manggar, East Kalimantan,

Borneo, Makassar Strait,

Pacific Ocean, Indonesia

01°12’55.20"S,

116°58’27.50"E

Fyler (2009) • •

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 Bankfreshwater 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 specimensAcanthotaenia 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 frommitochondrially 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 • • •





PA16-026 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 • - •










PA16-050 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 • - •














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 • • -













93 Appendix

20.0


Fontitrygon margarita

Fontitrygon margaritella

Bathyoshia brevicaudata











Hypanus americanus
























Hypanus sabinus




Hypanus dipterurus




23

26

3

25

3176

20

1

1

1

11

3

1

1

2

19

1

2

16

2

3

27

2

1

3

5

4

21

46

2

1

17

34

2

1

12

Bathyoshia centroura

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 sequencesunder 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

7.0

Dasyatis brevis






Hypanus dipterurus











Neotrygon kuhlii













Hemitrygon akajei













1

2

3

25

1

1

39

2

1

14

1

1

1

16

13

1

3

1

2

1

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

7.0




Dasyatis brevis


























Hemitrygon akajei












Neotrygon kuhlii





21

19

1

1

1

1

4

17

18

20

14

3

1

5

2

Figure A.18: Phylogenetic relationship between hosts, based in mitochondrially encoded ATP synthasemembrane subunit 6 (MT-ATP6) gene, of present work and GenBank sequences under parsimony as theoptimality criteria. Contents between parentheses refer to host accesion code. Goodman-Bremer valuesimplemented. 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

Molecularcode

Hologenophorescode

Acanthobothrium species Host species Collection location Coordinates

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


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




















Molecularcode

Hologenophorescode


























Molecularcode

Hologenophorescode









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

















Molecularcode

Hologenophorescode
























Acanthobothrium Blanchard, 1948 from the Northwest Atlantic … · 2019. 3. 1. · o escotismo...

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