Inês Correia Rosa Ecologia De Atherina Spp. Ao Longo Do Gradiente Salino Do Canal De Mira (Ria De Aveiro)

Departamento de Biologia 2008

Inês Correia Rosa Ecologia de Atherina spp. ao longo do gradiente salino do Canal de Mira (Ria de Aveiro)

dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ecologia, Biodiversidade e Gestão de Ecossistemas, realizada sob a orientação científica do Doutor Vítor Almada, Professor Catedrático do ISPA e do Doutor Fernando Gonçalves, Professor Auxiliar com Agregação do Departamento de Biolo gia da Universidade de Aveiro

o júri presidente Doutor Carlos Manuel Martins Santos Fonseca professor auxiliar convidado da Universidade de Aveiro

Doutor Vítor Manuel Carvalho Almada professor catedrático do Instituto Superior de Psicologia Aplicada

Doutor Fernando José Gonçalves professor auxiliar com agregação da Universidade de Aveiro

Doutor Henrique Nogueira Cabral professor auxiliar da Universiadde de Lisboa

Doutor Bruno Branco Castro investigador de pós-doutoramento do CESAM e da Universidade de Aveiro

agradecimentos Ao Prof. Doutor Vítor Almada por toda a sabedoria que me transmitiu, por me ter disponibilizado todos os recursos que eu necessitava e pelo esforço que fez para que o presente trabalho estivesse terminado dentro de tão pouco tempo.

Ao Prof. Doutor Fernando José Mendes Gonçalves por todo o apoio dado desde o início e pela simplificação dos problemas que por vezes surgiam. Obrigada por me ter integrado tão bem na sua equipa e, pr incipalmente, obrigada por acreditar em mim e nas minhas capacidades.

Ao Bruno, pelo apoio incondicional em todos os momentos, não só pelos aspectos práticos como saídas de campo, análise estatística, discussão dos resultados e revisão da tese, mas princ ipalmente pelos aspectos humanos: a amizade, os conselhos, a paciência, o incentivo, a sinceridade, a preocupação e constante presença. Um abreijo para ti, Bruno!

Ao Doutor José Vingada pela ajuda com o material de pesca eléctrica, pela captura de alguns dos meus peixes e por algumas informações úteis a este trabalho.

Ao senhor Aldiro pela “sabedoria de pescador”, pelas fotos e pela boa disposição.

A todos os meus colegas dos laboratórios onde trabalhei durante este ano: à Joana Robalo pela atenção que me dispensou, à Sónia por me ter aj udado no trabalho prático, e à malta do LEAD por tudo o que me ensinaram, pelos conselhos, pelos sorrisos e conversas de desanuviar, pelos almoços, pelo apoio e preocupação: tenho orgulho de verdade em fazer parte desta equipa.

A todos os meus amigos biólogos que sabem o que custa ser-se investigador e que sempre me apoiaram e se preocuparam, em especial à Li, Nilza, Lena, Catarina, ao João, ao Chico, à Guida, à Nini, à Joana e à Marta.

E aos meus amigos que não são biólo gos, mas que estão presentes em cada pedacinho da minha vida: Cataréu (mil obrigados pelas guaridas e calhandrices) e restante Casa Amarela (saudades...), Diana, Dri e Andreia (mil sorrisos), Casaleiro (tanta coisa...), Maria João (partilha), Susa (vais-me fazer tanta falta). Ao Mário e ao Rui pela ajuda na pescaria.

À minha família, pai, mano, avô, tios e primos, e principalmente à Mãe que tanta paciência tem comigo e que me tenta compreender e ajudar incansavelmente.

Aos meus professores do conservatór io que compreenderam o facto de eu ter de faltar bastante às aulas. Um agradecimento especial ao Sérgio, que mais que professor é um amigo: obrigada por toda a paciência ao longo destes anos e por não desistires de mim.

resumo As espécies Atherina boyeri Risso, 1810 e Atherina presbyter Cuvier, 1829 , apesar de apresentarem características merísticas e morfométricas distintas, são por vezes difíceis de distinguir (nomeadamente em casos onde os peixes são pequenos ou estão danificados). E stas duas espécies apresentam também comportamento e ecologia distintos: A. presbyter é tipicamente marinha, podendo estar presente nas áreas mais salinas dos estuários, A. boyeri pode completar todo o seu ciclo de vida em água doce ou salobra sendo o seu comportamento nos estuários desconhecido. Pouco se sabe acerca da biologia destes peixes em Portugal, incluindo informação sobre as populações estuarinas e conservação. A amostragem foi efectuada no Canal de Mira (um dos braços principais da Ria de Aveiro, Portugal) em intervalos de 3 meses (Verão 2007- Primavera 2008) e foram determinados alguns parâmetros biométricos como comprimento total, GSI e alguns factores de condição. A Ria de Aveiro foi escolhida como caso de estudo de modo a determinar a influência do habitat , nomeadamente do gradiente de salinidade na distribuição e ecologia da população de peixe-rei. A distinção entre A. presbyter e A. boyeri foi feita com base em caracteres morfológicos e moleculares. O presente trabalho permitiu concluir que A. presbyter está presente em locais mais perto da boca do estuário enquanto A. boyeri tem preferência pelos locais no final do canal. As espécies coexistem em zonas intermédias (sainidade 5, 5 a 22), com clara dominância de A. presbyter sobre A. boyeri. A presente população de peixe-rei reproduz-se durante a P rimavera e Verão e os juvenis apenas se reproduzem na época reprodutora seguinte após o seu nascimento. Foi também possível concluir que há uma grande discrepância relativamente aos critérios meríst icos sugeridos pela bibliografia usados para distinguir as duas espécies, pelo que é aqui proposto uma complementaridade entre critérios morfológicos e genéticos.

abstract Atherina boyeri Risso, 1810 and Atherina presbyter Cuvier, 1829, although distinct in meristic and morphometric characters, are sometimes difficult to differentiate, especially when small or injured fish are involved. They also exhibit distinct ecology and behaviour: while A. presbyter is typically marine, entering the more saline sections of estuaries, A. boyeri may complete its life cycle in fresh or brackish waters, although its behaviour in estuaries is unknown. Little is known on the biology of both species in Portugal, including the estuarine popula tions, and basic information needed for their conservation is missing. A sampling campaign was carried out in Canal de Mira (one of the main arms of Ria de Aveiro, Portugal) every three months (summer 2007 – spring 2008), and biometric features such as to tal length, GSI, and condition factors were determined. To avoid misidentifications, specimens were diagnosed with the aid of morphological and molecular characters. Ria de Aveiro was chosen as a case study to determine the influence of habitat , namely the salinity gradient, in the distribution and ecology of the sand smelts assemblage. We observed that A. presbyter occurred mostly downstream, while A. boyeri preferred the upper reaches of the estuary and both species co- occurred in the intermediate zones (salinity 5.5 to 22), with A. presbyter being dominant in this zone. Reproduction of Canal de Mira assemblage occurred during spring and summer, and juveniles only reproduced in the next reproductive season after their birth. We also concluded that there is a large overlap in scale counts between the two species, so a complementarity between morphological and genetic criteria is here proposed.

Às minhas avós

“E ela [avó] talvez se alegre no lugar onde estiver, e eu sinto saudades, muitas de quando tinha a minha avó.” José Luís Peixoto, Cal

Índice

Introdução Geral 8 Bibliografia 16

Capítulo 1 22 Ecological differences between Atherina presbyter and Atherina boyeri along a salinity gradient: meristic and genetic evidence

Abstract 23 Introduction 23 Materials and Methods 26 Results 29 Discussion 41 References 47

Conclusão Geral 53 Bibliografia 56

Introdução Geral

Desde sempre, um dos objectivos primordiais da Ecologia foi compreender os mecanismos que determinam a distribuição e abundância dos organismos vivos. O conceito de nicho ecológico transmite a ideia de que uma espécie ocorre em determinado local quando as condições ambientais se enquadram nos limites de tolerância fisiológica da espécie e se encontram disponíveis os recursos de que os organismos dessa espécie dependem (Pires, 1999). Neste contexto, ecossistemas bastante instáveis ao nível de factores físicos, químicos e biológicos, como os habitats estuarinos, são de extremo interesse, no que diz respeito à distribuição e abundância dos organismos vivos que nele habitam. Estuários e lagunas 1 são ecossistemas de interface que comportam ambiente continental e marinho, recebendo inputs biogeoquímicos do ecossistema terrestre, rios e mares (Lopes and Silva, 2006). Estes ecossistemas são áreas bastante ricas em recursos alimentares para os peixes (e restante fauna), onde a elevada produtividade é mantida devido aos elevados níveis de nutrientes nos sedimentos e na coluna de água (Lopes and Silva, 2006). No entanto, tal produtividade não faz dos estuários um habitat extremamente rico em diversidade ictiológica, mas sim com grande abundância de taxa individuais, já que as mudanças frequentes e abruptas de salinidade, temperatura da água, oxigénio dissolvido e turbidez, exigem considerável tolerância fisiológica às comunidades estuarinas (Whitfield, 1999). As alterações de salinidade são de maior ou menor magnitude conforme o balanço entre o influxo de água doce e o regime de marés, podendo ser graduais, como geralmente ocorre em estuários temporariamente fechados, ou repentinas, como é frequente em estuários influenciados por marés (Whitfield, 1999). A capacidade de se ajustar a estas flutuações é a mais essencial adaptação dos peixes estuarinos. A ictiofauna estuarina é geralmente dividida em duas categorias de história de vida: i) espécies residentes ou sedentárias, que passam a totalidade do seu ciclo de vida no estuário; ii) espécies migradoras, que podem ser marinhas ou de

1 Lagunas ( sensu stricto ) correspondem ao isolamento de porções de domínio marinho por uma ou mais barreiras litorais móveis recentes, com pequena(s) interrupção(ões), onde as águas provenientes dos meios fluvial e/ou marinho se encontram e misturam

(Pombo, 1998).

Introdução Geral

água doce, e que usam os estuários para alimentação, reprodução, abrigo ou como maternidade (Koutrakis et al. , 2004). As espécies mais bem adaptadas aos ecossistemas estuarinos são as espécies sedentárias, já que suportam as condições flutuantes deste habitat ao longo de todo o seu ciclo de vida, nem que para isso tenham de fazer migrações dentro do próprio estuário (Rebelo, 1993). As restantes espécies frequentam os estuários apenas em determinados momentos do seu ciclo de vida por motivos específicos de cada ciclo (a menor transparência da água, a intensa vegetação e o acentuado recorte das margens nestas áreas, por exemplo, criam locais de abrigo contra eventuais predadores aos organismos juvenis mais frágeis (Pombo, 1998)). Num estuário, os factores que constituem ameaça aos peixes são variados e frequentemente diferem de uma área biogeográfica para outra. Estes factores podem incluir degradação do habitat , manipulações hidrológicas, poluição de origem industrial, doméstica ou agrícola, alterações climáticas globais, contaminações genéticas e outros impactos de espécies introduzidas (alterações na composição de macrófitas e fitoplâncton, excesso de competidores e predadores, alterações nas propriedades físicas e químicas da água, introdução de doenças e parasitas (Holcik, 1991)), construção de açudes, captação de água para fins agrícolas, e excesso de exploração piscatória (Almaça, 1995; Pires, 1999; Whitfield, 1999). As medidas directas para conservação de espécies de peixes e stocks incluem, para além da designação de áreas protegidas, a conservação de habitat , controlo sobre os métodos, esforço, eficiência e sazonalidade de pesca, outras formas de legislação, controlo de poluição e translocações benignas. Destas medidas, o factor mais importante é, sem dúvida, a conservação dos habitats , uma vez que um ecossistema aquático saudável suporta, invariavelmente, populações de peixes saudáveis (Whitfield, 1999). Muito carece por fazer no que respeita ao conhecimento sobre a ecologia e o estado de conservação de algumas espécies piscícolas em Portugal. Assim, qualquer estudo centrado nas comunidades ictiológicas que frequentam quer os habitats estuarinos quer os de água doce, é importante no sentido de compreendermos o actual estado de conservação das espécies e habitats , bem como os factores que os ameaçam. As espécies da família Atherinidae existentes nas águas portuguesas (peixes-rei, piarda ou camarão-buxo), por exemplo, são classificadas pelo Instituto de Conservação da Natureza na categoria de

Introdução Geral

Informação Insuficiente (Cabral and Oliveira, 2005), devido essencialmente à falta de dados sobre os efectivos populacionais. Assim, é de extremo interesse obter informações acerca da ecologia e conservação destes organismos. Em Portugal, a família é representada por três espécies incluídas no género Atherina : A. hepsetus Linnaeus, 1758, A. boyeri Risso, 1810 e A. presbyter Cuvier, 1829. A primeira espécie tem uma ocorrência bastante esporádica nas águas portuguesas, sendo tipicamente Mediterrânica; A. boyeri e A. presbyter são os peixe-rei mais comuns. A sistemática destas duas espécies tem sido bastante controversa: as espécies exibem uma morfologia e anatomia bastante similar, quer no tamanho adulto quer no tamanho da primeira maturação, pelo que a classificação taxonómica através das características morfométricas e merísticas comuns (e.g. comprimento da cabeça, número de escamas na linha longitudinal e número de vértebras) é ambígua e gera algumas dúvidas (Pombo et al. , 2005; Francisco et al. , 2006) (Fig. 1). Esta dificuldade taxonómica é acentuada pelo facto das espécies poderem coexistir no mesmo habitat . Bamber et al. (1983), através do estudo de características morfométricas e merísticas, concluíram que os caracteres utilizados anteriormente para distinguir A. presbyter de A. boyeri (morfologia prémaxilar, dentição ectopterigóide, número de escamas da linha lateral e número total de vértebras) não eram válidos. Nesse estudo, as diferenças foram consideradas como o resultado de diferentes pressões abióticas a actuarem num único fenótipo, bem como do isolamento geográfico e selecção natural. No entanto, estudos contemplando evidências relativas à biologia (Creech, 1992) e genética (Creech, 1991; Francisco et al. , 2006; Almada et al. , submitted ) de várias populações, demonstraram diferenças entre A. boyeri e A. presbyter , consistentes com a existência de duas espécies. Estudos recentes (Klossa-Kilia et al. , 2002; Trabelsi et al. , 2002; Astolfi et al. , 2005; Klossa-Kilia et al. , 2007; Francisco et al. , in press ) provam ainda que A. boyeri sensu é polifilético donde emergem 3 distintas linhagens: uma de água doce/salobra ( A.boyeri) e duas tipicamente marinhas presentes no Mar Mediterrâneo (forma não pontuada e forma pontuada).

Introdução Geral

Fig. 1: Adultos de Atherina presbyter (cima) e A. boyeri (baixo).

Os organismos da espécie A. boyeri são peixes teleósteos eurihalinos que habitam essencialmente áreas estuarinas, mas que também ocorrem em sistemas dulçaquícolas (Clavero et al. , 2006). Já A. presbyter é uma espécie marinha costeira que entra ocasionalmente nos estuários e lagunas costeiras, nomeadamente durante a fase juvenil, sendo rara a sua ocorrência na fase adulta (Pombo, 1998; Francisco et al. , 2006). Assim, apesar de possuírem preferências abióticas diferentes podem ocorrer no mesmo espaço ao mesmo tempo. Quanto à distribuição geográfica, A. boyeri é comum nas lagunas e rios em torno do Mediterrâneo e mares adjacentes, e ocorre em rios, estuários e lagoas na margem nordeste do Atlântico ao longo da costa Oeste de Espanha e Portugal, tendo também sido reportadas algumas populações isoladas na costa Inglesa e Holandesa. Já A. presbyter encontra-se distribuída desde as Ilhas Britânicas e sul do Mar do Norte até às Ilhas Canárias, Mauritânia e Cabo Verde, havendo também registos da espécie a oeste do Mar Mediterrâneo (Francisco et al. , 2006). Ambas as espécies apresentam sexos separados, onde as gónadas são testículos nos machos e um único ovário nas fêmeas. Os testículos são uma estrutura branca e lobada, mais evidente quanto mais maduro é o indivíduo; o ovário é arredondado com um peritoneu preto (Moreno et al. , 2005) (Fig.2). Os ovos não ocorrem em ninhadas e são depositados na vegetação em águas salobras (Bamber et al. , 1985; Bartulovic et al. , 2006). O desenvolvimento inicial é rápido, surgindo uma pós-larva ao fim de 15-16 dias, pronta para uma alimentação exógena activa à base de pequenos zooplanctontes. À medida que crescem e melhoram a sua capacidade natatória, os indivíduos vão-se alimentando de animais maiores e de maior diversidade. Os adultos de A. boyeri são generalistas na selecção de presas, podendo ser classificados como carnívoros oportunistas (Bartulovic et al. , 2004b; Bartulovic et al. , 2004c) com preferência por pequenos crustáceos (planctónicos (Trabelsi et al. , 1994) ou

Introdução Geral bentónicos (Rosecchi and Crivelli, 1992; Vizzini and Mazzola, 2002; Vizzini and Mazzola, 2005)), podendo alimentar-se secundariamente de larvas de insectos (Rosecchi and Crivelli, 1992; Chrisafi et al. , 2007), anelídeos (Trabelsi et al. , 1994), moluscos (larvas de bivalves) e ovos de peixe (Chrisafi et al. , 2007). Quanto à dieta de A. presbyter , Jennings et al (1997) referem a preferência da espécie por zooplâncton, no entanto, outros estudos, concluem que a sua dieta é essencialmente à base de invertebrados bentónicos (Pinnegar and Polunin, 2000), o que pode sugerir a diversidade de dieta de A. presbyter . Deste modo, estes peixes desempenham um importante papel nas teias alimentares estuarinas, tanto como predadores como presas, sendo dos conectores mais importantes entre os organismos planctónicos, bentónicos e os indivíduos ocasionais (e.g. insectos) (Pajuelo and Nespereira, 2001; Bartulovic et al. , 2004c). A importância de A. boyeri estende-se ao interesse piscatório, estando registada como um recurso comercialmente importante nas costas mediterrânicas da Espanha, Croácia e Grécia (Andreu-Soler et al. , 2003a; Bartulovic et al. , 2004a; Koutrakis et al. , 2004).

Fig. 2 : Gónada masculina (esquerda) e feminina (direita) de peixe-rei.

Apesar da importância ecológica comum a ambas as espécies, são poucos os estudos efectuados sobre A. presbyter , referindo-se unicamente a aspectos biológicos como morfologia (e.g. Bamber et al. , 1983), idade e crescimento (e.g. Lorenzo and Pajuelo, 1999; Moreno and Morales-Nin, 2003), reprodução (e.g. Bamber et al. , 1985; Pajuelo and Nespereira, 2001; Moreno et al. , 2005), sistemática (e.g. Bamber and Henderson, 1985; Creech, 1991; 1992) e regime alimentar (e.g. Turnpenny et al. , 1981; Moreno and Castro, 1995). Em oposição, são numerosos os estudos efectuados em A. boyeri em variados locais do Atlântico e Mediterrâneo e sobre vários aspectos (e.g. Berrebi and Brittondavidian, 1980; Gon and Bentuvia, 1983; Palmer and Culley, 1983; Trabelsi et al. , 1994;

Introdução Geral

Tomasini et al. , 1996; Leonardos and Sinis, 2000; Tutman et al. , 2000; Leonardos, 2001; Congiu et al. , 2002; Tomasini and Laugier, 2002; Vizzini and Mazzola, 2002; Andreu-Soler et al. , 2003a; Andreu-Soler et al. , 2003b; Bartulovic et al. , 2004b; a; Bartulovic et al. , 2004c; Koutrakis et al. , 2004; Koutrakis et al. , 2005; Vizzini and Mazzola, 2005; Bartulovic et al. , 2006). Os estudos específicos sobre estas duas espécies cingem-se às investigações de Pombo et al. (2005) e Francisco et al. (2006, in press ), sendo o último relacionado com aspectos filogeográficos dos peixe-rei. Tal como no estudo de Pombo et al. (2005), a amostragem do presente trabalho foi efectuada na Ria de Aveiro. A Ria é considerada uma laguna costeira estuarina (Fig. 3); é um ecótono que define um ambiente dinâmico particular, com influência fluvial e marinha, baixa profundidade, elevada turbidez, substrato de natureza lodosa, flutuações sazonais de temperatura, gama de salinidade e oxigénio elevadas, rico em nutrientes, altamente produtivo e com grande abundância de vegetação submersa, o que favorece a sua colonização por espécies ictiológicas diversas (Pombo, 1998). Este sistema lagunar tem sido bastante afectado por influências antropogénicas (poluição atmosférica de causa industrial e poluição hídrica, nomeadamente por influência de herbicidas e Fig. 3: Ria de Aveiro efluentes urbanos (Reis, 1985)), mas também por alterações climáticas que afectam a distribuição das espécies, causando a sua migração para Norte (Pombo et al. , 2002). A grande produtividade e diversidade de habitat conferem a esta laguna um grande interesse científico e económico. No contexto particular do presente trabalho, a Ria de Aveiro é particularmente importante já que, segundo Pombo (2005), as espécies A. boyeri e a A. presbyter, são dos grupos ecológicos mais representativos desta laguna. Além do mais, o Canal de Mira (ao qual se cingiu a amostragem) apresenta um gradiente de salinidade bastante claro desde a boca do estuário até ao fim do canal, comportando-se como um estuário dentro do próprio estuário. A Ria de Aveiro é,

Introdução Geral portanto, um bom local de estudo para colmatar a falta de informação sobre os peixe-rei e perceber de que modo estas espécies se comportam ao longo de um gradiente de salinidade permitindo responder a questões como: As populações de A. presbyter e A. boyeri são uma população homogénea ou mista? E se as duas espécies coexistirem, há preferências salinas de uma e outra espécie? E tem a salinidade influência na distribuição populacional e fisiologia de cada espécie?

Introdução Geral

BIBLIOGRAFIA

Almaça, C. (1995). Fresh-water fish and their conservation in Portugal Biological Conservation 72 , 125-127. Almada, V. C., Domingues, V., Cabral, H., ALMADA, F., Robalo, J. & Francisco, S. M. ( submitted ). A rapid and inexpensive molecular technique to descriminate between Atherina boyeri and A. presbyter around the Iberian Peninsula and its potential applications on ecology and larval identification. Andreu-Soler, A., Oliva-Paterna, F. J., Fernandez-Delgado, C. & Torralva, M. (2003a). Age and growth of the sand smelt, Atherina boyeri (Risso 1810), in the Mar Menor coastal lagoon (SE Iberian Peninsula). Journal of Applied Ichthyology 19 , 202-208. Andreu-Soler, A., Oliva-Paterna, F. J. & Torralva, M. (2003b). Estrategia de crecimiento de Atherina boyeri Risso, 1810 (Pisces: Atherinidae) en la laguna costera del Mar Menor (SE Península Ibérica). Munibe (Ciencias Naturales-Natur Zientziak 54 , 95-112. Astolfi, L., Dupanloup, I., Rossi, R., Bisol, P. M., Faure, E. & Congiu, L. (2005). Mitochondrial variability of sand smelt Atherina boyeri populations from north Mediterranean coastal lagoons. Marine Ecology-Progress Series 297 , 233-243. Bamber, R. N., Glover, R., Henderson, P. A. & Turnpenny, A. W. H. (1983). Diplostomiasis in the Sand Smelt, Atherina-Presbyter (Cuvier), Population at Fawley-Power-Station. Journal of Fish Biology 23 , 201-210. Bamber, R. N. & Henderson, P. A. (1985). Morphological Variation in British Atherinids, and the Status of Atherina-Presbyter Cuvier (Pisces, Atherinidae). Biological Journal of the Linnean Society 25 , 61-76. Bamber, R. N., Henderson, P. A. & Turnpenny, A. W. H. (1985). The Early Life- History of the Sand Smelt (Atherina-Presbyter). Journal of the Marine Biological Association of the United Kingdom 65 , 697-706. Bartulovic, V., Glamuzina, B., Conides, A., Dulcic, J., Lucic, D., Njire, J. & Kozul, V. (2004a). Age, growth, mortality and sex ratio of sand smelt, Atherina boyeri Risso, 1810 (Pisces : Atherinidae) in the estuary of the Mala Neretva River (middle-eastern Adriatic, Croatia). Journal of Applied Ichthyology 20 , 427-430. Bartulovic, V., Glamuzina, B., Conides, A., Dulcic, J., Lucic, D., Njire, J. & Kozul, V. (2004b). Age, growth, mortality and sex ratio of sand smelt, Atherina boyeri

Introdução Geral

Risso, 1810 (Pisces: Atherinidae) in the estuary of the Mala Neretva River (middle- eastern Adriatic, Croatia). Journal of Applied Ichthyology 20 , 427-430. Bartulovic, V., Glamuzina, B., Conides, A., Gavrilovic, A. & Dulcic, J. (2006). Maturation, reproduction and recruitment of the sand smelt, Atherina boyeri Risso, 1810 (Pisces:Atherinidae) in the estuary of Mala Neretva River (southeastern Adriatic, Croatia). Acta Adriat. 47 (1) , 5-11. Bartulovic, V., Lucic, D., Conides, A., Glamuzina, B., Dulcic, J., Hafner, D. & Batistic, M. (2004c). Food of sand smelt, Atherina boyeri Risso, 1810 (Pisces : Atherinidae) in the estuary of the Mala Neretva River (middle-eastern Adriatic, Croatia). Scientia Marina 68 , 597-603. Berrebi, P. & Brittondavidian, J. (1980). Enzymatic Survey of 4 Populations of Atherina-Boyeri Based on Electrophoresis and the Occurrence of a Microsporidiosis. Journal of Fish Biology 16 , 149-157. Cabral, M. J. & Oliveira, M. (2005). Livro Vermelho dos Vertebrados de Portugal . Lisboa. Chrisafi, E., Kaspiris, P. & Katselis, G. (2007). Feeding habits of sand smelt (Atherina boyeri, Risso 1810) in Trichonis Lake (Western Greece). Journal of Applied Ichthyology 23 , 209-214. Clavero, M., Blanco-Garrido, F. & Prenda, J. (2006). Monitoring small fish populations in streams: A comparison of four passive methods. Fisheries Research 78 , 243-251. Congiu, L., Rossi, R. & Colombo, G. (2002). Population analysis of the sand smelt Atherina boyeri (Teleostei Atherinidae), from Italian coastal lagoons by random amplified polymorphic DNA. Marine Ecology-Progress Series 229 , 279-289. Creech, S. (1991). An Electrophoretic Investigation of Populations of Atherina- Boyeri Risso, 1810 and a-Presbyter Cuvier, 1829 (Teleostei, Atherinidae) - Genetic-Evidence in Support of the 2 Species. Journal of Fish Biology 39 , 807- 816. Creech, S. (1992). A Multivariate Morphometric Investigation of Atherina-Boyeri Risso, 1810 and a-Presbyter Cuvier, 1829 (Teleostei, Atherinidae) - Morphometric Evidence in Support of the 2 Species. Journal of Fish Biology 41 , 341-353. Francisco, S. M., Cabral, H., Vieira, M. N. & Almada, V. C. (2006). Contrasts in genetic structure and historical demography of marine and riverine populations of

Introdução Geral

Atherina at similar geographical scales. Estuarine, Coastal and Shelf Science 69 , 655-661. Francisco, S. M., Congiu, L., Stefanni, S., Castilho, R., Brito, A., Ivanova, P., Levy, A., Cabral, H., Kilias, G., Doadrio, I. & Almada, V. C. ( in press). Phylogenetic relationships of the North-eastern Atlantic and Mediterranean forms of Atherina (Pisces, Atherinidae). Molecular Phylogenetics and Evolution . Gon, O. & Bentuvia, A. (1983). The Biology of Boyer Sand Smelt, Atherina-Boyeri Risso in the Bardawil Lagoon on the Mediterranean Coast of Sinai. Journal of Fish Biology 22 , 537-547. Holcik, J. (1991). Fish introductions in Europe with particular reference to its central and eastern part. Canadian Journal of Fisheries and Aquatic Sciences 48 , 13-23. Jennings, S., Renones, O., MoralesNin, B., Polunin, N. V. C., Moranta, J. & Coll, J. (1997). Spatial variation in the N-15 and C-13 stable isotope composition of plants, invertebrates and fishes on Mediterranean reefs: Implications for the study of trophic pathways. Marine Ecology-Progress Series 146 , 109-116. Klossa-Kilia, E., Papasotiropoulos, V., Tryfonopoulos, G., Alahiotis, S. & Kilias, G. (2007). Phylogenetic relationships of Atherina hepsetus and Atherina boyeri (Pisces : Atherinidae) populations from Greece, based on mtDNA sequences. Biological Journal of the Linnean Society 92 , 151-161. Klossa-Kilia, E., Prassa, M., Papasotiropoulos, V., Alahiotis, S. & Kilias, G. (2002). Mitochondrial DNA diversity in Atherina boyeri populations as determined by RFLP analysis of three mtDNA segments. Heredity 89 , 363. Koutrakis, E. T., Kamidis, N. I. & Leonardos, I. D. (2004). Age, growth and mortality of a semi-isolated lagoon population of sand smelt, Atherina boyeri (Risso, 1810) (Pisces : Atherinidae) in an estuarine system of northern Greece. Journal of Applied Ichthyology 20 , 382-388. Koutrakis, E. T., Tsikliras, A. C. & Sinis, A. I. (2005). Temporal variability of the ichthyofauna in a Northern Aegean coastal lagoon (Greece). Influence of environmental factors. Hydrobiologia 543 , 245-257. Leonardos, I. & Sinis, A. (2000). Age, growth and mortality of Atherina boyeri Risso, 1810 (Pisces: Atherinidae) in the Mesolongi and Etolikon lagoons (W. Greece). Fisheries Research 45 , 81-91.

Introdução Geral

Leonardos, I. D. (2001). Ecology and exploitation pattern of a landlocked population of sand smelt, Atherina boyeri (Risso 1810), in Trichonis Lake (western Greece). Journal of Applied Ichthyology 17 , 262-266. Lopes, J. F. & Silva, C. (2006). Temporal and spatial distribution of dissolved oxygen in the Ria de Aveiro lagoon. Ecological Modelling 197 , 67-88. Lorenzo, J. M. & Pajuelo, J. G. (1999). Age and growth of the sand smelt Atherina (Hepsetia) presbyter Cuvier, 1829 in the Canary Islands (Central-east Atlantic). Fisheries Research 41 , 177-182. Moreno, T. & Castro, J. J. (1995). Community structure of the juvenile of coastal pelagic fish species in the Canary-Islands waters Scientia Marina 59 , 405-413. Moreno, T., Castro, J. J. & Socorro, J. (2005). Reproductive biology of the sand smelt Atherina presbyter Cuvier, 1829 (Pisces: Atherinidae) in the central-east Atlantic. Fisheries Research 72 , 121-131. Moreno, T. & Morales-Nin, B. (2003). Age determination and validation on otoliths of the sand-smelt Atherina presbyter (Cuvier, 1829) (Pisces: Atherinidae) from the central-east Atlantic. Fisheries Research 62 , 77-87. Pajuelo, J. G. & Nespereira, J. M. L. (2001). Aspectos de lá biología reproductora del pejerrey o guelde blanco Atherina presbyter Cuvier, 1829 (Atherinidae) en Gran Canaria (islas Canarias). Bol. Inst. Esp. Oceanogr. 17 (3 e 4) , 239-244. Palmer, C. J. & Culley, M. B. (1983). Aspects of the Biology of the Sandsmelt Atherina-Boyeri Risso, 1810 (Teleostei, Atherinidae) at Oldbury-Upon-Severn, Gloucestershire, England. Estuarine Coastal and Shelf Science 16 , 163-172. Pinnegar, J. K. & Polunin, N. V. C. (2000). Contributions of stable-isotope data to elucidating food webs of Mediterranean rocky littoral fishes. Oecologia 122 , 399- 409. Pires, A. (1999). Aspectos da Ecologia de peixes e macroinvertebrados em rios intermitentes da bacia do Guadiana. In Zoologia e Antropologia , p. 177. Lisboa: Faculdade de Ciências da Universidade de Lisboa. Pombo, L. (1998). A Ictiofauna da Ria de Aveiro - Estrutura, Dinâmica e Populações. In Biologia , p. 126. Aveiro: Universidade de Aveiro. Pombo, L. (2005). Diversidade, dinâmica de populações e capacidade de produção ictiológica numa laguna costeira - a Ria de Aveiro. In Biologia , p. 272. Aveiro: Universidade de Aveiro.

Introdução Geral

Pombo, L., Elliott, M. & Rebelo, J. E. (2002). Changes in the fish fauna of the Ria de Aveiro estuarine lagoon (Portugal) during the twentieth century. Journal of Fish Biology 61 , 167-181. Pombo, L., Elliott, M. & Rebelo, J. E. (2005). Ecology, age and growth of Atherina boyeri and Atherina presbyter in the Ria de Aveiro, Portugal. Cybium 29 , 47-55. Rebelo, E. (1993). A Ictiofauna da Ria de Aveiro e o Período Lagunar do Ciclo de Vida do Robalo, Dicentrarchus labrax Linnaeus, 1758. In Biology , p. 180. Aveiro: Universidade de Aveiro. Reis, A. (1985). Recursos Biológicos da Ria de Aveiro. Jornadas Da Ria de Aveiro I. Rosecchi, E. & Crivelli, A. J. (1992). Study of a sand smelt ( Atherina boyeri Risso 1810) population reproducing in fresh water. Ecology of Freshwater Fish 1, 77-85. Tomasini, J. A., Collart, D. & Quignard, J. P. (1996). Female reproductive biology of the sand smelt in brackish lagoons of southern France. Journal of Fish Biology 49 , 594-612. Tomasini, J. A. & Laugier, T. (2002). Male reproductive strategy and reserve allocation in sand smelt from brackish lagoons of southern France. Journal of Fish Biology 60 , 521-531. Trabelsi, M., Gilles, A., Fleury, C., Maamouri, F., Quignard, J.-P. & Faure, E. (2002). Atherina punctata and Atherina lagunae (Pisces, Atherinidae), new species found in the Mediterranean Sea. 2. Molecular investigations of three Atherinid species. Comptes Rendus Biologies 325 , 1119-1128. Trabelsi, M., Kartas, F. & Quignard, J. P. (1994). Comparaison du régime alimentaire d'une population marine et d'une population lagunaire d' Atherina boyeri des côtes tunisiennes. Vie Milieu 44 (2) , 117-123. Turnpenny, A. W. H., Bamber, R. N. & Henderson, P. A. (1981). Biology of the sand-smelt ( Atherina presbyter valenciennes ) around Fawley power-station. Journal of Fish Biology 18 , 417-427. Tutman, P., Glamuzina, B., Skaramuca, B., Kozul, V., Glavic, N. & Lucic, D. (2000). Incidence of spinal deformities in natural populations of sandsmelt, Atherina boyeri (Risso, 1810) in the Neretva river estuary, middle Adriatic. Fisheries Research 45 , 61-64.

Introdução Geral

Vizzini, S. & Mazzola, A. (2002). Stable carbon and nitrogen ratios in the sand smelt from a Mediterranean coastal area: feeding habits and effect of season and size. Journal of Fish Biology 60 , 1498-1510. Vizzini, S. & Mazzola, A. (2005). Feeding ecology of the sand smelt Atherina boyeri (Risso 1810) (Osteichthyes, Atherinidae) in the western Mediterranean: evidence for spatial variability based on stable carbon and nitrogen isotopes. Environmental Biology of Fishes 72 , 259-266. Whitfield, A. K. (1999). Ichthyofaunal assemblages in estuaries: A South African case study. Reviews in Fish Biology and Fisheries 9, 151-186.

Capítulo 1

Ecological differences between Atherina presbyter and Atherina boyeri along a salinity gradient: meristic and genetic evidence

Capítulo 1

Ecological differences between Atherina presbyter and Atherina boyeri along a salinity gradient: meristic and genetic evidence

ABSTRACT Atherina boyeri Risso, 1810 and Atherina presbyter Cuvier, 1829, although distinct in meristic and morphometric characters, are sometimes difficult to differentiate, especially when small or injured fish are involved. They also exhibit distinct ecology and behaviour: while A. presbyter is typically marine, entering the more saline sections of estuaries, A. boyeri may complete its life cycle in fresh or brackish waters, although its behaviour in estuaries is unknown. Little is known on the biology of both species in Portugal, including the estuarine populations, and basic information needed for their conservation is missing. A sampling campaign was carried out in Canal de Mira (one of the main arms of Ria de Aveiro, Portugal) every three months (summer 2007 – spring 2008), and biometric features such as total length, GSI, and condition factors were determined. To avoid misidentifications, specimens were diagnosed with the aid of morphological and molecular characters. Ria de Aveiro was chosen as a case study to determine the influence of habitat , namely the salinity gradient, in the distribution and ecology of the sand smelts assemblage. We observed that A. presbyter occurred mostly downstream, while A. boyeri preferred the upper reaches of the estuary and both species co-occurred in the intermediate zones (salinity 5.5 to 22), with A. presbyter being dominant in this zone. Reproduction of Canal de Mira assemblage occurred during spring and summer, and juveniles only reproduced in the next reproductive season after their birth. We also concluded that there is a large overlap in scale counts between the two species, so a complementarity between morphological and genetic criteria is here proposed.

INTRODUCTION Atherinidae (sand smelts or silversides) is a family of marine and freshwater teleost fish which occurs worldwide in tropical and temperate habitats. In the

Capítulo 1

European Atlantic Coast, the family is represented by the species Atherina boyeri Risso, 1810 and Atherina presbyter Cuvier, 1829, which are rather similar regarding their morphology, and anatomy. However they exhibit distinct geographical distributions and ecology (Pombo et al. , 2005). The distribution of A. boyeri is mostly centered on the Mediterranean and Black seas, although it also occurs on brackish and fresh waters around the North-Eastern Atlantic (from west coast of Spain and Portugal to some isolated populations that have been reported on the coast of England and the Netherlands). Although presenting a similar latitudinal range (North Africa to British Isles), A. presbyter is mainly an Atlantic species, which has also been reported in the Western Mediterranean Sea (Francisco et al. , 2006). In terms of their ecology A boyeri is a euryhaline fish that mainly inhabits estuaries, coastal lagoons, salt marshe, rivers and lakes over a wide range of salinities, from freshwater to hypersaline conditions (Andreu-Soler et al. , 2003a); A. presbyter is a small pelagic fish species that inhabits inshore marine waters and, occasionally, enters estuaries and coastal lagoons (Pajuelo and Lorenzo, 2000; Francisco et al. , 2006). In an estuary, A. boyeri is considered a resident species, being able to complete its life cycle inside the estuary (Whitfield, 1999), while A. presbyter is a marine juvenile migrant species, which uses estuaries and coastal lagoons primarily as a nursery ground. Often, A. presbyter returns seasonally to the estuary, but most of its adult life is spent at the sea (Pombo et al. , 2005). The sand smelt plays an important role in estuarine food webs as it is one of the most important links between planktonic, benthic and terrestrial organisms (such as insects which regularly enter the estuarine food webs in certain periods of the year), and carnivorous fish species (Bartulovic et al. , 2004c). Additionally, sand smelt fisheries are considered commercially important in the Mediterranean coast of Croatia (Bartulovic et al. , 2004b), Spain (Andreu-Soler et al. , 2003a), and Greece (Koutrakis et al. , 2004). The population biology of A. boyeri has been extensively investigated in various different locations (e.g. Berrebi and Brittondavidian, 1980; Gon and Bentuvia, 1983; Palmer and Culley, 1983; Trabelsi et al. , 1994; Tomasini et al. , 1996; Leonardos and Sinis, 2000; Tutman et al. , 2000; Leonardos, 2001; Congiu et al. , 2002; Tomasini and Laugier, 2002; Vizzini and Mazzola, 2002; Andreu-Soler et al. , 2003a; Andreu-Soler et al. , 2003b; Bartulovic et al. , 2004b; a; Bartulovic et al. , 2004c; Koutrakis et al. , 2004;

Capítulo 1

Koutrakis et al. , 2005; Vizzini and Mazzola, 2005; Bartulovic et al. , 2006). In contrast, published information on A. presbyter is very scarce, mainly focusing on morphology (e.g. Bamber et al. , 1983), age and growth (e.g. Lorenzo and Pajuelo, 1999; Moreno and Morales-Nin, 2003), reproduction (e.g. Bamber et al. , 1985; Pajuelo and Nespereira, 2001; Moreno et al. , 2005), systematics (e.g. Bamber and Henderson, 1985; Creech, 1991; 1992) and feeding aspects (e.g. Turnpenny et al. , 1981; Moreno and Castro, 1995). In the case of the Portuguese populations of sand smelt, there is a worrying lack of information; this is very serious, considering that these species may be in danger due to the several threats that affect brackish and inland waters, such as pollution, sand extraction, inadequate fish passes in dams, and the successive introductions of exotic fishes (Almaça, 1995; Pires, 1999). It is therefore urgent to obtain as much information as possible about sand smelt species in Portuguese brackish and inland waters. Ria de Aveiro is one of the most peculiar coastal ecosystems in Portugal and it represents an area of considerable economic importance for the region: i) in the primary sector (agriculture, fisheries, aquaculture, and salt-production); ii) in the secondary sector, as a dominant area for industry in the region; iii) in the tertiary sector, especially for tourism (Pombo et al. , 2004; Pombo et al. , 2007). The heterogeneous topographic and abiotic attributes of Ria de Aveiro (shallowness, high turbidity, nature of substrata, temperature, fluctuation, salinity and oxygen), coupled with its and high biotic productivity (despite high pollution levels), make it a very important icthyologic system since it supports abundant sedentary species, provides a suitable nursery area to marine migratory species, and is sought by many occasional species particularly in their juvenile stages (Rebelo, 1992). More details on the icthyological community in Ria de Aveiro can be found in Arruda et al. , 1988; Rebelo, 1992; Rebelo, 1993; Pombo, 1998; Pombo et al. , 2002; 2004; Pombo, 2005; Pombo et al. , 2007. This ecosystem is subjected to an intense fluctuation of its abiotic parameters, making it an excellent study site to assess the influence of habitat in the distribution and ecology of its fish assemblage. Additionally, according to Pombo (2005), A. boyeri and A. presbyter are among the most important ecological groups of this lagoon. This study was undertaken to understand the distribution of A. boyeri and A. presbyter in Canal de Mira (Ria de Aveiro), in order to clarify if they coexist spatially in this particular ecosystem and how the salinity gradient influences their

Capítulo 1

distribution. To avoid misidentifications, specimens were diagnosed with the aid of morphological and molecular characters (see methods). Additionally, basic information on growth, reproduction and somatic condition of sand smelts was assessed, as well as the relationship of the meristic features with the salinity gradient.

MATERIAL AND METHODS

Study site Ria de Aveiro is a shallow coastal lagoon situated on the northwest coast of Portugal (40º38’N, 8º44’W), consisting of a complex network of tidal channels, tidal flats, salt marshes and supra-tidal sand isles that encompass an area of approximately 530km 2, making it the largest coastal lagoon system in Portugal (Duarte et al. , 2007). The average depth of the lagoon is about 1m, except in the navigation channels where dredging operations are frequently carried out (Dias et al. , 2000a; 2001). Tidal propagation and residence time are described in detail in Dias et al. (2000b; 2001) and Lopes & Dias (2007), respectively. Further details on the bathymetry, current fields and water quality of the Ria de Aveiro lagoon are described in Lopes et al. (2005). Looking at the main physical and chemical state variables of Ria de Aveiro, Lopes & Dias (2007) concluded that, excluding the areas close to the lagoon mouth, where some stratification patterns in the salinity and the temperature vertical profiles are observed, the lagoon is a well mixed system. This system is influenced by a maritime, temperate climate, which has a well-defined seasonal variation in the air temperature and rainfall (Pombo et al. , 2007), producing a seasonal fluctuation of temperature, salinity, dissolved oxygen and turbidity in Ria de Aveiro (Pombo et al. , 2004). Our study was conducted in Canal de Mira, a 20km elongated shallow arm within the heterogeneous lagoon (Dias et al. , 2001). This channel was chosen for several reasons: i) it behaves like an estuary within another estuary (the lagoon), displaying a clear salinity gradient from its mouth to the end of the channel; ii) it bears less anthropogenic pressure than the other arms of Ria de Aveiro; iii) its reduced depth and sandy substrate allow easy handling of the fishing gear during the catches.

Capítulo 1

Sampling strategy and procedures Fish ( Atherina spp) were collected between August 2007 and May 2008 at three-month intervals. Samples were collected at low tide, along the salinity/spatial gradient. Salinity was measured in situ with a portable conductivity meter (WTW LF 330/SET). In each sampling campaign, 5-6 stations were sampled, depending on the success of the captures in the upper reaches. Fish were caught using a similar sampling effort at each station (two trawls). Our sampling gear consisted of a traditional bag seine net (“chincha”) operated by boat and/or by hand (depending on the water depth). The “chincha” gear used was almost rectangular in shape and composed by a central bag (a ‘cod end’, 295cm long and 145cm wide), two lateral wings (each 12m long, the width decreasing along the net, reaching 50cm at the edge). Both ends were extended by ropes (6m each), while net structure was assured by floating buoys at the top and ceramic weights at the bottom (for the whole extension of the net). The stretched mesh sizes in the gear were 19mm at the wings, 17mm at the cod mouth, 16 mm at the cod sleeve, and 10mm at the cod end. The net was deployed by fixing one of the ends to the margin while the remainder was trawled in a semi- circle thus retaining within the cod-end all the fish from an area of about 43.31m 2. The net efficiency was estimated at 90% (Pombo, 1998; Pombo et al. , 2004). All atherinids captured were sorted and immediately stored on ice, until further processing was possible (see below).

Fish processing and genetic analysis In the laboratory, the left pectoral fin of all fish was removed and stored in 70% ethanol (when the left pectoral fin was absent, the right one was removed). In cases where the individuals were too small (less than 4cm), the whole specimen was placed in ethanol. Preserved material was used for subsequent DNA extraction and analysis. Differentiation between A. boyeri and A. presbyter was primarily performed based on scale counts (when possible): according to Whitehead et al (1984), A. boyeri has (39) 44-48 (49) scales along its longitudinal line while A. presbyter has 52-57. All individuals were weighed (fresh and eviscerated weight, Fw and Ew, respectively), and measured (total and standard length, TL and SL, respectively). We corrected weight measurements of specimens whose pectoral fin had been

Capítulo 1

removed by back-calculating the weight of the excised fin based on the weight of the remaining fin. Weight of the liver (Lw), gonad (Gw) and gut (GTw) was also recorded, except when it was not possible to differentiate the gonad (immature individuals). Total genomic DNA was extracted from the pectoral fin using Extract-N- Amp TM Tissue PCR Kit (Sigma-Aldrich) following the recommendations of the manufacturer. This same kit (in a posterior phase) was also used to amplify a 350- bp fragment belonging to the 12S rDNA ribossomic mitochondrial DNA region, using the primer sequences described by Almada et al. (2002): 5’-AAC TGG GAT TAG ATA CCC CAC-3' (forward) and 5'-GGG AGA GTG ACG GGC GGT GTG-3 (reverse). PCR reactions were carried out in a Biorad Gene-Cycler TM (total volume 20µl) using the following cycling parameters: i) an initial denaturation step of 3 minutes at 91ºC; ii) 40 cycles of 1 minute at 91ºC, 1 minute at 52ºC and 1 minute at 72ºC; iii) and a final elongation step of 10 minutes at 72ºC. The amplified products were assayed with both HinfI and MboII restriction enzymes (Fermentas, #ER0801 and #ER0822, respectively), following manufacturer instructions, and were loaded and visualised on 1,5% agarose gels. Based on the RFLP criteria proposed by Almada et al (submitted ), specimens were unambiguously identified as A. boyeri (MboII recognizes a restriction site that only occurs in this species), or A. presbyter (HInfI recognizes a restriction site that only occurs in this species).

Data analysis In order to allow statistical inferences between seasons along the salinity gradient, three discrete salinity classes were defined: zero, <22, and 22. Gonadosomatic index (GSI) was determined as (Gw/Fw)x100. Condition factors were also calculated: K=(Fw/TL 3)x1000, K liver=(Lw/TL 3)x1000, K gut=(GTw/TL 3)x1000, and eviscerate K=(Ew/TL 3)x1000. See above for codes. In all analyses, we considered individuals with GSI smaller than 2 and total length less than 8cm ( A. presbyter ) or with GSI less than 2 and smaller than 6cm (A. boyeri ) as juveniles. Juvenile and adults of each species were treated separately. Two-way ANOVAs were performed in order to determine differences across salinity class and time of year for all the parameters calculated for juvenile A.

Capítulo 1 presbyter . Due to the absence of juveniles in the 22 salinity class during the autumn, this season was excluded from the statistical analysis. Differences between seasons were tested with a post-hoc Tukey test. When interactions between factors (salinity class and time of year) were found, a one-way ANOVA was performed in order to determine differences across salinity class for each season. Similarly, three-way ANOVAs were performed in the adult data matrix, using sex as an additional factor. Again, differences between seasons were determined with Tukey test. When interactions between factors (sex, salinity class and time of year) were found, the data matrix was decomposed in a two-way ANOVA, allowing the determination of differences across salinity class and sex for each season. Due to the low number of A. boyeri individuals, it was not possible to robustly compare the different parameters calculated across the salinity class, time of year and sex. Correlation analysis was used to assess the significance of the linear relationship between salinity and the studied parameters (body length, GSI, condition factors). Sex ratios (F:M) for both species were compared using 2 tests. All statistical analyses used a significance level of 0.05.

RESULTS

Pooling all individuals where scale-counting was possible, we observed 38 to 49 scales along the longitudinal line of A. boyeri (identity confirmed through genetic analysis) and 42 to 58 scales in A. presbyter (identity confirmed through genetic analysis). This means that the inferior limit in A. presbyter is considerably different from the bibliographic information (see discussion) and that a rather large overlap in this diagnostic character exists between both species (42-49 scales; see Figure 1). This was true even after excluding specimens where scale counting could not be accurately performed.

Capítulo 1

40 Average Average numberof scales A.presbyter A. boyeri 35 0 5 10 15 20 25 30 35 Salinity Figure 1: Average number of scales along the longitudinal line of A. boyeri and A. presbyter according to the salinities where they were captured. The area delimited in the graph represents the overlap zone between both species in this diagnostic character.

In Canal de Mira (Ria de Aveiro), A. boyeri was found in fresh and brackish water up to salinities of 22. Although absent in the freshwater domain (salinity < 5), A. presbyter was present in a wide range of salinity (5.5 - 33.7). Both species coexisted at salinities between 5.5 and 22 (Figures 1 and 2). During the whole sampling period, the number of A. presbyter caught was always higher than A. boyeri . The former species exhibited an irregular pattern across seasons along the salinity gradient, while the latter was consistently more abundant at the site with lowest salinity (Figure 2).

Capítulo 1

70 A. presbyter 60 A. boyeri

30 Abundance 20

0 0 5 10 15 20 25 30 35 Salinity

70 Summer Autumn 60

700

60 Winter Spring Abundance 50

0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Salinity Figure 2: Abundance (nr. fish/catch) of A. boyeri and A. presbyter according to the salinities where they were captured during the whole sampling period (top panel) and in each season (bottom panel). The area delimited in the graph (top panel) represents the interface zone between both species relatively to salinity.

A. presbyter

An independence 2 test demonstrates that the sex ratio (F:M) varied between seasons ( 2=42.3; d.f=3; p<0.001). In the spring, sex ratio was 1:2 (F:M), but throughout the rest of the year there were more females than males (sex ratio varied from 2:1 to 3.6:1; see Figure 3 and Table 1). All of these sex ratios are

Capítulo 1

significantly different from 1:1 (independent 2 tests for each season; see Table 1). The frequency of immature individuals was highest (ca. 42%) in autumn and winter, being residual in the spring (Figure 3). The size distribution of A. presbyter captured in Canal de Mira is shown in Figure 3. During summer, individuals smaller than 50mm were not captured and two frequency peaks were observed: the 50-80mm cohort and the 90-115mm cohort. In the present work, small juveniles were first captured in the autumn, corresponding to fish that were born during the spring and summer of that year. Their presence was still observable in the winter, probably because of reduced growth rates due to low temperatures. In spring, almost no juveniles were caught, suggesting successful growth and maturation of the cohort born in the previous summer.

Immature 20 Summer Female n=117 Male 15

0 20 Autumn n=114 15

0 20 Winter n=118

Frequency(%) 15

0 20 Spring n=162 15

0 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 Size class (mm) Figure 3: Seasonal variation in the size distribution of A. presbyter .

Capítulo 1

Table 1: Sex ratio of A. presbyter in each season and corresponding χ2 significance.

Female Male Total Chi-Sq P (d.f.=1) Sex Ratio Summer 71 20 91 28.58 <0.001 3.55 Autumn 42 21 63 7.00 0.008 2.00 Winter 48 22 70 9.66 0.002 2.18 Spring 63 96 159 6.85 0.009 0.66

GSI as a function of body size shows two distinct clusters, for both females and males (Figure 4): smaller individuals, who present low GSI (except in the spring), and larger (> 8 cm) specimens, who display high GSI (except in the autumn). We included the former cluster of organisms into the group of juvenile individuals, provided they satisfied both these criteria: total length < 8 cm and GSI < 2. Graphical analysis (Figure 4) shows that individuals matured earlier in spring, while GSI was very low in the autumn for all size classes (after the reproductive season) including mature individuals.

Capítulo 1

16 Summer 14 12 10 8 6 4 2 0 16 Autumn 14 12 10 8 6 4 2 0 GSI 16 Winter 14 12 10 8 6 4 2 0 16 Spring 14 12 10 8 6 4 2 0 4 6 8 10 12 4 6 8 10 12 Total length (cm) Figure 4: GSI of A. presbyter males (left) and females (right) as a function of the total length of individuals. Filled and empty circles represent adults and juveniles, respectively.

Overall, variation in body size and condition of juveniles was mostly attributable to season, as seen by its contribution to total variation (Table 2, two- way ANOVA). Some differences between salinity classes within each season were found (Table 2, one-way ANOVAs), but they were inconsistent. Juveniles from winter were found to be significantly smaller and displayed a lower K eviscerate than the ones captured in spring and summer. Additionally, juveniles caught in the summer exhibited an overall better condition (K, K liver, K gut) than in winter and spring (Figure 5, Table 2, two-way ANOVA followed by Tukey test for season).

Capítulo 1

Table 2: Summary of two-way and one-way ANOVAs performed in the juvenile A. presbyter data matrix for each parameter (TL-Total length, K, K liver, K gut, and K evisc-K eviscerate). One-way ANOVAs were performed for each season when significant interactions between factors were present in the preceding two-way ANOVA. DF corresponds to the degrees of freedom of the numerator (MS factor) and denominator (MS residual) of the F test (df numerator, df denominator).

Two-way ANOVA One-way ANOVA

Source DF F p Season DF F p

TL Salinity 1, 161 11.01 0.001 Summer 1, 56 6.29 0.015 Season 2, 161 55.10 <0.001 Winter 1, 76 47.92 <0.001 Salinity*Time 2, 161 23.98 <0.001 Spring 1, 29 1.36 NS K Salinity 1, 155 0.29 NS

Season 2, 155 74.15 <0.001

Salinity*Time 2, 155 1.39 NS

K liver Salinity 1, 151 0.08 NS Summer 1, 56 13.39 0.001 Season 2, 151 12.63 <0.001 Winter 1, 69 10.35 0.002 Salinity*Time 2, 151 12.78 <0.001 Spring 1, 26 0.31 NS K gut Salinity 1, 155 0.05 NS Summer 1, 56 10.65 0.002 Season 2, 155 12.18 <0.001 Winter 1, 70 13.34 <0.001 Salinity*Time 2, 155 12.11 <0.001 Spring 1, 29 0.36 NS K evisc Salinity 1, 155 0.10 NS

Season 2, 155 4.84 < 0.001

Salinity*Time 2, 155 1.35 NS

Season was, by far, the most important factor in the variation of body size and reproductive state of A. presbyter adults (Table 3, see contribution to total variation in three-way ANOVA). Salinity also influenced total length of A. presbyter adults, but as for juveniles, these differences were inconsistent between seasons (Table 3, two-way ANOVAs). Adult fish captured during summer and spring were significantly smaller and exhibited higher GSI than the ones captured during autumn and winter (Figure 5, Table 3, three-way ANOVA followed by Tukey test for season). To a lesser extent, sex also influenced GSI values and, at least in autumn and spring, female GSI was higher than male GSI. Variation in condition factors (Table 3, three-way ANOVA) was always attributable to sex (K, K liver, K gut, K eviscerate), with only occasional contributions of season (K liver, K gut) or salinity (K eviscerate).

Capítulo 1

Summer Autumn Winter Spring

4 Totallength (cm) 12 10 8 6 GSI 4 2 0 12

K 8

4 0.4

0.3

0.2 K liver 0.1

0.0 0.4

0.3

Kgut 0.2

0.1

6 Kevisc 4 Male Female 2 Juvenile 22 22 22 22 22 22 22 22 to ve to ve to ve to ve up abo up abo up abo up abo

Salinity Figure 5: Total length, GSI and condition factors (K, K liver, K gut and K eviscerate) of A. presbyter in all sampling seasons and in the two salinity classes considered (up to 22 and above 22). Error bars represent SD.

Table 3: Summary of three-way and two-way ANOVAs performed in the adult A. presbyter data matrix for each parameter (TL-Total length, GSI, K, K liver, K gut, and K evisc-K eviscerate). Two-way ANOVAs were performed for each season when significant interactions between factors (Sal.-Salinity) were present in the preceding three-way ANOVA. Sal. Stands for salinity class (<22 and 22) DF

Capítulo 1 corresponds to the degrees of freedom of the numerator (MS factor) and denominator (MS residual) of the F test (df numerator, df denominator).

Three-way ANOVA Two-way ANOVA

Source DF F p TL Sal. 1, 244 9.22 0.003 Summer Autumn Season 3, 244 15.69 <0.001 Source DF F p Source DF F p Sex 1, 244 2.06 NS TL Sal. 1, 55 2.61 NS Sal. 1, 26 <0.001 NS Sal.*Season 3, 244 8.35 <0.001 Sex 1, 55 0.29 NS Sex 1, 26 2.73 NS Season*Sex 3, 244 3.51 <0.001 Sal.*Sex 1, 55 0.20 NS Sal.*Sex 1, 26 1.35 0.001 Sal.*Sex 1, 244 2.62 NS GSI Sal. 1, 55 0.03 NS Sal. 1, 25 0.34 NS Sal.*Season*Sex 3, 244 0.59 NS Sex 1, 55 3.83 NS Sex 1, 25 5.96 0.022 GSI Sal. 1, 242 2.57 NS Sal.*Sex 1, 55 0.33 NS Sal.*Sex 1, 25 2.30 NS Season 3, 242 86.84 <0.001 K Sal. 1, 55 0.41 NS Sal. 1, 26 0.03 NS Sex 1, 242 8.31 0.004 Sex 1, 55 13.18 <0.001 Sex 1, 26 8.83 0.006 Sal.*Season 3, 242 2.42 <0.001 Sal.*Sex 1, 55 0.60 NS Sal.*Sex 1, 26 7.82 0.01 Season*Sex 3, 242 3.35 0.02 K liver Sal. 1, 55 4.70 0.034 Sal. 1, 26 1.41 NS Sal.*Sex 1, 242 2.54 NS Sex 1, 55 31.91 <0.001 Sex 1, 26 3.37 NS Sal.*Season*Sex 3, 242 0.58 NS Sal.*Sex 1, 55 1.53 NS Sal.*Sex 1, 26 1.40 NS K Sal. 1, 244 0.21 NS K gut Sal. 1, 53 5.50 0.023 Sal. 1, 26 0.36 NS Season 3, 244 1.19 NS Sex 1, 53 4.59 0.038 Sex 1, 26 2.36 NS Sex 1, 244 25.34 <0.001 Sal.*Sex 1, 53 0.90 NS Sal.*Sex 1, 26 1.90 NS Sal.*Season 3, 244 0.08 NS K evisc Sal. 1, 55 0.07 NS Sal. 1, 26 0.43 NS Season*Sex 3, 244 3.49 0.016 Sex 1, 55 5.81 0.019 Sex 1, 26 2.16 NS Sal.*Sex 1, 244 10.55 0.001 Sal.*Sex 1, 55 0.62 NS Sal.*Sex 1, 26 2.43 NS Sal.*Season*Sex 3, 244 3.71 0.012 K liver Sal. 1, 244 3.10 NS Winter Spring Season 3, 244 33.46 <0.001 Source DF F p Source DF F p Sex 1, 244 83.52 <0.001 TL Sal. 1, 36 0.93 NS Sal. 1, 127 44.34 <0.001 Sal.*Season 3, 244 4.64 0.004 Sex 1, 36 1.73 NS Sex 1, 127 15,63 <0.001 Season*Sex 3, 244 0.84 NS Sal.*Sex 1, 36 0.02 NS Sal.*Sex 1, 127 1.23 NS Sal.*Sex 1, 244 5.97 0.015 GSI Sal. 1, 36 6.10 0.018 Sal. 1, 126 19.49 <0.001 Sal.*Season*Sex 3, 244 0.37 NS Sex 1, 36 1.10 NS Sex 1, 126 19.68 <0.001 K gut Sal. 1, 242 0.01 NS Sal.*Sex 1, 36 0.85 NS Sal.*Sex 1, 126 <0.001 NS Season 3, 242 10.18 <0.001 K Sal. 1, 36 0.06 NS Sal. 1, 126 0.29 NS Sex 1, 242 11.85 0.001 Sex 1, 36 0.16 NS Sex 1, 126 8.54 0.004 Sal.*Season 3, 242 5.31 0.001 Sal.*Sex 1, 36 0.06 NS Sal.*Sex 1, 126 0.76 NS Season*Sex 3, 242 1.12 NS K liver Sal. 1, 36 5.05 0.031 Sal. 1, 126 9.15 0.003 Sal.*Sex 1, 242 5.43 0.021 Sex 1, 36 19.23 <0.001 Sex 1, 126 119.95 <0.001 Sal.*Season*Sex 3, 242 0.52 NS Sal.*Sex 1, 36 0.20 NS Sal.*Sex 1, 126 5.02 0,027 K evisc Sal. 1, 244 0,78 NS K gut Sal. 1, 36 6.86 0.013 Sal. 1, 126 <0.001 NS Season 3, 244 0.62 NS Sex 1, 36 0.11 NS Sex 1, 126 22.09 <0.001 Sex 1, 244 8.53 0.004 Sal.*Sex 1, 36 1.27 NS Sal.*Sex 1, 126 0.78 NS Sal.*Season 3, 244 0.63 NS K evisc Sal. 1, 36 0.28 NS Sal. 1, 126 <0.001 NS Season*Sex 3, 244 2.55 NS Sex 1, 36 0.39 NS Sex 1, 126 2.01 NS

Sal.*Sex 1, 244 7.88 0.005 Sal.*Sex 1, 36 0.54 NS Sal.*Sex 1, 126 0.43 NS

Sal.*Season*Sex 3, 244 2.73 0.044

Differences in salinity were not consistent. In most cases, significantly higher values of K, K liver, K gut and K eviscerate were observed in females (Figure 5, Table 3, two-way ANOVAs). In the specific case of K liver, no significant differences were found between summer, autumn, and winter, but a significantly

Capítulo 1

lower value of K liver was recorded in spring (Table 3, three-way ANOVA followed by Tukey test for season). A similar analysis showed that K gut was significantly higher in summer than in the rest of the year.

A.boyeri

As previously said, A. boyeri was found mostly in the upper reaches of the estuary and it was the only atherinid present in freshwater (zero salinity). Unfortunately, the reduced number of specimens caught and/or the absence of juveniles or males during some seasons excludes a detailed statistical analysis as performed with the A. presbyter data matrix. The individuals caught had an average body length (TL) of 6.0 ± 1.54cm, ranging from 2.3 cm to 10.6 cm; the largest female was 8.5cm long while the largest males attained 10.6cm. Sex ratio (F:M) was not significantly different from 1:1 ( 2=0,103; d.f=1; p=0,748), pooling all individuals from all sampling seasons. During summer, no males were caught, so we could not test whether sex ratio varied across seasons.

No immature individuals of this species were recorded in autumn and spring, while in summer and winter they represented the most frequent group in the A. boyeri population (Figure 6). The reduced number of individuals caught in some sampling seasons prevents analyses of age groups or cohorts (Figure 6).

Capítulo 1

25 Summer n= 27 20

0 25 Fall 20 n= 4 15

0 25 Winter 20 n= 38 Frequency Frequency (%) 15

0 20 Spring 15 n= 57

5 Immature Female Male 0 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 Size class (mm) Figure 6: Seasonal variation in the size distribution of A. boyeri .

Similarly to what was done with A. presbyter , small fish with low GSI were also considered juvenile; in the case of A. boyeri , this was true for individuals smaller than 6 cm and, simultaneously, with GSI < 2 (see Figure 7).

Capítulo 1

Males Females

16 Summer 14 12 10 8 6 4 2 0 16 Winter 14 12 10 GSI 8 6 4 2 0 16 Spring 14 12 10 8 6 4 2 0 4 6 8 10 12 4 6 8 10 12 Total length (cm) Figure 7: GSI of A. boyeri males (left) and females (right) as a function of the total length of individuals. Autumn was excluded due to low number of captures, while no males were caught in summer. Filled and empty circles represent adults and juveniles, respectively.

Figure 8 displays the seasonal variation of some parameters of the Canal de Mira population of A. boyeri . Graphical analysis suggests juvenile fish were larger in the winter, but in an overall better condition during the summer (particularly for K and K gut). Adult fish displayed similar trends among sexes, being most noticeable a two-fold increase of GSI in spring.

Capítulo 1

Female 10 Male 0.4 Juvenile

8 0.3

6 0.2 K liver

4 0.1 Total length(cm)

2 0.0 10 0.8

8 0.6 6

GSI 0.4 4 K gut

2 0.2

0 0.0 10 8

8 6

K 6 4 K evisc

4 2

2 0 er n er ng er n er g m um int ri m um int rin m ut W Sp m ut W Sp Su A Su A

Figure 8: Seasonal variation in total length, GSI and condition factors (K, K liver, K gut and K eviscerate) of A. boyeri . Error bars represent SD.

Some variation was also due to the salinity gradient (Figure 9). Adult individuals were restricted to salinities up to 11.5, while juvenile fish were found in salinities from 0 to 22. This is supported by a significant correlation (negative) between in situ salinity and total length (r=-0.363, p<0.001; Figure 9), when pooling all individuals. Condition factors did not significantly correlate with salinity (except for K gut - r=-0,366, p=0.031 and K eviscerate - r=-0,378, p=0.025 – of juveniles), while GSI was significantly correlated (negatively) in female (r=-0.435, p<0.001; Figure 9) A. boyeri .

Capítulo 1

Female 10 Juvenile Male

4 Total length(cm)

0 16

GSI 8

0 0 5 10 15 20 Salinity Figure 9: Total length and GSI for A. boyeri as a function of the salinities where specimens were captured.

DISCUSSION The present study confirms that discrimination between A. boyeri and A. presbyter based only on the number of scales along the longitudinal line is inadequate in many cases. This is the most commonly employed diagnostic feature of atherinid species in ecological studies, following Whitehead et al. (1984), who states that A. boyeri has (39) 44-48 (49) scales, while A. presbyter has 52-57 scales along its longitudinal line. That criterion was very different from the counts obtained in the present work for A. presbyter (42-58 scales); Thus, there is a large overlap in scale counts between the two species, which precludes accurate morphological identification in this overlap area (42-49 scales) without genetic tools. Additionally, scale counting is not always possible in cases of juveniles and injured individuals, so a complementarity between morphological

Capítulo 1

(Whitehead et al. , 1984) and genetic criteria (Almada et al. , submitted ) is necessary. In Canal the Mira, there was a clear preference of A. boyeri for fresh and brackish water, being absent in sites where salinity was superior to 22. On the contrary, A. presbyter preferred areas with higher salinities, and closer to the mouth of the estuary, being also present in brackish environments (down to salinities of 5.5). In a way, these results confirm the ecology of A. boyeri as a euryhaline resident species. However, regarding A. presbyter , they suggest that this species might spend more time in the estuary than only during the breeding season, since individuals in different stages of life cycle were captured in the canal, with a clear preference for the more saline areas. Our results also show a clear predominance of A. presbyter over A. boyeri throughout the entire year. Pombo et al. (2005), in a sampling campaign conducted in 1999, captured in the same channel 143 A. boyeri and 104 A. presbyter. These differences in results may be simply explained by misidentification (see above) of sand-smelts during the study (only morphological characters were used); however, we cannot exclude regression of the Ria de Aveiro population of A. boyeri , which is more affected by man- or climate-induced changes, due to its resident nature. In fact, this lagoon is strongly affected by anthropogenic influences (Figueiredo da Silva et al. , 2002), which have increased in the last years due to growth of local human populations and recreational activities (including fishing). Generally, A. presbyter has a spawning period from early spring to the beginning of summer (Pajuelo and Lorenzo, 2000; Pajuelo and Nespereira, 2001), but this depends strongly on the latitude - cases have been found where it reproduced from January to June with some individuals spawning in September and October (Moreno et al. , 2005). Such a long breeding season is generally characteristic of multiple spawners (Tomasini et al. , 1996), which is the case of A. presbyter . In our study, during summer and spring, small juveniles were not captured at all. This absence might be due to a bias in the fishing gear used (e.g. too large mesh). A large cohort of small individuals only appeared in autumn, which grew slowly during the low winter temperatures. In spring, two separate cohorts occurred: the cohort born in the previous summer, mostly composed of males, and a cohort of larger organisms with more than one year of age and probably with less than 4+ (Pombo et al. (2005) reported in Canal de Mira only one

Capítulo 1

individual with 4+ with 15.1cm). However, to be more exact, age should be determined with precision using scale or otholith readings. The presence of individuals with large GSI during spring and summer (see Figures 3 and 4) suggests that this population reproduces during spring and summer, which is consistent with its strategy as a multiple spawner (Pajuelo and Nespereira, 2001; Moreno et al. , 2005). During autumn and winter there was a reduction of the number of adults, which is in agreement with the conclusions of Henderson et al. (1984): older adults tend to decrease their proportion when juveniles appear. This pattern could be due to death of larger organisms, since individuals with 3+, 10.9-12.5cm, and 4+, 15.1cm are rare (Lorenzo and Pajuelo, 1999; Pombo et al. , 2005). During most of the year, there was a clear predominance of A. presbyter females over males. In spring, the opposite trend was observed. Moreno et al. (2005), in a study carried out in the Canary Islands, consistently observed a prevalence of females (1:1,34). The inversion during spring obtained in the present study could be related to the beginning of the breeding season. In the population studied by Moreno et al. (2005), females were significantly larger than males, something which did not happen in the present study, except in spring. There was also seasonality in the GSI of A. presbyter adults. Minimum GSI was reached during autumn (0.35 for females and 0.16 for males) immediately after the spawning period. GSI values increased until spring where they reached their maximum during the year (15.0 for females and 11.2 for males), coinciding with the breeding season. Apparently, the general condition of A. presbyter in Canal de Mira was influenced by sampling season only in its early stages of life, while adults were capable of maintaining an overall good condition, particularly females, which seemed to be well adapted to the brackish environment. In the case of juveniles, somatic condition was minimum during the winter and increased as the conditions (food, temperature) improved (see also discussion on energy input and reserves down), reaching a maximum in summer. Our results show that A. presbyter in Canal de Mira were well nourished, particularly during summer, which is in agreement with availability of zooplankton and benthic macroinvertebrates, the main prey of atherinids, in Canal de Mira, (according to Morgado (1997) and Cunha (1990) they predominate during spring

Capítulo 1 and summer and have their minimum abundance in winter). Our results also suggest a better capacity of females to take profit of the estuarine food sources. Energy is primarily provided by food resources and, to face environmental variability, fish can store energy in their body as a way of buying a degree of independence from the environment (Tomasini and Laugier, 2002). In this context, lipids and aminoacids taken from food are stored in liver and muscle cells, so K liver and K eviscerate, respectively, can give us information about the energy reserves of an organism as well as its energy allocation strategies (Wootton, 1999). During the whole year, females in Canal de Mira displayed more energetic reserves than males. Spring was the season where A. presbyter had less hepatic reserves and since spring corresponds to the spawning season, this decrease in K liver suggests that liver reserves are allocated to gonad maturation. In line with this interpretation it is important to remember that this gland produces vitellogenin, a substance which is the precursor of yolk proteins that are later used as the nutrient material by the developing embryos and larvae (Shi et al. , 2006). It has repeatedly been reported to be a female-specific protein (Shi et al. , 2006), which might help to explain their strong decrease of hepatic reserves during spring. However, the same happens for males and juveniles, which may be justified by the fact that this hormone may be physiologically associated with defense reactions in both female and male fish (Shi et al. , 2006). A different scenario happens with muscular reserves, since season only influenced juvenile fish: there was a decrease from summer to winter (where minimum K eviscerate is attained) and a recovery towards spring. This suggests that, for more fragile sand-smelts such as juveniles, visceral fat content is mainly used to meet overwinter maintenance. These results are coincident with those obtained by Tomasini and Laugier (2002) for A. boyeri ; they concluded that, through winter, the strategic choice of younger fish was to allocate energy to maintenance at the expense of reproductive effort in order to survive. Regarding A. boyeri , there was a large seasonal variation in the population of Canal de Mira. However, the low number of captures compromised a more conclusive analysis. No males were found in the summer and very few specimens were found in autumn, but catches were considerably more successful in winter and spring, when rainfall produced the lowest salinities in the upper reaches of Canal de Mira. Clearly, A. boyeri from Canal de Mira seemed to prefer freshwater,

Capítulo 1

which is also corroborated by the absence of adults at salinities above 11.5 and by significant negative correlations of salinity with adult GSI and juvenile condition (nutritional state and muscular reserves). It is unclear whether this is an intrinsic feature of this population or a consequence of competition for resources with A. presbyter . When both species co-occurred (brackish interface), the latter species always dominated in terms of abundance. The A. boyeri population of Canal de Mira was balanced concerning the proportion of males and females (sex ratio 1:1). This conclusion is similar to most of the studies where A. boyeri sex ratio has been determined (Palmer and Culley, 1983; Fernandez-Delgado et al. , 1988; Leonardos and Sinis, 2000; Bartulovic et al. , 2006). The literature points out 10cm as the maximum TL ever recorded for A. boyeri (Koutrakis et al. , 2004), which is attributed to an individual with 4+. In our study, a fish with 10.6cm was captured, possibly with the same age; however, as for A. presbyter, to be more exact, age should be determined with precision using scale or otholith readings. Maximum GSI was clearly reached during spring, which is in agreement with other studies with A. boyeri (Gon and Bentuvia, 1983; Palmer and Culley, 1983; Fernandez-Delgado et al. , 1988; Rosecchi and Crivelli, 1992; Tomasini et al. , 1996; Leonardos and Sinis, 2000; Tomasini and Laugier, 2002; Andreu-Soler et al. , 2006; Bartulovic et al. , 2006). This suggests that the spawning season occurs during spring and/or summer. The former studies attribute a long spawning period (2 months to 6 months through spring and summer) to this species and suggest that A. boyeri is also a multiple spawner. After the breeding season, a quiescent period occurs from the end of summer to the beginning of winter (Fernandez-Delgado et al. , 1988; Rosecchi and Crivelli, 1992; Andreu-Soler et al. , 2006), which can be seen in the very low values of GSI of the Canal de Mira population during that period.

Capítulo 1

REFERENCES

Almaça, C. (1995). Fresh-water fish and their conservation in Portugal Biological Conservation 72 , 125-127. Almada, V., Almada, F., Henriques, M., Santos, R. S. & Brito, A. (2002). On the phylogenetic affinities of Centrolabrus trutta and Centrolabrus caeruleus (Perciformes: Labridae) to the genus Symphodus: molecular, meristic and behavioural evidences. Arquipélago 19A: 85-92 , 85-92. Almada, V. C., Domingues, V., Cabral, H., ALMADA, F., Robalo, J. & Francisco, S. M. ( submitted ). A rapid and inexpensive molecular technique to descriminate between Atherina boyeri and A. presbyter around the Iberian Peninsula and its potential applications on ecology and larval identification. Andreu-Soler, A., Oliva-Paterna, F. J., Fernandez-Delgado, C. & Torralva, M. (2003a). Age and growth of the sand smelt, Atherina boyeri (Risso 1810), in the Mar Menor coastal lagoon (SE Iberian Peninsula). Journal of Applied Ichthyology 19 , 202-208. Andreu-Soler, A., Oliva-Paterna, F. J. & Torralva, M. (2003b). Estrategia de crecimiento de Atherina boyeri Risso, 1810 (Pisces: Atherinidae) en la laguna costera del Mar Menor (SE Península Ibérica). Munibe (Ciencias Naturales-Natur Zientziak 54 , 95-112. Andreu-Soler, A., Oliva-Paterna, F. J. & Torralva, M. (2006). Seasonal variations in somatic condition, hepatic and gonad activity of sand smelt Atherina boyeri (Teleostei, Atherinidae) in the Mar Menor coastal lagoon (SE Iberian Peninsula). Folia Zoologica 55 , 151-161. Bamber, R. N., Glover, R., Henderson, P. A. & Turnpenny, A. W. H. (1983). Diplostomiasis in the Sand Smelt, Atherina-Presbyter (Cuvier), Population at Fawley-Power-Station. Journal of Fish Biology 23 , 201-210. Bamber, R. N. & Henderson, P. A. (1985). Morphological Variation in British Atherinids, and the Status of Atherina-Presbyter Cuvier (Pisces, Atherinidae). Biological Journal of the Linnean Society 25 , 61-76. Bamber, R. N., Henderson, P. A. & Turnpenny, A. W. H. (1985). The Early Life- History of the Sand Smelt (Atherina-Presbyter). Journal of the Marine Biological Association of the United Kingdom 65 , 697-706.

Capítulo 1

Bartulovic, V., Glamuzina, B., Conides, A., Dulcic, J., Lucic, D., Njire, J. & Kozul, V. (2004a). Age, growth, mortality and sex ratio of sand smelt, Atherina boyeri Risso, 1810 (Pisces : Atherinidae) in the estuary of the Mala Neretva River (middle-eastern Adriatic, Croatia). Journal of Applied Ichthyology 20 , 427-430. Bartulovic, V., Glamuzina, B., Conides, A., Dulcic, J., Lucic, D., Njire, J. & Kozul, V. (2004b). Age, growth, mortality and sex ratio of sand smelt, Atherina boyeri Risso, 1810 (Pisces: Atherinidae) in the estuary of the Mala Neretva River (middle- eastern Adriatic, Croatia). Journal of Applied Ichthyology 20 , 427-430. Bartulovic, V., Glamuzina, B., Conides, A., Gavrilovic, A. & Dulcic, J. (2006). Maturation, reproduction and recruitment of the sand smelt, Atherina boyeri Risso, 1810 (Pisces:Atherinidae) in the estuary of Mala Neretva River (southeastern Adriatic, Croatia). Acta Adriat. 47 (1) , 5-11. Bartulovic, V., Lucic, D., Conides, A., Glamuzina, B., Dulcic, J., Hafner, D. & Batistic, M. (2004c). Food of sand smelt, Atherina boyeri Risso, 1810 (Pisces : Atherinidae) in the estuary of the Mala Neretva River (middle-eastern Adriatic, Croatia). Scientia Marina 68 , 597-603. Berrebi, P. & Brittondavidian, J. (1980). Enzymatic Survey of 4 Populations of Atherina-Boyeri Based on Electrophoresis and the Occurrence of a Microsporidiosis. Journal of Fish Biology 16 , 149-157. Congiu, L., Rossi, R. & Colombo, G. (2002). Population analysis of the sand smelt Atherina boyeri (Teleostei Atherinidae), from Italian coastal lagoons by random amplified polymorphic DNA. Marine Ecology-Progress Series 229 , 279-289. Creech, S. (1991). An Electrophoretic Investigation of Populations of Atherina- Boyeri Risso, 1810 and a-Presbyter Cuvier, 1829 (Teleostei, Atherinidae) - Genetic-Evidence in Support of the 2 Species. Journal of Fish Biology 39 , 807- 816. Creech, S. (1992). A Multivariate Morphometric Investigation of Atherina-Boyeri Risso, 1810 and a-Presbyter Cuvier, 1829 (Teleostei, Atherinidae) - Morphometric Evidence in Support of the 2 Species. Journal of Fish Biology 41 , 341-353. Cunha, M. M. P. R. d. (1990). Caracterização da comunidade de macroinvertebrados bênticos e estudo das condições ambientais na zona do Areão (ria de Aveiro, canal de Mira) In Biology , p. 93 pp. Aveiro: Universidade de Aveiro.

Capítulo 1

Dias, J. M., Lopes, J. F. & Dekeyser, I. (2000a). Tidal propagation in Ria de Aveiro Lagoon, Portugal. Physics and Chemistry of the Earth, Part B: Hydrology, Oceans and Atmosphere 25 , 369-374. Dias, J. M., Lopes, J. F. & Dekeyser, I. (2001). Lagrangian transport of particles in Ria de Aveiro lagoon, Portugal. Physics and Chemistry of the Earth, Part B: Hydrology, Oceans and Atmosphere 26 , 721-727. Dias, J. M. A., Boski, T., Rodrigues, A. & Magalhaes, F. (2000b). Coast line evolution in Portugal since the Last Glacial Maximum until present - a synthesis. Marine Geology 170 , 177-186. Duarte, H., Menezes Pinheiro, L., Curado Teixeira, F. & Monteiro, J. (2007). High- resolution seismic imaging of gas accumulations and seepage in the sediments of the Ria de Aveiro barrier lagoon (Portugal). Geo-Marine Letters 27 , 115-126. Fernandez-Delgado, C. J. A., Hernando, M. H. & M., B. (1988). Life history patterns of the sand smelt Atherina boyeri Risso 1810 in the estuary of the Guadalquivir River, Spain. Estuar. Coast. Shelf Sci. 27 , 697-706. Figueiredo da Silva, J., Duck, R. W., Hopkins, T. S. & Rodrigues, M. (2002). Evaluation of the nutrient inputs to a coastal lagoon: the case of the Ria de Aveiro, Portugal. Hydrobiologia 475-476 , 379-385. Francisco, S. M., Cabral, H., Vieira, M. N. & Almada, V. C. (2006). Contrasts in genetic structure and historical demography of marine and riverine populations of Atherina at similar geographical scales. Estuarine, Coastal and Shelf Science 69 , 655-661. Gon, O. & Bentuvia, A. (1983). The Biology of Boyer Sand Smelt, Atherina-Boyeri Risso in the Bardawil Lagoon on the Mediterranean Coast of Sinai. Journal of Fish Biology 22 , 537-547. Henderson, P. A., Turnpenny, A. W. H. & Bamber, R. N. (1984). Long-Term Stability of a Sand Smelt (Atherina-Presbyter Cuvier) Population Subject to Power- Station Cropping. Journal of Applied Ecology 21 , 1-10. Koutrakis, E. T., Kamidis, N. I. & Leonardos, I. D. (2004). Age, growth and mortality of a semi-isolated lagoon population of sand smelt, Atherina boyeri (Risso, 1810) (Pisces : Atherinidae) in an estuarine system of northern Greece. Journal of Applied Ichthyology 20 , 382-388.

Capítulo 1

Koutrakis, E. T., Tsikliras, A. C. & Sinis, A. I. (2005). Temporal variability of the ichthyofauna in a Northern Aegean coastal lagoon (Greece). Influence of environmental factors. Hydrobiologia 543 , 245-257. Leonardos, I. & Sinis, A. (2000). Age, growth and mortality of Atherina boyeri Risso, 1810 (Pisces: Atherinidae) in the Mesolongi and Etolikon lagoons (W. Greece). Fisheries Research 45 , 81-91. Leonardos, I. D. (2001). Ecology and exploitation pattern of a landlocked population of sand smelt, Atherina boyeri (Risso 1810), in Trichonis Lake (western Greece). Journal of Applied Ichthyology 17 , 262-266. Lopes, J. F. & Dias, J. M. (2007). Residual circulation and sediment distribution in the Ria de Aveiro lagoon, Portugal. Journal of Marine Systems In Press, Corrected Proof . Lopes, J. F., Dias, J. M., Cardoso, A. C. & Silva, C. I. V. (2005). The water quality of the Ria de Aveiro lagoon, Portugal: From the observations to the implementation of a numerical model. Marine Environmental Research 60 , 594- 628. Lorenzo, J. M. & Pajuelo, J. G. (1999). Age and growth of the sand smelt Atherina (Hepsetia) presbyter Cuvier, 1829 in the Canary Islands (Central-east Atlantic). Fisheries Research 41 , 177-182. Moreno, T. & Castro, J. J. (1995). Community structure of the juvenile of coastal pelagic fish species in the Canary-Islands waters Scientia Marina 59 , 405-413. Moreno, T., Castro, J. J. & Socorro, J. (2005). Reproductive biology of the sand smelt Atherina presbyter Cuvier, 1829 (Pisces: Atherinidae) in the central-east Atlantic. Fisheries Research 72 , 121-131. Moreno, T. & Morales-Nin, B. (2003). Age determination and validation on otoliths of the sand-smelt Atherina presbyter (Cuvier, 1829) (Pisces: Atherinidae) from the central-east Atlantic. Fisheries Research 62 , 77-87. Morgado, F. M. R. (1997). Ecologia do zooplâncton da ria de Aveiro: caracterização espaço-temporal, transporte longitudinal e dinâmica tidal, nictemeral e lunar. In Biology p. 427 pp. Aveiro: Universidade de Aveiro. Pajuelo, J. G. & Lorenzo, J. M. (2000). Biology of the Sand Smelt, Atherina presbyter (Teleostei: Atherinidae), Off the Canary Islands (central-east Atlantic). Environmental Biology of Fishes 59 , 91-97.

Capítulo 1

Pajuelo, J. G. & Nespereira, J. M. L. (2001). Aspectos de lá biología reproductora del pejerrey o guelde blanco Atherina presbyter Cuvier, 1829 (Atherinidae) en Gran Canaria (islas Canarias). Bol. Inst. Esp. Oceanogr. 17 (3 e 4) , 239-244. Palmer, C. J. & Culley, M. B. (1983). Aspects of the Biology of the Sandsmelt Atherina-Boyeri Risso, 1810 (Teleostei, Atherinidae) at Oldbury-Upon-Severn, Gloucestershire, England. Estuarine Coastal and Shelf Science 16 , 163-172. Pires, A. (1999). Aspectos da Ecologia de peixes e macroinvertebrados em rios intermitentes da bacia do Guadiana. In Zoologia e Antropologia , p. 177. Lisboa: Faculdade de Ciências da Universidade de Lisboa. Pombo, L. (1998). A Ictiofauna da Ria de Aveiro - Estrutura, Dinâmica e Populações. In Biologia , p. 126. Aveiro: Universidade de Aveiro. Pombo, L. (2005). Diversidade, dinâmica de populações e capacidade de produção ictiológica numa laguna costeira - a Ria de Aveiro. In Biologia , p. 272. Aveiro: Universidade de Aveiro. Pombo, L., Elliott, M. & Rebelo, J. E. (2002). Changes in the fish fauna of the Ria de Aveiro estuarine lagoon (Portugal) during the twentieth century. Journal of Fish Biology 61 , 167-181. Pombo, L., Elliott, M. & Rebelo, J. E. (2004). The structure and functioning of fish communities in the Ria de Aveiro, Portugal - temporal and spatial variations and the influence of abiotic factors. Cahiers De Biologie Marine 45 , 197-211. Pombo, L., Elliott, M. & Rebelo, J. E. (2005). Ecology, age and growth of Atherina boyeri and Atherina presbyter in the Ria de Aveiro, Portugal. Cybium 29 , 47-55. Pombo, L., Rebelo, J. & Elliott, M. (2007). The structure, diversity and somatic production of the fish community in an estuarine coastal lagoon, Ria de Aveiro (Portugal). Hydrobiologia 587 , 253-268. Rebelo, J. E. (1992). The Ichthyofauna and Abiotic Hydrological Environment of the Ria-De-Aveiro, Portugal. Estuaries 15 , 403-413. Rosecchi, E. & Crivelli, A. J. (1992). Study of a sand smelt ( Atherina boyeri Risso 1810) population reproducing in fresh water. Ecology of Freshwater Fish 1, 77-85. Schultz, E. T. & Conover, D. O. (1997). Latitudinal differences in somatic energy storage: Adaptive responses to seasonality in an estuarine fish (Atherinidae: Menidia menidia). Oecologia 109 , 516-529. Shi, X., Zhang, S. & Pang, Q. (2006). Vitellogenin is a novel player in defense reactions. Fish & Shellfish Immunology 20 , 769-772.

Capítulo 1

Tomasini, J. A., Collart, D. & Quignard, J. P. (1996). Female reproductive biology of the sand smelt in brackish lagoons of southern France. Journal of Fish Biology 49 , 594-612. Tomasini, J. A. & Laugier, T. (2002). Male reproductive strategy and reserve allocation in sand smelt from brackish lagoons of southern France. Journal of Fish Biology 60 , 521-531. Trabelsi, M., Kartas, F. & Quignard, J. P. (1994). Comparaison du régime alimentaire d'une population marine et d'une population lagunaire d' Atherina boyeri des côtes tunisiennes. Vie Milieu 44 (2) , 117-123. Turnpenny, A. W. H., Bamber, R. N. & Henderson, P. A. (1981). Biology of the sand-smelt ( Atherina presbyter valenciennes ) around Fawley power-station. Journal of Fish Biology 18 , 417-427. Tutman, P., Glamuzina, B., Skaramuca, B., Kozul, V., Glavic, N. & Lucic, D. (2000). Incidence of spinal deformities in natural populations of sandsmelt, Atherina boyeri (Risso, 1810) in the Neretva river estuary, middle Adriatic. Fisheries Research 45 , 61-64. Vizzini, S. & Mazzola, A. (2002). Stable carbon and nitrogen ratios in the sand smelt from a Mediterranean coastal area: feeding habits and effect of season and size. Journal of Fish Biology 60 , 1498-1510. Vizzini, S. & Mazzola, A. (2005). Feeding ecology of the sand smelt Atherina boyeri (Risso 1810) (Osteichthyes, Atherinidae) in the western Mediterranean: evidence for spatial variability based on stable carbon and nitrogen isotopes. Environmental Biology of Fishes 72 , 259-266. Whitehead, P. J. P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J. & Tortonese, E. (1984). Fishes of the North-Eastern Atlantic and the Mediterranean . Paris. Whitfield, A. K. (1999). Ichthyofaunal assemblages in estuaries: A South African case study. Reviews in Fish Biology and Fisheries 9, 151-186. Wootton, R. J. (1999). Ecology of Teleost Fishes . London.

Conclusão Geral

Conclusão Geral A população de peixe-rei do Canal de Mira da Ria de Aveiro é constituída por Atherina presbyter e Atherina boyeri . A primeira prefere locais de salinidade mais elevada e, como tal, mais perto da boca do estuário, no entanto é também encontrada em locais de água salobra. A. boyeri tem preferência por locais mais perto do fim do canal (água doce ou salobra). As espécies coexistem em salinidades entre 5,5 e 22, com uma clara dominância de A. presbyter em termos de abundância. Neste estudo é fornecida informação sobre alguns parâmetros biológicos da população de peixe-rei, do canal de Mira, incluindo o comprimento total, GSI e alguns factores de condição (somática, do fígado, do tubo digestivo e eviscerado), ao longo de um gradiente temporal e de salinidade. O gradiente sazonal é claro em ambas as espécies (ressalvando o facto de, para A. boyeri , o reduzido número de capturas não ter permitido uma análise estatística) e em praticamente todos os parâmetros, ocorrendo uma redução do crescimento, GSI, reservas energéticas, condição somática e nutrição durante os períodos de condições mais adversas (Outono e Inverno). Estas diferenças acompanham não só a sazonalidade característica deste habitat, como também o ciclo de vida das espécies em questão. Relativamente à influência da salinidade em A. presbyter e A. boyeri , não foi possível retirar conclusões devido aos resultados inconsistentes obtidos para a primeira espécie, e ao reduzido número de capturas de A. boyeri . No caso particular de A. presbyter , foram também detectadas diferenças entre machos e fêmeas do Canal de Mira, sendo que as fêmeas evidenciam melhor condição somática e mais reservas energéticas que os machos, sugerindo uma melhor capacidade de adaptação às condições de produtividade e flutuações do estuário. Nesta espécie, durante a maior parte do ano, predominam as fêmeas com excepção da Primavera possivelmente devido à aproximação da época reprodutora. A população de A. boyeri apresenta igual proporção de machos e fêmeas. O presente trabalho confirma também o facto de A. presbyter apresentar um longo período reprodutor (Primavera e Verão), característico de peixes com desovas múltiplas (Tomasini et al. , 1996). Para além disso, é possível concluir

Conclusão Geral que a população juvenil desta espécie no Canal de Mira reproduz-se no ano seguinte após o nascimento. A distinção entre estas duas espécies, até mais recentemente, era realizada apenas com base em caracteres morfológicos, tais como número de escamas ao longo da linha longitudinal e presença ou ausência de dentes nos ossos ectopterigóides (Whitehead et al. , 1984). No entanto Almada et al. (submitted ) propõe o uso de técnicas moleculares para a identificação destes peixes. O presente trabalho confirma a utilidade da aplicação conjunta destas técnicas, já que a contagem de escamas da linha longitudinal, além de poder ser falaciosa, nomeadamente para A. presbyter , é bastante difícil em indivíduos de pequenas dimensões e/ou danificados aquando da sua captura. Assim, é aqui proposto uma identificação usando conjuntamente caracteres morfológicos e técnicas moleculares. Para além de colmatar a falta de informação existente acerca das espécies da família Atherinidae nas águas portuguesas, nomeadamente ao nível de aspectos fisiológicos, o este trabalho reforça a importância de obter dados acerca dos efectivos populacionais dos peixe-rei. Tendo em conta os resultados obtidos no presente estudo, há um claro desequilíbrio entre as populações de A. presbyter e A. boyeri . Além do mais, para o caso particular desta última espécie, é urgente perceber se a população da Ria de Aveiro está ameaçada ou não, já que comparando com o número de capturas efectuadas há cerca de 10 anos (Pombo et al., 2005), a população pode estar em regressão. Propõe-se então um estudo mais rigoroso e contínuo ao nível da densidade e estrutura da população dos peixe-rei em todo o estuário. Trabalhos futuros poderão também passar pelo estudo da dieta e estado de maturação sexual das espécies em questão, permitindo obter-se informações complementares acerca da sua nutrição e conclusões mais específicas acerca do ciclo de vida dos peixes-rei na Ria de Aveiro.

Conclusão Geral

BIBLIOGRAFIA

Almada, V. C., Domingues, V., Cabral, H., ALMADA, F., Robalo, J. & Francisco, S. M. ( submitted ). A rapid and inexpensive molecular technique to descriminate between Atherina boyeri and A. presbyter around the Iberian Peninsula and its potential applications on ecology and larval identification. Pombo, L., Elliott, M. & Rebelo, J. E. (2005). Ecology, age and growth of Atherina boyeri and Atherina presbyter in the Ria de Aveiro, Portugal. Cybium 29 , 47-55. Tomasini, J. A., Collart, D. & Quignard, J. P. (1996). Female reproductive biology of the sand smelt in brackish lagoons of southern France. Journal of Fish Biology 49 , 594-612. Whitehead, P. J. P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J. & Tortonese, E. (1984). Fishes of the North-Eastern Atlantic and the Mediterranean . Paris.