i UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS - CAMPUS DE BOTUCATU PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ZOOLOGIA) CARACTERIZAÇÃO MORFOLÓGICA E MOLECULAR DE ESPÉCIES DO GÊNERO Synthesium (DIGENEA: BRACHYCLADIIDAE) EM ODONTOCETOS (CETACEA) Mariana Bertholdi Ebert Orientador: Prof. Tit. Reinaldo José da Silva Coorientadora: Dra. Juliana Marigo Dissertação apresentada ao Programa de Pós-graduação em Ciências Biológicas (Zoologia) do Instituto de Biociências da Universidade Estadual Paulista, UNESP, Campus de Botucatu-SP, como parte dos requisitos para obtenção do título de Mestre em Ciências Biológicas (Zoologia). Botucatu, SP 2017 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE-CRB 8/5651 Ebert, Mariana Bertholdi. Caracterização morfológica e molecular de espécies do gênero Synthesium (Digenea: Brachycladiidae) em odontocetos (Cetacea) / Mariana Bertholdi Ebert. - Botucatu, 2017 Dissertação (mestrado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Reinaldo José da Silva Coorientador: Juliana Marigo Capes: 20400004 1. Mamífero aquático. 2. Intestinos - Parasitos. 3. Helminto. 4. Morfologia. 5. Taxonomia numérica. Palavras-chave: Delphinidae; Identificação molecular; Morfologia; Parasitos intestinais; Taxonomia. ii Dedico este trabalho a meus pais, Estela e Arlindo, inspirações da minha vida. iii “Deus ao mar o perigo e o abismo deu, Mas nele é que espelhou o céu.” (Fernando Pessoa) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Agradecimentos! ! ! v ! AGRADECIMENTOS- - - Este%trabalho%seria%impossível%sem%a%colaboração%de%algumas%pessoas%e%instituições% que,% de% diversas% formas,% deram% sua% contribuição% em% diferentes% etapas.% Destas,% manifesto%um%agradecimento%especial:%% Aos%meus%pais% pelo% infinito% amor,% respeito,% cumplicidade% e% confiança.% Sem%esses% pilares,%nada%seria%possível.% À%Juliana%Marigo,%pela%confiança%e%por%me%ensinar%tanto.%Que%sorte%a%minha%de%ter% uma%amiga%e%fada%madrinha%como%coorientadora!% Ao% Professor% Reinaldo% José% da% Silva,% por% ser% um% orientador% sempre% presente.% Agradeço% todos%os% ensinamentos,% apoio%e%disponibilidade%em%sempre% tirar% todas% minhas%dúvidas.�% Aos% amigos% do% LAPAS% pelo% acolhimento,% conversas% bemKhumoradas% durante% os% cafézinhos%e%ensinamentos%de%todas%as%formas.%% Aos%colegas%do%IPeC%e%Cananéia%por%me%ensinarem%a%amar%o%que%se%faz.%Agradeço%a% todos%que%ajudaram%nas%animadas%coletas%de%amostras%e%monitoramentos%de%praia.% Agradeço%também%todas%as%palavras%de%carinho%e%incentivo%nessa%caminhada.% À%Maria%Isabel%Müller,%por%me%guiar%pelo%maravilhoso%mundo%da%biologia%molecular.% À%Natalia%FraijaKFernández,%pelas%valiosas%discussões%sobre%parasitos%de%cetáceos%e% por%ser%sempre%gentil,%e%à%Mercedez%Fernández%pela%disponibilidade%em%ajudar%e%pela% cessão%de%amostras.%%%%% Ao%Conselho%Nacional% de%Desenvolvimento%Científico% e%Tecnológico% (CNPq),% pela% bolsa% de%mestrado,% e% à%UNESP%pelo% suporte% tecnológico%para% o% desenvolvimento% desta%pesquisa.%% Ao%Leandro%Cagiano%pela%imagem%de%capa.% À%todos%aqueles%que%contribuíram%de%alguma%forma,%mesmo%sem%saber,%para%o%meu% crescimento%na%pesquisa%e%como%ser%humano.% Por%fim,%agradeço%aos%golfinhos%e%baleias%e%seus%“vermes”,%por%involuntariamente%me% servirem%de%objeto%de%estudo%e%me%darem%a%chance%de%realizar%meus%sonhos.% Gratidão%ao%universo%por%todos%os%ensinamentos%de%todas%as%formas!%%%% ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Sumário! ! ! vii ! ! SUMÁRIO! RESUMO!..............................................................................................................................................!1! ABSTRACT!.........................................................................................................................................!3! INTRODUÇÃO!GERAL!..................................................................................................................!5! Estudos!parasitológicos!.........................................................................................................!6! Cetáceos!........................................................................................................................................!6! Parasitismo!e!diversidade!de!helmintos!em!cetáceos!..............................................!7! Família!Brachycladiidae!........................................................................................................!8! Gênero!Synthesium!...................................................................................................................!8! Referências!bibliográficas!.................................................................................................!10! OBJETIVO!........................................................................................................................................!15! Objetivo!geral!..........................................................................................................................!16! Objetivos!específicos!............................................................................................................!16! A!NEW!Synthesium!SPECIES!(DIGENEA:!BRACHYCLADIIDAE)!FROM! BOTTLENOSE!DOLPHINS!Tursiops/truncatus!(CETACEA:!DELPHINIDAE)!IN! SOUTHWESTERN!ATLANTIC!WATERS!...........................................................................!17! Abstract!.....................................................................................................................................!18! Introduction!.............................................................................................................................!19! Material!and!Methods!..........................................................................................................!19! Results!........................................................................................................................................!22! Discussion!.................................................................................................................................!26! References!................................................................................................................................!28! PHYLOGENETIC!RELATIONSHIP!AMONG!SPECIES!OF!Synthesium! (DIGENEA:!BRACHYCLADIIDAE),!PARASITES!OF!DOLPHINS,!BASED!ON! MORPHOLOGICAL!AND!MOLECULAR!DATA!................................................................!40! Abstract!.....................................................................................................................................!41! Introduction!.............................................................................................................................!42! Material!and!Methods!..........................................................................................................!43! Results!........................................................................................................................................!45! Discussion!.................................................................................................................................!48! References!................................................................................................................................!52! /CONSIDERAÇÕES!FINAIS!.......................................................................................................!69! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Resumo! ! ! ! 2 RESUMO Trematódeos do gênero Synthesium infectam o intestino de diversas famílias de odontocetos em todo o mundo. No entanto, a identificação e o status taxonômico de Synthesium ainda são confusos e, principalmente, baseados em análises morfológicas. As informações genéticas ainda são excassas. O presente estudo teve como objetivo investigar a diversidade de parasitos intestinais do gênero Synthesium colhidos de diferentes odontocetos, através de comparações morfológicas e moleculares. Foram estudados parasitos de golfinhos encontrados mortos em praias ou acidentalmente capturados em redes de pesca de diferentes regiões. Durante a necropsia, o intestino delgado foi removido e armazenado a -20°C. Posteriormente, os intestinos foram abertos, lavados em água corrente sobre peneira de 150 µm e o conteúdo examinado sob estereomicroscópio. Os trematódeos encontrados foram fixados e preservados em etanol 70% para análises morfológicas e moleculares. Os trematódeos foram preparados em lâminas temporárias e as análises morfométricas diagnósticas foram feitas em um sistema computadorizado para análise de imagens. O DNA genômico dos trematódeos foi extraído e amplificado utilizando-se primers com alvo no DNA ribossomal (menor subunidade do DNA ribossomal - gene SSU - e região intergênica - ITS1) e DNA mitocondrial (nicotinamida dinucleotídeo dehidrogenase subunidade 3 - gene ND3 - e citocromo c oxidase subunidade I - gene COI). Os resultados morfológicos e moleculares apontam a existência de pelo menos quatro novas espécies de trematódeos do gênero Synthesium, sendo uma delas, Synthesium neotropicalis, descrita neste trabalho. As análises filogenéticas demonstram um complexo de espécies agregando S. tursionis e o posicionamento de S. delamurei, S. subtile e S. seymouri gera questionamentos sobre a correta inclusão destas espécies ao gênero, incentivando sua revisão através de abordagens morfológicas e moleculares. Os resultados obtidos representam uma importante contribuição para o conhecimento da helmintofauna de cetáceos. ! Palavras-chave: Parasitos intestinais, Delphinidae, Identificação molecular, Morfologia, Taxonomia. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Abstract! ! ! ! 4 ABSTRACT Trematodes of the genus Synthesium infect the intestines of a wide range of odontocete families worldwide. However, the identification and the taxonomic status of Synthesium are still confusing and mainly based on morphological analyses. Genetic information is still scarce. The present study aimed to investigate the diversity of intestinal parasites of the genus Synthesium collected from different odontocetes species, by means of morphological and molecular approaches. Parasites of dolphins found washed ashore or accidentally caught in fishing nets from different regions were studied. During necropsy, the small intestines were removed and stored at -20°C. The intestines were opened, washed in tap water over a 150 µm sieve and examined under a stereomicroscope. The recovered trematodes were fixed and preserved in 70% ethanol for morphological and molecular analyses. The parasites were prepared as temporary slides and diagnostic morphometric analyses were performed on a computerized imaging system. The genomic DNA of the trematodes was extracted and amplified using primers targeted at the ribosomal (small ribosomal subunit - SSU gene- and internal transcribed spacer 1 - ITS1) and mitochondrial DNA (NDH dehydrogenase subunit 3 - ND3 gene- and cytochrome c oxidase subunit 1 – COI gene). The morphological and molecular results indicate the existence of at least four new species of trematodes of the genus Synthesium. Phylogenetic analyses show a species complex aggregating S. tursionis and the phylogenetic position of S. delamurei, S. subtile and S. seymouri raises questionings about the correct inclusion of these species to the genus, suggesting a revision of the genus through morphological and specialy molecular approaches. The obtained results represent an important contribution to the knowledge of the helmintofauna of cetaceans. Keywords: Intestinal parasites, Delphinidae, Molecular identification, Morphology, Taxonomy. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Introdução!geral! ! ! ! 6 INTRODUÇÃO GERAL Estudos parasitológicos Parasitos apresentam uma grande biodiversidade e potencialmente infectam a maioria dos metazoários de vida livre (POULIN & MORAND, 2000). Parasitos podem influenciar no funcionamento dos ecossistemas e fornecer informações sobre a história evolutiva e demográfica de seus hospedeiros, aspectos estes de grande importância para a conservação das espécies (AZNAR et al., 2010). Parasitos em cetáceos têm sido envolvidos em diversos estudos relacionados à delimitação de estoque populacional (BALBUENA et al., 1995; MARIGO et al., 2015), estrutura social (BALBUENA et al., 1995), história da população (KALISZEWSKA et al., 2005) e condição de saúde de seus hospedeiros (AZNAR et al., 2005). Assim, um modelo parasito-hospedeiro fornece uma ferramenta adequada para estudar a relação ecológica, sistemática, evolutiva e biogeográfica entre organismos (BROOKS & HOBERG, 2000). Cetáceos Os cetáceos (golfinhos, botos e baleias) são mamíferos marinhos completamente adaptados à vida aquática (DI BENEDITTO et al., 2010). A ordem Cetacea Brisson, 1762 é dividida em três subordens, Archaeoceti Flower, 1833 (apenas fósseis), Mysticeti Cope, 1864 e Odontoceti Flower, 1867. Os Mysticeti (cetáceos com barbatanas, conhecidos como baleias verdadeiras) estão divididos em quatro famílias e 14 espécies, sendo que oito delas ocorrem no Brasil. Os Odontoceti (cetáceos que possuem dentes, conhecidos como golfinhos e botos) dividem-se em nove famílias e 69 espécies, das quais 35 ocorrem no litoral brasileiro (ICMBio, 2010; DI BENEDITTO et al., 2010). A distribuição dos cetáceos ao longo dos 8.000 km da costa brasileira é de extrema amplitude, ocorrendo de forma contínua. Isso se dá pela diversidade de ecossistemas presentes ao longo de nosso litoral (baías, manguezais, recifais etc), os quais representam habitats favoráveis para reprodução e alimentação destes animais (IBAMA, 2005). A maioria das espécies listadas no Plano de Ação de Mamíferos Aquáticos do Brasil (IBAMA, 2001) está classificada na categoria DD (Deficient Data) da IUCN Red List of Threatened Species (http://www.iucnredlist.org), indicando que não existem informações adequadas para avaliação do status de conservação, ameaças sofridas e ! 7 outras características das espécies, inclusive sobre a diversidade parasitária abrigada ou a relação parasito-hospedeiro. Muito desta carência se deve a inexistência de estudos e destinação adequada de material biológico para fins acadêmicos e, consequentemente, dados substanciais a serem empregados em projetos que visam à conservação destes animais (IBAMA, 2005). Parasitismo e diversidade de helmintos em cetáceos Os primeiros registros de helmintos em cetáceos datam do inicio de 1930, época esta em que a caça comercial era permitida (BAYLIS, 1932; PRICE, 1932). Atualmente, a maioria dos registros utilizam carcaças de cetáceos provenientes de encalhes ocasionais ou capturas acidentais em redes de pesca (RAGA et al., 2008). Recentemente, alguns estudos têm utilizado técnicas não invasivas através da colheita de amostras de fezes para identificação de helmintos (KLEINERTZ et al., 2014; HERMOSILLA et al., 2016). Estudos parasitológicos em cetáceos são limitados pela dificuldade de acesso tanto aos hospedeiros como seus parasitos em bom estado de conservação. Como consequência, há uma escassez de informações morfológicas e principalmente genéticas a respeito destes organismos (RAGA et al., 2008). Devido a esta insuficiência de informações disponíveis, principalmente em águas brasileiras, é importante o incentivo a estudos parasitológicos de caráter contínuo e padronizado. Estes trabalhos são inclusive citados junto aos projetos e ações prioritárias estabelecidas pelo Plano de Ação para os Mamíferos Aquáticos do Brasil (IBAMA, 2001). A helmintofauna de cetáceos inclui atualmente cerca de 174 espécies divididas em quatro grandes grupos taxonômicos: os nematoides são o grupo mais diverso (62 espécies), seguido por trematódeos digenéticos (54 espécies), cestoides (38 espécies) e acantocéfalos (20 espécies) (FRAIJA-FERNÁNDEZ et al., in press). As famílias Pseudaliidae Railliet & Henry, 1909, Anisakidae Skrjabin & Karokhin, 1945 e Tetrameridae Travassos, 1914 são as mais representativas entre os nematoides parasitos de cetáceos. Os cestoides estão representados principalmente pelas famílias Tetrabothriidae Linton, 1891, Diphyllobothriidae Lühe, 1910 e Phyllobothriidae Braun, 1900. A família Polymorphidae Meyer, 1931 abriga todas as espécies de acantocéfalos infectantes de cetáceos. Já os trematódeos digenéticos estão distribuídos em quatro famílias: Brauninidae Wolf, 1903, Notocotylidae Lühe, 1909, Heterophyidae Leiper, 1909 e Brachycladiidae Odhner, 1905. A família Brauninidae é ! 8 monotípica, incluindo somente Braunina cordiformis Wolf, 1903. A família Notocotylidae inclui as espécies do gênero Ogmogaster Jägerskiöld, 1891. A família Heterophyidae é representada por uma única espécie, Pholeter gastrophilus (Kossack, 1910) Odhner, 1914. A família Brachycladiidae é a mais representativa, incluindo 10 gêneros e 42 espécies (GIBSON, 2005; FRAIJA-FERNÁNDEZ et al., in press). Família Brachycladiidae Digenéticos da família Brachycladiidae ocorrem nos ductos hepáticos e pancreáticos, intestinos, pulmões e seios nasais de mamíferos marinhos em todo o mundo (FERNÁNDEZ et al., 1998a,b; GIBSON, 2005). A maior parte da sua diversidade é encontrada em cetáceos (35 das 42 espécies descritas), mas algumas espécies também infectam pinípedes e lontras marinhas (AZNAR et al., 2001; DAILEY, 2007). Pouco se sabe sobre a biologia da família Brachycladiidae. Nenhum hospedeiro intermediário ou paratênico foi identificado até o momento (FERNÁNDEZ, 1996). Análises filogenéticas e morfológicas indicam que a origem deste grupo e sua associação com mamíferos marinhos provavelmente resultou de eventos de mudanças entre hospedeiros a partir de um ancestral ocorrente em peixes, e que foi capaz de infectar odontocetos através da relação predador-presa (FERNÁNDEZ et al., 1998b; BRAY et al., 2005; FRAIJA-FERNÁNDEZ et al., 2015). A organização taxonômica da família também é bastante controversa, incluindo muitas transferências de espécies e sinonímias. As identificações são principalmente baseadas em análises morfológicas de helmintos adultos e, recentemente, algumas espécies têm sido reavaliadas através de estudos moleculares (MARIGO et al., 2011). Atualmente são reconhecidas duas subfamílias; Brachycladiinae Odhner 1905 incluindo Brachycladium Looss, 1899, Hunterotrema McIntosh 1960, Oschmarinella Skrjabin 1947, Synthesium Stunkard & Alvey 1930, Campula Cobbold 1858, Zalophotrema Stunkard & Avey 1929, Orthosplanchnus Odhner 1905 e Odhneriella Skrjabin 1915; e Nasitrematinae Ozaki, 1935, que agrega os gêneros Nasitrema Ozaki 1935 e Cetitrema Skrjabin 1970 (GIBSON, 2005). Gênero Synthesium Parasitos do gênero Synthesium infectam o intestino, sobretudo a porção anterior do duodeno, de praticamente todas as famílias de odontocetos (17 espécies registradas ! 9 dentro das famílias Pontoporiidae Gray, 1846, Delphinidae Gray, 1821, Monodontidae Gray, 1821 e Phocoenidae Gray, 1825), sugerindo um longo período de adaptações parasito-hospedeiros (FERNÁNDEZ, 1996). O gênero Synthesium sofreu diferentes rearranjos taxonômicos nos últimos anos, com várias sinonímias entre gêneros morfologicamente relacionados como Leucasiella Krotov & Delyamure 1952, Hadwenius Price, 1932 e Odhneriella Skrjabin, 1915 (GIBSON, 2005, 2014). YAMAGUTI (1958) reconheceu Leucasiella como sinônimo de Hadwenius. Posteriormente, GIBSON (2005) considerou Hadwenius como sinônimo júnior de Synthesium. Atualmente são reconhecidas oito espécies deste gênero; três aparentam especificidade de hospedeiros sendo Synthesium seymouri (Price, 1932) Marigo, Vicente, Valente, Measures & Santos, 2008, parasito de belugas Delphinapterus leucas (Pallas, 1776), Synthesium delamurei Raga & Balbuena, 1988, parasito de baleias piloto de peitorais longas Globicephala melas Traill, 1809, e Synthesium pontoporiae (Raga, Aznar, Balbuena & Dailey, 1994) Marigo, Vicente, Valente, Measures & Santos, 2008 encontrado em toninhas Pontoporia blainvillei Gervais & d'Orbigny, 1844. Synthesium nipponicum Yamaguti, 1951 é exclusivo de delfinídeos da família Phocoenidae, parasitando o golfinho de Dall Phocoenoides dalli True, 1885 e o golfinho do proto Phocoena phocoena Linnaeus, 1758. O restante das espécies, Synthesium subtile Skrjabin, 1959, Synthesium mironovi Krotov & Delyamure, 1952, Synthesium elongatum Ozaki, 1935 e Synthesium tursionis (Marchi, 1873) Stunkard & Alvey, 1930 são generalistas e apresentam uma grande variedade de hospedeiros registrados (FERNÁNDEZ, 1996; GIBSON, 2005, 2014). Devido à dificuldade na obtenção de espécimes preservados e a fixação inadequada dos parasitos, muitos caracteres morfológicos são comprometidos (GIBSON, 2005), principalmente porque as espécies deste gênero são bastante similares e as diferenças morfológicas muitas vezes são sutis, o que dificulta a identificação e classificação através de abordagens morfológicas tradicionais. Alem disso, alguns estudos apontam variação morfológica intraespecífica em diferentes populações de odontocetos (LAMOTHE-ARGUMEDO, 1988; FERNANDÉZ et al., 1994, 1995). O uso de métodos de identificação molecular para este gênero ainda é recente e os resultados estimulam novas investigações (MARIGO et al., 2011, 2015). Um estudo integrando caracteres morfológicos e moleculares de espécies de Synthesium, coletados de diferentes odontocetos hospedeiros, gera informações ! 10 importantes para a identificação e caracterização correta das espécies deste gênero, contribuindo para um panorama mais preciso a respeito da biodiversidade e do status taxonômico de Synthesium e, em consequência, sua posição filogenética dentro da família Brachycladiidae. Referências bibliográficas AZNAR, F.J.; BALBUENA J.A.; FERNÁNDEZ, M. & RAGA, J.A. Living together: The parasites of marine mammals. In: Evans P. and Raga J.A. (eds). Marine mammals: Biology and Conservation. New York, USA: Kluwer Academic, Plenum Publishers, p. 385-421, 2001. AZNAR, F.J.; PERDIGUERO, D.; PÉREZ DEL OLMO, A.; REPULLÉS, A.; AGUSTÍ, C. & RAGA, J.A. 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Phylogenetic relationships of the family Campulidae (Trematoda) based on 18S rRNA sequences. Parasitology, v. 117, p. 383-391, 1998b. FRAIJA-FERNÁNDEZ, N.; FERNÁNDEZ, M.; RAGA, J.A. & AZNAR F.J Helminth diversity of cetaceans: An update. Advances in Marine Biology, v. 1, in press. FRAIJA-FERNÁNDEZ, N.; OLSON, P.D.; CRESPO, E.A.; RAGA, J.A.; AZNAR F.J. & FERNÁNDEZ, M. Independent host switching events by digenean parasites of cetaceans inferred from ribosomal DNA. International Jounal for Parasitology, v. 45, p. 167-73, 2015. ! 12 GERACI, J.R. & LOUNSBURY, V.J. Marine Mammals Ashore: A Field Guide for Strandings. Second Edition. Baltimore: National Aquarium in Baltimore, 371 p., 2005. GIBSON, D. Family Brachycladiidae Odhner, 1905. In: Jones A, Bray RA and Gibson DI (eds) Keys to the Trematoda Vol. 2. CABI Publishing and The Natural History Museum, Wallingford, p. 641-652, 2005. GIBSON, D. Synthesium Stunkard & Alvey, 1930. World Register of Marine Species at http://www.marinespecies.org/aphia.php?p=taxdetails&id=238045. Acessado em 22 de Junho de 2016, 2014. HERMOSILLA, C.; SILVA, L.M.R.; KLEINERTZ, S.; PRIETO, R.; SILVA M.A & TAUBERT, A. Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods. Parasitological Research, v. 115, p. 889-896, 2016. IBAMA. Mamíferos Aquáticos do Brasil: Plano de Ação - Versão II. Brasília: IBAMA, 2001. IBAMA. Protocolo de conduta para encalhes de mamíferos aquáticos: Rede de encalhes de mamíferos aquáticos do Nordeste. Recife: Edições IBAMA, 298 p., 2005. ICMBIO. Plano de ação nacional para a conservação dos mamíferos aquáticos: pequenos cetáceos. Brasília: Instituto Chico Mendes de Conservação da Biodiversidade, 132 p., 2010. KALISZEWSKA, Z.A.; SEGER, J.; ROWNTREE, V.J.; BARCO, S.G.; BENEGAS, R.; BEST, P.B.; BROWN, M.W.; BROWNELL JR, R.L.; CARRIBERO, A.; HARCOURT, R.; KNOWLTON, A.R.; MARSHALL-TILAS, K.; PATENAUDE, N.J.; RIVAROLA, M.; SCHAEFF, C.M.; SIRONI, M.; SMITH, W.A. & YAMADA T.K. Population histories of right whales (Cetacea: Eubalaena) inferred from mitochondrial sequence diversities and divergences of their whale lice (Amphipoda: Cyamus). Molecular Ecology, v. 14, p. 3439-3456, 2005. KLEINERTZ, S.; HERMOSILLA, C.; ZILTENER, A.; KREICKER, S.; HIRZMANN, J.; ABDEL-GHAFFAR, F. & TAUBERT, A. Gastrointestinal parasites of free- living Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Northern Red Sea, Egypt. Parasitology Research v. 113, p. 1405-1415, 2014. ! 13 LAMOTHE-ARGUMEDO, R. Trematodos de mamíferos III. Hallazgo de Synthesium tursionis (Marchi 1873) Stunkard & Alvey 1930 en Phocaena sinus (Phocoenidae) en el Golfo de California. Anales del Instituto de Biologia de la Universidade de México, Serie Zoologia, v. 58, p. 11-20, 1998. MARIGO, J.; VICENTE, A.C.P.; VALENTE, A.L.S.; MEASURES, L. & SANTOS, C.P. Redescription of Synthesium pontoporiae n. comb. with notes on S. tursionis and S. seymouri n. comb. (Digenea: Brachycladiidae Odhner, 10905). Journal of Parasitology, v. 94, p. 505-14, 2008. MARIGO, J.; THOMPSON, C.C.; SANTOS, C.P. & IÑIGUEZ, A.M. The Synthesium Brachycladiidae Odhner, 1905 (Digenea) association with hosts based on nuclear and mitochondrial genes. Parasitology International, v. 60, p. 530-533, 2011. MARIGO, J.; CUNHA, H.A.; BERTOZZI, C.P.; SOUZA, S.P.; ROSAS, F.C.W.; CREMER, M.J.; BARRETO, A.S.; DE OLIVEIRA, L.R.; CAPPOZZO, H.L.; VALENTE, A.L.S.; SANTOS, C.P. & VICENTE, A.C.P. Genetic diversity and population structure of Synthesium pontoporiae (Digenea, Brachycladiidae) linked to its definitive host stocks, the endangered Franciscana dolphin, Pontoporia blainvillei (Pontoporiidae) off the coast of Brazil and Argentina. Journal of Helminthology, v. 89, p. 19-27, 2015. OZAKI, Y. Trematode parasites of Indian porpoise Neophocaena phocaenoides Gray. Journal of Sciences of Hiroshima University Series B, v. 3, p. 1-24, 1935. POULIN, R. & MORAND, S. The diversity of parasites. The Quarterly Review of Biology, v. 75, p. 277-293, 2000. PRICE, E. The trematode parasite of marine mammals. Proceedings of the United States National Museum, London, 1932. RAGA, J.A. & BALBUENA, J.A. Leucasiella delamurei sp. n. (Trematoda: Campulidae), a parasite of Globicephala melaena (Traill, 1809) (Cetacea: Delphinidae) in the Western Mediterranean Sea. Helminthologia, v. 25, p. 95- 102, 1988. RAGA, J.A.; AZNAR, J.; BALBUENA, J.A. & DAILEY, M.D. Hadwenius pontoporiae sp. n. (Digenea: Campulidae) from the intestine of Franciscana (Cetacea: Pontoporiidae) in Argentinian waters. Helminthological Society of Washington, v. 61, p. 45-9, 1994. ! 14 RAGA, J.A.; FERNÁNDEZ, M.; BALBUENA, J. & AZNAR, F.J. Parasites. In W. Perrin, B. Würsig, & Thewissen (Eds.), Enciclopedia of Marine Mammals, p. 821-830, 2008. YAMAGUTI, S. Studies on the helminth fauna of Japan. Part 45. Trematodes of marine mammals. Arbeiten aus der Medizinischen Fakultat Okayama, v. 7, p. 83-294, 1951. YAMAGUTI, S. Systema helminthum Vol I The digenetic trematodes of vertebrates Parts I and II. Interscience, New York and London, 1958. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Objetivos! ! ! ! 16 OBJETIVO Objetivo Geral Investigar a diversidade de espécies do gênero Synthesium, parasitos de odontocetos, ao longo da costa brasileira. Objetivos específicos •! Avaliar os parasitos do gênero Synthesium provenientes de diferentes espécies de odontocetos hospedeiros com base em dados morfológicos e moleculares; •! Descrever espécies de digenéticos pertencentes ao gênero Synthesium através de comparações morfológicas e reconstruções filogenéticas e moleculares; •! Investigar a posição filogenética do gênero Synthesium dentro da família Brachycladiidae. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! A!new!Synthesium!species!(Digenea:!Brachycladiidae)! from!bottlenose!dolphins!Tursiops/truncatus!(Cetacea:! Delphinidae)!in!Southwestern!Atlantic!water! !! ! ! 18 A new Synthesium species (Digenea: Brachycladiidae) from bottlenose dolphins Tursiops truncatus (Cetacea: Delphinidae) in Southwestern Atlantic waters1 ABSTRACT A new species of Synthesium from the bottlenose dolphin Tursiops truncatus in South Brazilian waters is described. Morphological and molecular identification were performed and phylogenetic analyses were carried out using the ribosomal SSU and ITS1 and the mitochondrial ND3 and COI genes. The main characteristics of the new species are the subterminal round-shaped oral sucker, the anterior distribution of vitellaria reaching the level of the ovary and the oval-shaped testes. The results obtained with the molecular markers supported the inclusion of the specimens into the genus Synthesium. The nucleotide divergence detected for the mitochondrial genes among the new species and others of the same genus supported the erection of a new species. This is the 9th species assigned to the genus and the third Synthesium species recorded in the South Atlantic Ocean. KEYWORDS: Tursiops truncatus; Brachycladiidae; Synthesium; Morphology; Molecular identification; South Atlantic Ocean. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1!The final publication is available at Springer via http://dx.doi.org/10.1007/s00436-017-5421-2 ! ! 19 INTRODUCTION The bottlenose dolphin Tursiops truncatus Montagu, 1821 is a cosmopolitan species found primarily in coastal and inshore regions in tropical and temperate oceans worldwide (Jefferson et al. 1993). This species is continuously distributed along the Brazilian coast, in the Southwestern Atlantic Ocean (SWA), inhabiting a variety of natural environments (Pinedo et al. 1992). In southern Brazil, bottlenose dolphins are observed in coastal waters, bays and offshore of the Santa Catarina and Rio Grande do Sul states (Cremer et al. 2009; Fruet et al. 2014). The helminth fauna associated with T. truncatus has been surveyed in several geographical regions, mostly in the Northern Hemisphere (Dailey 1976; Raga et al. 1985; Fernández et al. 1994; Aznar et al. 2006, 2007; Kuwamura et al. 2007; Fauquier et al. 2009; Quiñones et al. 2013; Reboredo-Fernández et al. 2015), the Caribbean Sea (Mignucci-Giannoni et al. 1998; Colón-Llavina et al. 2009; Oliveira et al. 2011) and slightly in the South Atlantic (Tomo et al. 2010; Romero et al. 2014). Some studies regarding the diversity and composition of the helminth communities of different odontocete species, including bottlenose dolphins, are also recorded in Brazilian waters (Santos et al. 1996; Marigo et al. 2008, 2011; Carvalho et al. 2010). Synthesium Stunkard and Alvey, 1930 is a genus of digenetic parasites within the family Brachycladiidae Odhner, 1905 (see Gibson 2005 for a taxonomical review), commonly found in the intestines of a wide range of odontocete families (Pontoporiidae, Delphinidae, Monodontidae and Phocoenidae) in most parts of the world (Fernández et al. 1998a). Their taxonomy is still confused due to the difficulty in obtaining preserved specimens from decomposed or frozen hosts, misleading morphological interpretations (Gibson 2005) and compromising molecular identifications. In this study, we describe a new species of the genus Synthesium Stunkard & Alvey, 1930, family Brachycladiidae Odhner, 1905, collected from bottlenose dolphins T. truncatus off the Brazilian coast, SWA. The diagnosis is based on morphological and molecular analyses. MATERIAL AND METHODS Parasite sampling ! 20 The small intestines of three adult T. truncatus, two males and one female, stranded at the São Francisco do Sul district, Santa Catarina state, south Brazil (26º14'36"S, 48º38'17"O), between 2012 and 2014, were analyzed. During the necropsy, the gastrointestinal tracts were removed and stored at -20 °C for later examination. After thawing, the intestines were cut open, washed in tap water over a 150 µm sieve and the contents examined under a stereo microscope. Trematodes were cleaned in tap water, fixed and preserved in 70% ethanol for both morphological and molecular analyses. Morphological analyses The morphology of 35 specimens of the new species was studied by light microscopy. The worms were stained with chloridric carmine, dehydrated in a graded ethanol series, cleared with eugenol and mounted as temporary preparations. Three specimens of the new species were also histologically analyzed. The helminthes were placed in 2-hydroxyethyl-methacrylate (7022 18500 Leica historesin embedding kit). Transverse and longitudinal serial sections with a 4 µm thickness were made (Microtome Leica, model RM2165). These sections were stained with hematoxylin- eosin (HE). Morphometric analyses were done according to Fernández et al. (1995) in a computerized system for image analysis (Qwin Lite 3.1, Leica Microsystems, Wetzlar, Germany). Drawings were made with the aid of a camera lucida. Molecular Characterization Molecular analyses were conducted on five specimens collected from the three T. truncatus. Additionally, we obtained DNA from three specimens identified as Synthesium tursionis (Marchi, 1873) Stunkard & Alvey, 1930 collected from the small intestines of an adult Guiana dolphin Sotalia guianensis (Van Bénéden, 1864) stranded on the São Paulo coast of Brazil in 2012, and two specimens identified as Synthesium delamurei (Raga & Balbuena, 1988) collected from a long-finned pilot whale Globicephala melas (Traill, 1809) stranded on the Mediterranean coast of Spain in 2007. Genomic DNA was extracted from each worm using the DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol in a final volume of 30 µl. DNA fragments were amplified using primers for the ribosomal small subunit (SSU), internal transcribed spacer 1 (ITS1), NDH dehydrogenase subunit 3 (mtND3) and ! 21 cytochrome c oxidase subunit 1 (mtCOI). The primers and the cycling conditions are shown in Table 1. PCR amplifications were carried out using 3 to 5 µl of genomic DNA, 1.0 µl (for SSU, mtND3 and mtCOI genes) or 1.25 µl (for ITS1) of each set of primers and Ready- to-Go PCR beads (Pure Taq™Ready-to-Go™PCR beads, GE Healthcare). The solution consisted of stabilizers, BSA, dATP, dCTP, dGTP, dTTP, ~2.5 units of puReTaq DNA polymerase and reaction buffer. With the reconstituted bead to a final volume of 25 µl, the concentration of each dNTP was 200 µM in 10 mM Tris-HCl, (pH 9.0 at room temperature), 50 mM KCl and 1.5 mM MgCl2. Aliquots (3 µl) of individual PCR products were separated by electrophoresis using agarose gels (1%), stained with gel red (1 µl) (Biotium Inc) and visualized using ultraviolet transillumination. Gel images were captured electronically and analyzed using the program MULTI-ANALYST (v.1.1, Bio-Rad). PCR amplicons were purified using a QIAquick PCR Purification Kit (Qiagen) following the manufacturer’s instructions. Automated sequencing was directly performed on the purified PCR products using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) for cycle sequencing. The sequences were run on an Applied Biosystems ABI 3500 DNA genetic sequencer. Contiguous sequences from each molecular marker were assembled and edited in Sequencher v. 5.2.4 (Gene Codes, Ann Arbor, MI). All sequences were subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov) to confirm their identity. All alignments were made using the MUSCLE software implemented in Geneious version 7.1.3 (Kearse et al. 2012). Phylogenetic analyses New SSU, ITS1, mtND3 and mtCOI sequences were used for pairwise estimates of divergence. Only the mtND3 sequences were used for phylogenetic inferences due to the considerable number of other Brachycladiidae sequences available in GenBank. The genetic divergence between the sequences was calculated within the aligned portion using the Kimura-2-parameter distance model (Kimura 1980) in the MEGA6 program (Tamura et al. 2013). The phylogeny of mtND3 was inferred by a Neighbor-Joining analyses (NJ) using the MEGA6 program, Bayesian Inference (BI) carried out in the BEAST program ! 22 (Drumond et al. 2012) and Maximum Likelihood (ML) performed with PhyML v3.0 (Guindon et al. 2010). The NJ analyses were performed using the Kimura-2-parameter model and 2000 bootstrap replicates. Prior to the ML analyses, the best fitting models of nucleotide substitution were determined based on the Akaike Information Criteria (AIC) using jModelTest 2.1.1 (Posada 2008) as TPM3uf+I+G. Supports for ML were determined by performing 100 bootstrap replicates. The BI analyses were run with a GTR nucleotide substitution model available in the BEAST program. BI Markov Chain Monte Carlo (MCMC) chains were run for 300 million generations, the log-likelihood scores were plotted, and only the final 70% of the trees were used to produce the consensus tree by setting the "burn-in" parameter at 3x107 generations. The phylogenetic trees were generated and edited in FigTree v1.3.1 (Rambaut 2009). The species, hosts and accession numbers used in this study are summarized in Table 2. RESULTS Description of Synthesium neotropicalis n. sp. The observations and measurements were based on 35 whole-mounted and 3 serially sectioned specimens (see Fig. 1). Measurements (length x width) are shown as the range, with the mean in parentheses followed by the standard deviation, and are expressed in millimeters, unless otherwise stated. Body elongated, slender, dorsoventrally flattened 11.43 - 29.61 (22.90 ± 5.42), maximum width behind posterior testes 0.36 - 0.73 (0.49 ± 0.08). Body spines not observed. Oral sucker subterminal, muscular, round-shaped, slightly oval 0.33 - 0.74 (0.56 ± 0.09) x 0.25 - 0.62 (0.44 ± 0.09). Ventral sucker muscular located in posterior region of the first third of body 0.31 - 0.59 (0.49 ± 0.07) x 0.33 - 0.61 (0.45 ± 0.09). Oral sucker / ventral sucker length ratio 1:1.14. Distance between suckers 2.32 - 7.83 (5.12 ± 1.60). Prepharynx variable in length, mostly long 0.38 - 2.18 (1.40 ± 0.53). Pharynx pyriform, strongly muscular 0.37 - 0.81 (0.50 ± 0.09) x 0.13 - 0.34 (0.20 ± 0.05). Esophagus very short or almost indistinguishable. Intestine H-shaped with anterior caeca reaching medial level of oral sucker and posterior caeca ending blindly close to posterior extremity of body. Uroproct absent. Genital pore preacetabular. Cirrus pouch long 1.84 - 3.31 (2.65 ± 0.42), extending well beyond ventral sucker, containing large seminal vesicle located at its extremity 0.33 - 1.07 (0.61 ± 0.20) x 0.10 - 0.30 (0.21 ± 0.04), pars prostatica and unarmed cirrus. Cirrus pouch opening into genital ! 23 pore. Ovary round to oval 0.14 - 0.45 (0.30 ± 0.08) x 0.09 - 0.30 (0.17 ± 0.05), postacetabular, pretesticular. Mehlis’ gland preovarian. Vitelline reservoir ovoid, close to ovary. Laurer´s canal not observed. Seminal receptacle absent. Distance between ovary and anterior testes 0.07 - 1.82 (0.66 ± 0.37). Testes oval-shaped, tandem, with anterior testes situated in posterior region of middle third of body and posterior testes situated in anterior region of posterior third of body. Anterior testes 0.35 - 0.80 (0.60 ± 0.11) x 0.13 - 0.37 (0.30 ± 0.05) slightly smaller than posterior testes 0.37 - 1.06 (0.71 ± 0.15) x 0.14 - 0.45 (0.30 ± 0.07). Intertesticular distance 0.63 - 3.19 (1.74 ± 0.61). Distance between posterior testes and extremity of body 2.77 - 9.12 (6.00 ± 1.67). Vitellaria arranged in acinous bunches, profuse, commencing anteriorly to ovary and extending to posterior extremity of body. Distance between anterior margin of vitellaria and ovary 0.25 - 2.48 (1.13 ± 3.54). Distance between anterior margin of vitellaria and ventral sucker 2.00 - 11.80 (4.21 ± 2.42). Distance between anterior margin of vitellaria and extremity of body 5.64 - 16.10 (11.29 ± 2.93). Uterus coils intercecally, widening into unarmed metraterm before opening into genital pore. Eggs oval slightly flattened at opercular pole, triangular in cross-section 47 - 66 (55) x 28 - 41 (34) µm. Taxonomic summary: Definitive host: Tursiops truncatus Montagu 1821, bottlenose dolphin. Site: Small intestine. Type locality: Babitonga Bay, São Francisco do Sul district, Santa Catarina state, Brazil, South Atlantic Ocean. Specimens deposited: Holotype and paratypes specimens were deposited at Coleção Helmintológica do Instituto Oswaldo Cruz - Fundação Oswaldo Cruz (CHIOC-Fiocruz) Rio de Janeiro, RJ, Brazil under number (will be deposited after the manuscript acceptance). Paratypes were deposited at Coleção Helmintológica do Instituto de Biociências de Botucatu - UNESP (CHIB-UNESP), Botucatu, SP, Brazil under number (will be deposited after the manuscript acceptance). Etymology: The specific epithet “neotropicalis” refers to the Neotropical region, location where the new species was first collected. Remarks A total of 1,174 Synthesium neotropicalis n. sp. were recovered from the intestine of the three T. truncatus hosts. The general morphology of the specimens ! 24 analyzed allowed its inclusion in the family Brachycladiidae and the subfamily Brachycladiinae Odhner, 1905 according to Gibson (2005). The following features placed the specimens into the genus Synthesium: body very elongated, well-developed pyriform pharynx, intestines with anterior and posterior caeca without diverticula, with posterior caeca ending blindly close to posterior extremity of body, ventral sucker in anterior third of the body, presence of cirrus sac, unarmed metraterm, testes in middle third or third quarter of body and vitellaria entirely in hindbody. The principal morphological features that differentiate Synthesium neotropicalis n. sp. from the other Synthesium species are a combination of characteristics such as the subterminal round-shaped oral sucker, the anterior distribution of vitellaria reaching the level of ovary and the oval-shaped testes. Synthesium neotropicalis n. sp. differs from S. seymouri, S. nipponicum, S. mironovi, and S. subtile in its shape (round versus cup-shaped) and position (subterminal versus terminal) of the oral sucker, the morphology of the pharynx (pyriform versus oval), the anterior distribution of vitellaria (at the level of the ovary in Synthesium neotropicalis n. sp.) and the egg size (smaller than in the other species). Synthesium neotropicalis n. sp. differs from Synthesium tursionis because the latter has a terminal cup-shaped oral sucker, lobed testes with shorter distance from the end of the body and anterior distribution of vitellaria reaching the seminal vesicle level. Synthesium neotropicalis n. sp. can also be distinguished from Synthesium elongatum because the latter presents lobe-shaped testes and vitellaria commencing at the seminal vesicle level. The new species resembles S. pontoporiae and S. delamurei in the shape and position of the oral sucker (round subterminal) and the shape of the testes (oval). However, S. pontoporiae shows a wider body size, vitellaria commencing at the seminal vesicle level and smaller oral and ventral suckers. Finally, S. delamurei presents gonads positioned close to the extremity of the body, anterior extent of vitellaria at the seminal vesicle level, and longer and wider testes and eggs compared to the new species. The morphological features differentiating all of the Synthesium species are summarized in Table 3. Phylogenetic Analyses Sequences of the SSU, ITS1, mtCOI and mtND3 genes were obtained from all specimens of the new species. We successfully sequenced the ITS1 and mtCOI genes ! 25 for the S. tursionis specimens. However, we could only obtain sequences of mtND3 for S. delamurei from the specimens utilized. Partial SSU alignment (816 bp) comprised a newly generated sequence from Synthesium neotropicalis n. sp. (accession number will be deposited after manuscript acceptance) and the sequences from S. pontoporiae (FJ357162) and S. tursionis (FJ357163) retrieved from GenBank. The alignment of the mtCOI gene (406 bp) and ITS1 (534 bp) included newly generated sequences of Synthesium neotropicalis n. sp. (will be deposited after manuscript acceptance (COI) xxxx and (ITS1) xxx) and S. tursionis (will be deposited after manuscript acceptance (COI) xxxx and (ITS1) xxx), as well as S. pontoporiae (JX644156; JX644084) retrieved from GenBank. The mtND3 alignment (276 bp) consisted of 13 Brachycladiidae sequences retrieved from GenBank, newly generated sequences from Synthesium neotropicalis n. sp. (accession number will be deposited after manuscript acceptance) and S. delamurei (accession number will be deposited after manuscript acceptance), as well as Tormopsolus orientalis (KT180219) as an outgroup (Table 2). The partial SSU pairwise distance analysis revealed that the Synthesium neotropicalis n. sp., S. tursionis and S. pontoporiae sequences included in the alignment are identical. The ITS1 pairwise distance analysis showed only 1 nucleotide (0.2% of divergence) difference between Synthesium neotropicalis n. sp. and S. pontoporiae, suggesting a very close relationship. The nucleotide divergence between Synthesium neotropicalis n. sp. and S. tursionis was also very small (4 nucleotides, 0.9% of divergence). Additionally, Synthesium pontoporiae and S. tursionis diverged from each other by 3 nucleotides (0.7% of divergence). The mtCOI pairwise distance analysis showed a nucleotide divergence between Synthesium neotropicalis n. sp. and S. pontoporiae of 9.1% (35 nucleotides) and between Synthesium neotropicalis n. sp. and S. tursionis of 14.6% (55 nucleotides). As for S. pontoporiae and S. tursionis, the divergence was 14.6% (55 nucleotides). The mtND3 pairwise distance analysis revealed that the sequences of Synthesium neotropicalis n. sp. differed from S. pontoporiae by 14 nucleotides (5.3% of divergence), from S. tursionis by 35 nucleotides (14% of divergence) and from S. delamurei by 49 nucleotides (20.8% of divergence). The difference between S. tursionis and S. delamurei was 53 nucleotides (22.4%) and between S. pontoporiae and S. delamurei was 51 nucleotides (21.8% of divergence). Additionally, the sequences of S. ! 26 pontoporiae and S. tursionis are different for 43 nucleotides (17.8% of divergence). The genetic divergence estimated among Brachycladiidae genera had a mean of 19.6%, ranging from 11.9% (Brachycladium atlanticum (Abril, Balbuena & Raga, 1991) Gibson, 2005 versus Campula oblonga Cobbold, 1858) to 27.4% (Synthesium pontoporiae versus Nasitrema globicephalae Neiland, Rice & Holden, 1970). The mtND3 phylogenetic analyses (Fig. 2) based on the BI, NJ and ML trees showed identical topologies with supported values in general. The ML analysis showed supported values on the terminal nodes except for the Synthesium neotropicalis n. sp and the S. pontoporiae clade. In all topologies, Synthesium neotropicalis n. sp. and Synthesium pontoporiae are sister taxa and grouped with S. tursionis in a monophyletic clade. However, S. delamurei is placed in a different clade apart from all other Synthesium species. DISCUSSION In the past, the genus Synthesium has undergone various taxonomic rearrangements with several synonymies between morphologically closely related genera such as Leucasiella Krotov & Delyamure, 1952, Hadwenius Price, 1932 and Odhneriella Skrjabin, 1915 (see Gibson 2005, 2014). Yamaguti (1958) recognized Leucasiella as a synonym of Hadwenius. Later, Gibson (2005) considered Hadwenius as a junior synonym of Synthesium. The genus Odhneriella remains accepted with some species transferred to Synthesium (Adams and Rausch 1989; Gibson 2005). Odhneriella can be easily distinguished from Synthesium due to the presence of a uroproct, large and elongate testes and a metraterm armed with spines (Gibson 2005). Synthesium neotropicalis n. sp. presented all the diagnostic characteristics to be included in the genus Synthesium, except for an armed cirrus. The presence of spines in the cirrus is a diagnostic characteristic of the genus Synthesium (Gibson, 2005). It is true that spines can be lost due to poor preservation, but we think that it is unlikely that we have missed the spines or, at least, their basal discs in the cirrus, given the large number of specimens we examined. Nevertheless, two other species assigned to the genus Synthesium, namely S. delamurei and S. mironovi, are described as possessing an unarmed cirrus. Originally these species were described as belonging to the genus Leucasiella due to the structure of the vitellaria and the lack of spines in the cirrus. Until now, no redescriptions of S. delamurei and S. mironovi have been carried out and a ! 27 morphological reassessment could reveal new observations that are different from their original descriptions. Because the genus Synthesium currently aggregates species with both an armed and unarmed cirrus, we think that spines in the cirrus must not be a relevant diagnostic characteristic and should be revised. In the South Atlantic Ocean only two Synthesium species have been reported parasitizing odontocetes until now: S. pontoporiae, hosted by the endemic and endangered dolphin Franciscana Pontoporia blainvillei (Gervais & d'Orbigny, 1844) from Argentinian and Brazilian waters (Raga et al. 1994; Silva and Cousin 2004; Marigo et al. 2002, 2008), and S. tursionis, found in S. guianensis and T. truncatus from Brazil (Marigo et al. 2008, 2010) and Argentina (Romero et al. 2014). Synthesium spp. have also been reported in the intestine of Cephalorhyncus commersoni (Lacépède, 1804), Lagenorhyncus obscurus (Gray, 1828) and Lagenorhyncus cruciger (Quoy & Gaimad, 1824) off Argentina (Dans et al. 1999; Berón-Vera et al. 2001; Fernández et al. 2003). Synthesium neotropicalis n. sp. is the third Synthesium species recorded in the South Atlantic Ocean. Regarding the molecular analyses, the sequences of the partial SSU did not exhibit any genetic variation between Synthesium neotropicalis n. sp., S. pontoporiae and S. tursionis. Thus, interspecific differences could not be inferred from this marker. The nucleotide variations in the ITS1 within our alignment were less than 1%. For the ITS region, previous studies have shown that the intraspecific divergence for species of digeneans is below 1% (Vilas et al. 2005; Nolan and Cribb 2005), with some exceptions (Galazzo et al. 2002; Jousson and Bartoli 2002). Nevertheless, the rate of change in a region of DNA may vary from one group to another. Kane and Rollinson (1994) found a 0.9% variation in the ITS region between species of Schistossoma. Also, Marigo et al. (2015) found identical ITS1 sequences among S. pontoporiae specimens, suggesting a single lineage of this species along the SWA coast. The nucleotide divergence observed in ITS1 of the species of Synthesium analyzed here was quite low and should be interpreted with caution rather it provides information at the species level for the genus. Although the ribosomal markers used in this study did not completely diagnose the Synthesium species employed here, the data generated will contribute to future phylogenetic analyses of the group. The genetic divergence found among the species analyzed in this study as Synthesium neotropicalis n. sp, S. pontoporiae, S. tursionis and S. delamurei ranged from 9.1 to 14.6% for mtCOI and from 5.3 to 22.4% for mtND3. Previous studies have ! 28 suggested that trematode species show maximum intraspecific divergences to be considered one single species at approximately 2% for mtDNA (Vilas et al. 2005). This is below the observed values for the Synthesium species analyzed here. For mtCOI specifically, the mean pairwise divergence between the congeners is 19% (ranging from 3.9 to 25%) (Moszczynska et al. 2009). Although this rate also might vary between the digenean groups our results corroborate these values. Moreover, the two recognized species coexisting along the Brazilian coast, namely S. pontoporiae and S. tursionis, presented interspecific genetic differences of 14.6% in mtCOI and 17.8% in mtND3. These results might be interpreted as a threshold to narrow the divergence between Synthesium species. Therefore, comparing the genetic divergence found between both species and Synthesium neotropicalis n. sp., we recognize the latter as a different taxon. The phylogenetic position of S. delamurei caught our attention because we expected it to be placed within the Synthesium clade. The mtND3 genetic divergence between S. delamurei and its congeners (22.4%) was also higher than among the other Synthesium species. From our results, the mean mtND3 genetic divergence between the genera of the Brachycladiidae family is 19.6%. Fernandéz et al. (2000) also found genetic divergence in mtND3 between brachycladiids from separate genera such as Nasitrema globicephalae and Synthesium tursionis to be 26.5%. The specimens used here were morphologically identified as S. delamurei according to Raga and Balbuena (1988). However, given the phylogenetic inference and genetic distance results, a detailed study should be conducted to elucidate whether S. delamurei is correctly assigned to the genus Synthesium. A combination of morphological traits and molecular tools using different genes is highly recommended for describing new species. The morphostructural and morphometrical features combined with the phylogenetic reconstruction and genetic distances presented here support the erection of a new parasite species infecting T. truncatus. 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(D) Detail of anterior extent of vitellaria, ovary and anterior testes – Scale bar = 200 µm. (E) Detail of eggs – Scale bar = 20 µm. Abbreviations: AC = Anterior Caeca, AT = Anterior Testis, CP = Cirrus Pouch, GP = Genital Pore, M = Metraterm, MG = Mehli’s Gland, O = Ovary, OS = Oral Sucker, P = Pharynx, PC = Posterior Caeca, PP = Prepharynx, PT = Posterior Testes, SV = Seminal Vesicle, U = Uterus, V = Vitellaria, VR = Vitelline Reservoir, VS = Ventral Sucker. ! 36 Fig 2 Bayesian tree based on the mtND3 sequences. The species names are followed by the GenBank accession numbers in parentheses. The support values at the branching points are shown as follows: Bayesian posterior probability/ neighbor-joining bootstrap/ maximum likelihood bootstrap. Dashes are shown for the branches not supported by the analyses (scored < 70%). The branch length scale bars indicate mean number of substitutions per site ! 37 Table 1 Primers, cycling conditions for PCR and respective bibliographic sources Primer Sequences 5'-3' Cycling conditions Source SSU 18SF Initial denaturation at 94°C for 5 min; 40 cycles of 94°C for 40 s, 55°C for 40 s and 72°C for 2 min; Final extension at 72°C for 10 min This study (adapt. from Fernández et al. 1998b) CGTATCTTTCAAATGTCTGCCC 18SR CCGATGACCTTGCTAAACC ITS1 ITS1F Initial denaturation at 94°C for 3 min; 30 cycles of 94°C for 1 min, 48°C for 1 min and 72°C for 1 min and 30 s; Final extension at 72°C for 7 min Marigo et al. 2015 GACGACCAAACTTGATCATT ITS1R TGCGCTCTTCATCGACACACGA COI COIPRA Initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 47°C for 30 s and 72°C for 1 min; Final extension at 72°C for 7 min Bessho et al. 1992 TGGTTTTTTGTGCATCCTGAGGTTTA COIPRB AGAAGAACGTAATGAAAATGAGCAAC ND3 ND3F Initial denaturation at 95°C for 5 min; 35 cycles of 95°C for 30s, 47°C for 30s and 72°C for 50s; Final extension at 72°C for 7 min Fernández et al. 1998a; Fernández et al. 2000 GCTTAATTKKTAAAGCYTTGRATTCTTACT ND3D CTACTAGTCCCACTCAACRTAACCYT ! 38 Table 2 Species, hosts, Genbank accession numbers and bibliographic sources of parasites sequences used for phylogenetic analyses Parasite species Host species (Common name) GenBank accession no. Source SSU rDNA Synthesium pontoporiae Pontoporia blainvillei (Franciscana) FJ357162 Marigo et al. unpub. data Synthesium neotropicalis n. sp. Tursiops truncatus (Bottlenose dolphin) * This study Synthesium tursionis Tursiops truncatus (Bottlenose dolphin) FJ357163 Marigo et al. unpub. data ITS1 rDNA Synthesium neotropicalis n. sp. Tursiops truncatus (Bottlenose dolphin) * This study Synthesium pontoporiae Pontoporia blainvillei (Franciscana) JX644084 Marigo et al. 2015 Synthesium tursionis Sotalia guianensis (Guiana dolphin) * This study ND3 mtDNA Brachycladium atlanticum Stenella coeruleoalba (Striped dolphin) AF034551 Fernández et al. 1998a Brachycladium atlanticum Stenella coeruleoalba (Striped dolphin) KT180217 Fraija-Fernández et al. 2016 Brachycladium goliath Balaenoptera acutorostrata (Minke whale) KR703278 Briscoe et al. 2016 Brachycladium sp. Balaenoptera acutorostrata (Minke whale) AF123439 Fernández et al. 2000 Campula oblonga Phocoena phocoena (Harbour porpoise) AF34554 Fernández et al. 1998a Campula oblonga Phocoena phocoena (Harbour porpoise) KT180214 Fraija-Fernández et al. 2016 Nasitrema globicephalae Globicephala melas (Long-finned pilot whale) AF034557 Fernández et al. 1998a Nasitrema delphini Delphinus delphis (Common dolphin) KT180216 Fraija-Fernández et al. 2016 Oschmarinella rochebruni Stenella coeruleoalba (Striped dolphin) KT180215 Fraija-Fernández et al. 2016 Oschmarinella rochebruni Stenella coeruleoalba (Striped dolphin) AF034556 Fernández et al. 1998a Orthosplanchnus fraterculus Enhydra lutris (Sea Otter) AF034555 Fernández et al. 1998a Synthesium pontoporiae Pontoporia blainvillei (Franciscana) FJ829472 Marigo et al. 2011 Synthesium neotropicalis n. sp. Tursiops truncatus (Bottlenose dolphin) * This study Synthesium tursionis Tursiops truncatus (Bottlenose dolphin) AF034552 Fernández et al. 1998a Synthesium delamurei Globicephala melas (Long-finned pilot whale) * This study Tormopsolus orientalis Seriola dumerili (Greater amberjack) KT180219 Fraija-Fernández et al. 2016 COI mtDNA Synthesium neotropicalis n. sp. Tursiops truncatus (Bottlenose dolphin) * This study Synthesium pontoporiae Pontoporia blainvillei (Franciscana) JX644156 Marigo et al. 2015 Synthesium tursionis Sotalia guianensis (Guiana dolphin) * This study *Accession numbers will be included after manuscript acc ! 39! Table 3 Mean (range) of morphological measurements of Synthesium neotropicalis n. sp. compared to other species of the genus. Values are given as length × width in millimeters unless otherwise stated S. neotropicalis n. sp. S. delamurei S. pontoporiae S. elongatum S. tursionis S. seymouri S. nipponicum S. mironovi S. subtile No. of Specimens (n=35) (n=4) (n=20) (n=2) (n=15) (n=10) (n=10) (n=2) (n=20) Host T. truncatus G. melas P. blainvillei N. phocoenoides T. truncatus D. leucas P. dalli; P. phocoena D. leucas O. orca; D. leucas; G. melas Body Length 22.9 (11.4 - 29.6) 12.1 (9.7 - 16.8) 5.0 (3.6-7.1) 13.0 - 18.0 14.3 (8.85 - 21.31) 34.1 (27.2-38.1) 14.0 (13-15.9) 8.9 - 12.9 33.3 (14.0 - 38.3) Max. Width 0.5 (0.4 - 0.7) 0.7 (0.6 - 0.8) 0.5 (0.3-0.7) 1 - 2.1 0.6 (0.5 - 0.8) 0.8 (0.6-1.0) 0.9 (0.8-1.0) 0.7 - 1.2 1.5 (1.2 - 2.0) Oral Sucker Position Subterminal Subterminal Subterminal Subterminal Terminal Terminal Terminal Terminal Terminal Oral Sucker 0.6 x 0.4 (0.3-0.7 x 0.3-0.6) 0,5 x 0.4 (0.4-0.5 x 0.3-0.5) 0.2 x 0.1 (0.1-0.2 x 0.1-0.2) 0.4 x 0.5 0.6 x 0.5 (0.4-0.7 x 0.4-0.7) 2.0 x 1.6 (1.8-2.6 x 1.3-2.1) 0.8 x 0.6 (0.8-0.9 x 0.6-0.7) 0.3-1.0 x 0.5-1.1 1.5 x 1.8 (1.4-2.2 x 1.2-1.8) Prepharynx Length 1.40 (0.4 - 2.2) 0.5 (0.3-0.7) 0.2 (0.7-0.3) 0.5 - 0.5 0.5 (0.2-0.8) 0.8 (0.4-1.3) 0.4 (0.3-0.6) 0.3 0.5 (0.05-1.8) Pharynx Shape Pyriform Pyriform Pyriform Pyriform Pyriform Oval Oval - Oval Pharynx 0.5 x 0.2 (0.4-0.8 x 0.1-0.3) 0.2 (0.1-0.2) 0.6 x 0.1 (0.1-0.3 x 0.03- 0.2) 0.4-0.6 x 0.3-0.3 0.5 x 0.2 (0.5-0.7 x 0.2-0.3) 1.0 x 0.8 (0.9-1.2 x 0.7-1.3) 0.3 x 0.3 (0.3-0.4 x 0.3-0.3) 0.3-0.4 x 0.3-0.4 0.7 x 0.7 (0.5-0.8 x 0.5-0.9) Ventral Sucker Position Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Anterior 1/3 Ventral Sucker 0.5 x 0.4 (0.3-0.6 x 0.3-0.6) 0.6 x 0.5 (0.5-0.8 x 0.4-0.6) 0.3 x 0.3 (0.2-0.4 x 0.2-0.4) 0.7 x 0.5 0.6 x 0.5 (0.4-0.8 x 0.4-0.7) 1.3 x 1.00 (1.1-1.5 x 0.8-1.4) 0.7 x 0.7 0.6-1.0 x 0.6- 0.8 0.8 x 0.9 (0.7-1.3 x 0.7-1.1) Cirrus Pouch Length 2.6 (1.8 x 3.3) 2.6 (2.0 - 3.4) 0.9 (0.7 - 1.1) 0.9 - 1.5 2.2 (1.4-2.9) 3.2 (1.3-4.1) 1.2 (1.0-1.4) - 4.4 (2.4 - 6.1) Testes Shape Oval Oval Oval Lobed Lobed Oval Oval Oval Oval Ovary 0.3 x 0.2 (0.1-0.4 x 0.1-0.3) 0.3 x 0.2 (0.3-0.4 x 0.1-0.2) 0.2 x 0.1 (0.1-0.3 x 0.06- 0.2) 0.3-0.4 x 0.2-0.3 0.2 x 0.2 (0.1-0.3 x 0.1-0.2) 0.3 x 0.2 (0.2-0.4 x 0.1-0.3) 0.22 x 0.22 (0.2-0.3 x 0.2-0.3) 0.1 - 0.3 0.4 x 0.3 (0.2-0.5 x 0.2-0.4) Gonads Position Medial 1/3 Posterior 1/3 Medial 1/3 Medial 1/3 Medial 1/3 Medial 1/3 Medial 1/3 Anterior 1/3 Medial 1/3 Anterior Testes 0.6 x 0.3 (0.3-0.8 x 0.1-0.4) 1.0 x 0.3 (0.8-1.2 x 0.2-0.4) 0.3 x 0.2 (0.2-0.5 x 0.1-0.2) - 0.7 x 0.3 (0.5-1.1 x 0.3-0.6) 1.5 x 0.7 (1.3-1.8 x 0.5-1.0) 0.7 x 0.3 (0.6-0.8 x 0.2-0.3) 0.6-0.9 x 0.2-0.3 1.2 x 0.6 (0.7-1.8 x 0.4-0.8) Posterior Testes 0.7 x 0.3 (0.4-1.0 x 0.1-0.4) 1.0 x 0.3 (0.9-1.1 x 0.3-0.4) 0.3 x 0.2 (0.2-0.5 x 0.1-0.3) - 0.7 x 0.4 (0.5-1.1 x 0.3-0.6) 1.6 x 0.7 (1.3-1.9 x 0.5-0.8) 0.7 x 0.3 (0.6-0.8 x 0.3-0.4) 0.5-1.0 x 0.2-0.5 1.3 x 0.6 (0.8-1.9 x 0.4-0.9) Anterior Extent of Vitellaria Ovary Seminal Vesicle Seminal Vesicle Uterine field Seminal Vesicle Anterior testes Anterior Testes Anterior Testes Seminal Vesicle Egg Size (µm) 55 x 34 (47–68 x 28-41) 67 x 40 (57-72 x 32-50) 53 x 26 (46-60 x 23-28) 47-55 x 25-31 53 x 29 (51-55 x 28-32) 91 x 55 (83-97 x 51-55) 70-82 x 35-45 72-90 x 33-37 89 x 49 (75-98 x 44-55) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Phylogenetic!relationship!among!species!of!Synthesium! (Digenea:!Brachycladiidae),!parasites!of!dolphins,!based! on!morphological!and!molecular!data! ! ! ! 41 Phylogenetic relationships among species of Synthesium (Digenea: Brachycladiidae), parasites of dolphins, based on morphological and molecular data ABSTRACT The identification of Synthesium species is difficult due to their morphological similarity. Morphological and molecular analyses based on ribosomal and mitochondrial markers were conducted for species delimitation of Synthesium collected from delphinid hosts. The comparative analysis combining molecular and morphological data suggests the erection of three distinct putative species of Synthesium parasitizing dolphins from the Brazilian and Spanish coasts. Furthermore, new molecular data on Synthesium tursionis evidence the presence of cryptic species. The phylogenetic position of Synthesium delamurei, Synthesium subtile, and Synthesium seymouri revels the need of taxonomic revision of the genus and potentially of the Brachycladiidae family. Keywords: Trematode; Molecular characterization; Taxonomy; Biodiversity; Odontocete ! 42 INTRODUCTION Species of Synthesium Stunkard & Alvey, 1930 are commonly found in the intestines of a wide range of odontocete (Cetacea) families (Pontoporiidae Gray, 1846, Delphinidae Gray, 1821, Monodontidae Gray, 1821 and Phocoenidae Gray, 1825) in most parts of the world (Fernández et al., 1998a). In the past years the genus Synthesium has undergone different taxonomic rearrangements with several synonymies between morphologically close related genera such as Leucasiela Krotov & Delyamure 1952, Hadwenius Price, 1932, and Odhneriella Skrjabin, 1915 (See Gibson, 2005, 2014 for complete review). To date, nine valid species are recognized: the type-species Synthesium tursionis (Marchi, 1873) Stunkard & Alvey, 1930, Synthesium pontoporiae (Raga, Aznar, Balbuena & Dailey, 1994) Marigo, Vicente, Valente, Measures & Santos, 2008, Synthesium seymouri (Price, 1932) Marigo, Vicente, Valente, Measures & Santos, 2008, Synthesium elongatum Osaki, 1935, S. nipponicum Yamaguti, 1951, Synthesium mironovi Krotov & Delyamure, 1952, Synthesium subtile Skrjabin, 1959, Synthesium delamurei (Raga & Balbuena, 1988) (Gibson, 2014) and Synthesium neotropicalis Ebert, Müller, Marigo, Valente, Cremer & Silva, 2017. Preserved specimens from decomposed or frozen hosts are problematic and usually leads to misidentifications (Gibson, 2005). Nevertheless, some Synthesium species present few morphological differences in adult stage, which compromises their correct identification. Such problems could be readily overcome by the use of molecular methods. Ribosomal and mitochondrial DNA regions can provide suitable markers for systematic studies of trematodes, helping to explore population variants and differentiation among species (Vilas et al., 2005; Nolan and Cribb, 2005). SSU rDNA and ND3 mtDNA sequences of Brachycladiidae have been used in previous phylogenetic studies (Fernández et al., 1998a,b, 2003; Marigo et al., 2011, 2015; Fraija-Fernandéz et al., 2016), whereas other markers such as ITS1 rDNA and COI mtDNA are still scarce (Marigo et al., 2011, 2015; Ebert et al., 2017) and only one study was performed up to date (Marigo et al., 2015). Thus, a molecular approach including different specimens of Synthesium may reveal a bulk of new species for this genus. In the present study, we used DNA sequences of ribosomal and mitochondrial genes along with morphological data to discuss species boundaries and consequent identification of Synthesium and their phylogenetic relationships within the family Brachycladiidae. ! 43 MATERIAL AND METHODS Parasite collection We obtained Synthesium spp. samples from 14 dolphins found washed ashore along the South and Southeastern Brazilian coast, Southwestern Atlantic Ocean (one Delphinus delphis Linnaeus, 1758, one Steno bredanensis Lesson, 1828, three Tursiops truncatus Montagu, 1821, eight Sotalia guianensis Van Bénéden, 1864 and one Stenella frontalis Cuvier, 1829). During necropsy, the dolphins’ gastrointestinal tracts were removed and stored at -20 °C until further examination. After thawing, the intestines were cut open, washed in tap water over a 150 µm sieve and the content examined under a stereo microscope. Trematodes were cleaned in tap water, fixed and preserved in 70% ethanol for both morphological and molecular analyses. Additional samples of S. delamurei collected from a Globicephala melas (Traill, 1809) and S. tursionis from a T. truncatus, both found stranded on the Mediterranean Coast of Spain, S. seymouri collected from three Delphinapterus leucas (Pallas, 1776) from Saint Lawrence bay, Canada, and S. subtile collected from a G. melas from the Faroe Islands were also obtained and used for DNA sequencing. To implement the phylogenetic analyses, other Brachycladiidae species were also obtained and sequenced. Table 1 provides a taxonomic listing of specimens analyzed, identification voucher, their hosts, and collection localities. Morphological examination and morphometric analyses We studied the morphological aspects of the specimens from all Brazilian host by means of light microscopy. The worms were stained with chloridric carmine, dehydrated through a graded ethanol series, cleared with eugenol and mounted as temporary preparations. Diagnostic morphometric measurements were made according to Fernández et al. (1995) in a computerized system for image analysis (Qwin Lite 3.1, Leica Microsystems, Wetzlar, Germany). ! 44 Extraction of gDNA, PCR amplification and Sequencing Total genomic DNA was extracted from whole worms using a Qiagen® DNeasyTM Blood & Tissue Kit following the manufacturer’s recommended protocols, with a final elution volume of 30 µl. Fragments were amplified using primers for the ribosomal internal transcribed spacer 1 (ITS1), and mitochondrial genes NDH dehydrogenase subunit 3 (mtND3) and cytochrome c oxidase subunit 1 (mtCOI). PCR amplifications (25 µl) were performed using Ready-to-Go™ (GE Healthcare) PCR beads (each containing ~2.5 units of pureTaq DNA polymerase, 10 mM Tris-HCl at pH 9.0, 50 mM KCl and 1.5 mM MgCl2 and 200 µM of each dNTP), 3 to 5 µl of genomic DNA and 1.0 µl of each PCR primer. Primers and cycling conditions are showed in Table 2. PCR amplicons were purified using QIAquick PCR Purification Kit (Qiagen) following the manufacturer’s instructions. Automated sequencing was performed directly on purified PCR products using BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) for cycle sequencing. Sequences were run onto an Applied Biosystems ABI 3500 DNA genetic sequencer. Alignments and phylogenetic analyses Contiguous sequences from each molecular marker were assembled and edited using Sequencher v. 5.2.4 (Gene Codes, Ann Arbor, MI). Each sequence was subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov) to confirm identity. Newly generated ITS1, mtND3 and mtCOI sequences were combined with other Brachycladiidae sequences retrieved from GenBank and aligned using MUSCLE software implemented on Geneious 7.1.3 (Kearse et al., 2012) with default settings. Tormopsolus orientalis Yamaguti, 1934 (Acanthocolpidae Lühe, 1906) was included as outgroup for ITS1 and ND3 alignments. For species delimitation, we used the pairwise distance model and the GMYC model. The pairwise distance was calculated within each marker aligned portion using Kimura-2-parameter (Kimura, 1980) in MEGA6 program (Tamura et al., 2013). For GMYC analyses applied for the mtCOI dataset, a lognormal relaxed molecular clock tree was estimated using BEAST v.1.8.2 program (Drumond et al., 2012). The nucleotide evolutionary model used to estimate the ultrametric tree was the GTR model. The Markov Chain Monte Carlo (MCMC) were run for 1 x 107 generations, and a tree was sampled ! 45 every 100th generation. The GMYC analysis was performed within the online platform GMYC web server (http://species.h-its.org/gmyc/) with a single threshold method. For phylogenetic inference, the obtained ITS1 and mtND3 datasets were analyzed individually by Maximum Likelihood (ML) and Bayesian Inference (BI), applying the model of nucleotide evolution GTR + I + G selected using PAUP v4a147 (Swofford, 2002) for both datasets. ML analyses were carried out using the program RAxML v. 8 (Stamatakis, 2014). The model parameters and bootstrap support values (1,000 repetitions) were estimated using RAxML. BI trees were generated using MrBayes v. 3.2 (Ronquist et al., 2012) running two independent MCMC runs of four chains for 1 x 107 generations and sampling tree topologies every 103 generations. Burn-in periods were set to the first 25,000 generations. MrBayes and RAxML analyses were carried out on CIPRES web portal (Miller et al., 2010). Phylogenetic trees were generated and edited in FigTree v1.3.1 (Rambaut, 2009). RESULTS Morphological analyses Using traditional morphology, we could identify two different species of Synthesium in our samples: S. tursionis (in S. guianensis samples) and S. neotropicalis (in all T. truncatus samples). In four samples (S. frontalis, S. guianensis 353 and 537, D. delphis, and S. bredanensis), we could only identify the specimens until the genus Synthesium. In these samples and comparing to other Synthesium species, some morphological differences could be noticed in total length of body, shape and position of oral sucker, distance between sucker, distribution of vitellaria, shape of testes and distance between posterior testes and posterior extremity of body. Considering these differences, we defined 3 morphotypes: Synthesium morphotype 1 found in S. guinanensis and S. frontalis; Synthesium morphotype 2, found in D. delphis; and Synthesium morphotype 3, found in S. bredanensis (Fig. 1). We also found co-infections with S. tursionis and Synthesium morphotype 1 in S. guianensis. Synthesium morphotype 1: body elongated, slender, dorsoventrally flattened with maximum width behind posterior testes. The oral sucker is subterminal, muscular and slightly oval. The ventral sucker is muscular and located in the posterior region of the first third of body. The intestine is H-shaped with the anterior caeca reaching the medial level of oral sucker and posterior caeca ending blindly close to posterior extremity of ! 46 body. The cirrus pouch is long and extends well beyond the ventral sucker. A large seminal vesicle is located at cirrus pouch’s extremity. Spines in cirrus are not observed. The ovary is round to oval. The testes are lobed, with the anterior testes presenting five lobes (in most specimens) and the posterior testes with six lobes (in most specimens). The initial position of vitellaria is at the pars prostatica region. Synthesium morphotype 2: body length relatively short (when comparing to most Synthesium described species), elongated, slender, dorsoventrally flattened with maximum width behind posterior testes. The oral sucker is subterminal, muscular and slightly oval. The cirrus pouch is long and extends well beyond the ventral sucker. A large seminal vesicle is located at cirrus pouch’s extremity. The cirrus is armed with spines. The ovary is round to oval. Distance between ventral sucker and ovary is very short. The testes are oval with irregular margin. The initial position of vitellaria is at the medial region of seminal vesicle. Synthesium morphotype 3: body length relatively long (when comparing to most Synthesium described species), slender, dorsoventrally flattened with maximum width behind posterior testes. The oral sucker is subterminal, muscular and slightly oval. The ventral sucker is muscular and located in the posterior region of the first third of body. The cirrus pouch is long and extends well beyond the ventral sucker with a large seminal vesicle located at its extremity. Spine in cirrus not observed. The ovary is round to oval. The testes are lobed, with the anterior testes presenting five lobes (in most specimens) and the posterior testes with six lobes (in most specimens). The initial position of vitellaria is at the posterior end of seminal vesicle. None of the morphotype were considered to be any of the nine Synthesium species already described. Table 3 provides the morphometric differences found in each morphotype considered and Table 4 provides the main morphological aspects that distinguish all Synthesium species and the resulted morphotypes. Molecular and Phylogenetic Analyses We obtained 64 sequences (18 ITS1 sequences; 24 mtND3 sequences; 22 mtCOI sequences) from 33 different Brachycladiidae specimens, of which 28 were Synthesium spp. specimens. The ITS1 alignment (551 bp) included 20 sequences (18 newly generated and 2 retrieved from GenBank). The mtND3 alignment (252 bp) consisted of 46 Brachycladiidae sequences (24 newly generated and 22 retrieved from GenBank). The ! 47 mtCOI alignment (413 bp) comprised 32 sequences from Brachycladiidae (22 newly generated and 10 retrieved from GenBank). Table 5 provides a listing of all sequences used to construct the datasets. The genetic distance analyses revealed that the Synthesium species and morphotypes present intraspecific divergence of less than 2% for mtCOI and mtND3, except between the Brazilian and Spanish S. tursionis specimens (9.0%), whereas the interspecific divergence showed higher genetic variation (minimum of 6.4% and maximum of 20,4% for mtCOI; minimum of 3,3% and maximum of 27,7% for mtND3), especially for the comparisons using S. delamurei, S. seymouri, and S. subtile (See Supplementary Table 1 and 2 for values). For ITS1 alignment, no intraspecific genetic distance between Synthesium specimens was observed, except between one S. tursionis specimen from Spain (StE3) compared to the all other S. tursionis (0.2%), and the interspecific distance ranged from 0.2% to 10.5% (See Supplementary Table 3 for values). The molecular phylogenies (BI and ML) based on ITS1 and mtND3 datasets (Fig. 2 and 3) yielded similar topologies with most bootstrap values strongly supported. All Synthesium species, except for S. delamurei and S. seymouri were placed in a monophyletic group divided in two well defined clusters; one comprising all S. pontoporiae, S. neotropicalis, and Synthesium morphotype 2 (Clade A); and another with all S. tursionis from the Brazilian coast, S. tursionis from the Mediterranean Sea, Synthesium morphotype 1, and Synthesium morphotype 3 (Clade B). Synthesium seymouri and S. delamurei clustered apart from each other within the other Breachycladiidae species utilized in this study. As for ITS1, the topology obtained grouped Oschmarinella rochebruni with the Synthesium species, Brachycladium atlanticum and Brachycladium goliath did not form a monophyletic clade, with Campula as sister taxon of B. atlanticum and S. delamurei is unresolved. The obtained results in the GMYC analysis for mtCOI (Fig. 4) suggested the recognition of 10 putative species, 2 of which contained a single individual (B. goliath and N. attenuatum), and the confidence limits for the estimated number of species ranged from 10 to 11. The GMYC analysis identified S. pontoporiae, S. neotropicalis, S. tursionis from the Brazilian coast, S. tursionis from the Spanish coast, S. delamurei, and S. seymouri + S. subtile as single entities. The analysis also identified two putative species that corresponded to Synthesium morphotypes 1 and 3. ! 48 DISCUSSION Most diagnostic characters of the three morphotype support the hypothesis that they belong to the genus Synthesium (e.g. shape of body, well developed pyriform pharynx, intestines with anterior and posterior caeca without diverticula, with posterior caeca ending blindly close to posterior extremity of body, ventral sucker in anterior third of the body, presence of cirrus sac, unarmed metraterm, testes in middle third or third