INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA - INPA...

INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA - INPA

PROGRAMA DE PÓS-GRADUAÇÃO DO INPA

PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA DE ÁGUA DOCE

E PESCA INTERIOR

COMPOSIÇÃO QUÍMICA DE OTÓLITOS DE TUCUNARÉ-AÇU

(Cichla temensis HUMBOLT, 1821) E SEU POTENCIAL COMO

MARCADOR AMBIENTAL EM POPULAÇÕES DE LAGOS DE

VÁRZEA E IGAPÓ NA AMAZÔNIA-BRASILEIRA

RANIERE GARCEZ COSTA SOUSA

Manaus, Amazonas

Fevereiro/2014

ii

RANIERE GARCEZ COSTA SOUSA

COMPOSIÇÃO QUÍMICA DE OTÓLITOS DE TUCUNARÉ-AÇU

(Cichla temensis HUMBOLT, 1821) E SEU POTENCIAL COMO

MARCADOR AMBIENTAL EM POPULAÇÕES DE LAGOS DE

VÁRZEA E IGAPÓ NA AMAZÔNIA-BRASILEIRA

Orientador: CARLOS EDWAR DE CARVALHO FREITAS, PhD.

Tese apresentada ao Programa de Pós-

Graduação em Biologia de Água Doce e Pesca Interior

do INPA, como parte dos requisitos para obtenção do

título de Doutor em Ciências Biológicas, área de

concentração Biologia de Água Doce e Pesca Interior.

Manaus, Amazonas

Fevereiro/2014

Fontes financiadoras: Projeto PRONEX, (Edital 023/2009) – FAPEAM; Programa Institucional de

Doutorado Sanduíche no Exterior – PDSE/CAPES; Instituto Nacional de Pesquisas na Amazônia –

INPA; Instituto de Inteligência Socioambiental Estratégica da Amazônia, I-PIATAM; Conselho

Nacional de Desenvolvimento Científico e Tecnológico – CNPq e Washington and Lee University –

WLU, U.S.A.

iii

Ficha catalográfica

S725 Sousa, Raniere Garcez Costa

Composição química de otólitos de tucunaré-açu (cichla

temensis humbolt, 1821) e seu potencial como marcador ambiental

em populações de lagos de várzea e igapó na Amazônia-brasileira /

Raniere Garcez Costa Sousa. --- Manaus: [s.n.], 2014.

xiii, 91 f. : il. color.

Tese (Doutorado) --- INPA, Manaus, 2014.

Orientador : Carlos Edwar de Carvalho Freitas.

Área de concentração : Biologia de Água Doce e Pesca Interior.

1. Microquímica de otólitos. 2. Tucunaré-açu. 3. Ecologia de

populações. I. Título.

CDD 597.58

Sinopse: Este estudo investiga o uso de isótopos de estrôncio e

elementos traços como marcadores ambientais em otólitos de

tucunarés-açu Cichla temensis, para a identificação dos locais de

nascimento de indivíduos jovens e adultos. Também, baseado na

microquímica dos otólitos e geologia, avalia a distribuição espacial,

padrões metapopulacionais e de filopatria de indivíduos adultos em

tributários da Bacia do Médio Rio Negro.

Palavras-chave: Microquímica de otólitos; isótopos e elementos

traços; movimentos de dispersão; ecologia de populações.

iv

Dedicatória

À Joyce Lara Garcez e meus filhotes, pelo amor

e carinho, fontes de motivação para continuar

sempre otimista na minha jornada.

v

AGRADECIMENTOS

A Deus pela oportunidade da vida.

Ao Professor Carlos Edwar de Carvalho Freitas, meu amigo e orientador nesta

tese, por sua integridade, paciência e companheirismo, os quais me incentivaram a

persistir e não fraquejar nos momentos difíceis, e, principalmente, pela imensa

dedicação na orientação dessa pesquisa, fato impossível de ser mensurado.

Ao Professor Robert Humston, meu supervisor durante o doutorado sanduiche

na Washington and Lee University, o qual me ajudou a desenvolver os modelos

estatísticos e de análises químicas de otólitos.

Ao Instituto Nacional de Pesquisas da Amazônia - INPA, através do Programa

de Pós Graduação em Biologia de Água doce e Pesca Interior - BADPI - pela ímpar

oportunidade de capacitação profissional que me foi concedida.

Aos órgãos de fomento: Conselho Nacional de Desenvolvimento Científico e

Tecnológico - CNPq e Programa Institucional de Doutorado Sanduíche no Exterior -

PDSE/CAPES, os quais, através de suas bolsas de estudos e auxílios foram de

fundamental importância para a provisão da minha subsistência durante este trabalho,

quando do meu estágio tanto aqui no Brasil como no exterior. Também ao Instituto

Brasileiro de Meio Ambiente – IBAMA - pelas licenças concedidas para a coleta e

transporte de materiais em campo.

A todos os integrantes do Instituto de Inteligência Socioambiental Estratégica da

Amazônia - I-PIATAM - especialmente aos amigos Caroline Campos, Antônio Oliveira,

Michel Catarino, Flávia Souza e Hostília Campos, os quais estiveram sempre dispostos

a ajudar-me em momentos pouco auspiciosos.

Ao amigo e compadre Júlio Alberto Dias Siqueira, por sua valiosa e rara

amizade, bem como pelo apoio concedido nas coletas de dados que tive que

empreender.

A todos os auxiliares de campo, mateiros e pescadores que foram parte

imprescindível deste trabalho, cujos nomes não foram anotados. Mas que anonimamente

deram suas parcelas de contribuição.

vi

Aos meus filhotes, Adrielly, Ranielly e Raniere, bênçãos do Criador em minha

vida, nos quais encontrei estímulo para lutar e motivação para perseverar na minha

caminhada.

À minha querida esposa Joyce Lara Araújo da Fonseca Garcez, que sempre

esteve ao meu lado como uma guerreira, principalmente quando, por meses, tive que

ausentar-me do lar em excursões ao exterior do país. Que Deus me conceda a graça de

tê-la por toda a vida.

vii

Salmos 37:5

Entrega o teu caminho ao SENHOR

confia Nele, e o mais Ele fará.

Salmos de Davi

Bíblia Sagrada

viii

RESUMO

Neste estudo foi avaliada a aplicação da geoquímica de otólitos como marcador

ambiental da origem de nascimento de tucunarés-açu Cichla temensis (Humboldt,

1833), jovens menores que um ano (YOY) e de indivíduos adultos, na bacia do Rio

Negro. Inicialmente, foram medidas as variações de isótopos de estrôncio e elementos

traços em otólitos de exemplares jovens em diferentes locais de coletas e comparados os

resultados coma geologia do local. Os resultados indicam que padrões na microquímica

dos otólitos dos peixes jovens, particularmente na razão isotópica 87Sr/86Sr,

correspondem aos padrões da base geológica do local de nascimento. Isto permitiu uma

classificação correta de 99% dos peixes juvenis com seus rios de origem, usando uma

tabela de correlação da análise de função discriminante linear (LDFA). Os resultados

mostram também que é possível usar inferências de mapas geológicos e assinatura

geoquímica dos otólitos para traçar movimentos de indivíduos adultos entre o Rio

Negro e seus tributários. A análise LDFA mostrou que a microquímica da parte central

dos otólitos dos tucunarés adultos classificou corretamente cada indivíduo com seu local

de nascimento, com 41,9% de acurácia. Ainda, a variação da razão isotópica 87Sr/86Sr da

linha transversal (do centro para a borda) da superfície dos otólitos dos tucunarés-açus

adultos, indicou a existência de populações isoladas e mistas de tucunarés-açu na área

do estudo. Os resultados também sugerem que os tucunarés adultos realizam

movimentos de dispersão entre os tributários estudados indicando um padrão

metapopulacional e filopátrico, percebidos com o retorno desses indivíduos aos seus

locais de nascimento, e pela permanência de outros nesses locais, mostrando fidelidade

ao sítio de origem. O presente estudo proporciona novas informações a respeito da

distribuição espacial do tucunaré-paca ou tucunaré-açu C. temensis em seu ambiente

natural, que poderiam ser usadas como apoio efetivo nas estratégias de gestão pesqueira

e fornecer informações básicas para trabalhos futuros em investigações sobre a história

inicial de vida e ecologia espacial de peixes na bacia Amazônica.

Palavras-chave: Manejo pesqueiro; geoquímica; assinatura digital química em otólitos;

isótopos de estrôncio; filopatria.

ix

ABSTRACT

This study examined the application of otolith geochemistry as a natural marker of natal

origins in peacock bass Cichla temensis (Humboldt, 1833) young-of-the-year (YOY)

and adults in the Negro River basin. Initially were assessed variations in strontium

isotopes and trace elements in otoliths of young individuals from different sampling

locations comparing the results with local geology. The results suggests that patterns in

otolith microchemistry of YOY, particularly 87Sr/86Sr isotopes, correspond to patterns in

origin bedrock geology. This approach allowed us to correctly classify 99% of juvenile

fish to their natal streams using a cross-validation table from the linear discriminant

function analysis (LDFA). The results also indicates that is possible to use inferences

from geologic maps to track movements of adults fish between Negro River and its

tributaries. The LDFA analyses shows that the microchemistry from the central part of

the adults peacock bass otolith, classified correctly each individual for its birth location

with 41.9% of accuracy. Also, variation in 87Sr/86Sr isotopic rate from the transect line

(from the core to the border) on the otolith surface of adults peacock bass, indicated the

existence of isolated and mixed populations in the study area. The results also suggests

that adult peacock bass ensure dispersal movements between studied tributaries showing

a metapopulational and philopatric pattern, noticed through the individuals returned to

their birth locations, and by the permanence of some individuals in those locations,

showing loyalty to their origin place. Therefore, the present report offers new

information about spatial distribution of C. temensis in their natural environment, which

could support effective strategies of fishery management, and provides the basic

information for future work to investigate the early life history and spatial ecology of

fresh water fish in the Amazon basin.

Keywords: Fish management; geochemistry; otolith chemical fingerprints; strontium

isotopes; philopatry.

x

SUMÁRIO

Introdução geral ............................................................................................................. 1

Biologia do tucunaré-açu Cichla temensis ....................................................................... 3

Mobilidade de dispersão de C. temensis ........................................................................... 4

Otólitos ............................................................................................................................. 5

Migração e microquímica de otólitos ............................................................................... 7

Distribuição da razão isotópica de 87Sr/86Sr no ambiente................................................. 7

Problema enfocado no estudo ........................................................................................ 9

Hipóteses ........................................................................................................................ 10

Objetivo geral ................................................................................................................ 11

Objetivos específicos ..................................................................................................... 12

Organização da Tese .................................................................................................... 12

Metodologia geral ......................................................................................................... 13

Área de estudo ................................................................................................................ 13

Coleta e análise dos dados .............................................................................................. 15

Capítulo I. Otolith geochemistry in young-of-the-year peacock bass Cichla temensis for

investigating natal dispersal in the Rio Negro (Amazon - Brazil) river system ............. 21

Abstract ......................................................................................................................... 23

Introduction .................................................................................................................. 24

Materials and Methods ................................................................................................ 25

Study Area ...................................................................................................................... 25

Sample collection ........................................................................................................... 29

Otolith preparation and analysis ..................................................................................... 29

Statistical analysis .......................................................................................................... 30

Geologic analyses ........................................................................................................... 31

xi

Results ............................................................................................................................ 32

Discussion ...................................................................................................................... 41

Acknowledgments ......................................................................................................... 42

Capítulo II. Philopatry and metapopulation patterns for the peacock bass Cichla

temensis of the middle Negro River Basin (Amazonas - Brazil): an otolith chemical

analysis. .......................................................................................................................... 43

Abstract ......................................................................................................................... 45

Introduction .................................................................................................................. 46

Material and Methods .................................................................................................. 48

Study area ....................................................................................................................... 48

Sample collections and preparations .............................................................................. 50

Strontium isotope and elemental analysis ...................................................................... 51

Water data collection and analysis ................................................................................. 51

Otoliths statistical analyses ............................................................................................. 52

Results ............................................................................................................................ 53

Young-of-year (YOY) and adult peacock bass otolith core microchemical analyses .... 53

Adult peacock bass otolith core chemical analysis ........................................................ 56

87Sr/86Sr isotope ratio analysis from otolith transects of adults peacock bass ................ 59

Water chemical analysis ................................................................................................. 62

Discussion ...................................................................................................................... 63

Cichla temensis movements, reproduction and phylopatric behaviour synchronism. ... 66

Hydrological barrier and C. temensis metapopulation structure .................................... 67

Peacock bass fisheries management proposition ............................................................ 72

Acknowledgments ......................................................................................................... 73

Conclusões ..................................................................................................................... 74

Referências Bibliográficas ........................................................................................... 75

xii

LISTA DE FIGURAS

Introdução geral

Figura 1. Exemplar de tucunaré-açu Cichla temensis, capturado na região do Rio

Padauari em 2012............................................................................................................17

Figura 1. Exemplar de tucunaré-açu Cichla temensis, capturado na região do Rio

Padauari em 2012...........................................................................................................19

Figura 3. Localização dos locais amostrais na região do Médio Rio Negro, município de

Barcelos-Amazonas…………………………………………………………………….27

Figura 4. Biometria e extração dos otólitos dos peixes adultos.......................................29

Figura 5. Material utilizado para a separação e montagem dos otólitos..........................30

Figura 6. Lixamento dos otólitos.....................................................................................30

Figura 7. Banheira ultrassônica para limpeza dos otólitos..............................................31

Figura 8. Acondicionamento dos otólitos em sacolas e caixas esterilizadas para

transporte……………………………………………………………………………….31

Figura 9. Coletor Múltiplo de Espectrometria de Massas por Plasma Indutivamente

Acoplado..........................................................................................................................32

Figura 10. Queima a laser da superfície do otólito..........................................................32

Capítulo I

Figure 1. Location and geological conditions of the study área......................................40

Figure 2. Mean isotopic and trace element concentration ratios in the otoliths of YOY

fish groups.......................................................................................................................49

Figure 3. Microchemical isotopic and trace element ratios from the otoliths of YOY C.

temensis distributed within groups and between areas....................................................52

Capítulo II

Figure 1. Study area and geographic distribution of sampling sites in the middle Negro

River, Barcelos Municipality...........................................................................................63

Figure 2. A scatter-plot displaying the distribution of Cichla temensis’ YOY and adult

otolith core chemical fingerprints....................................................................................68

Figure 3. Adult Cichla temensis peacock bass’ otolith core 87Sr/86Sr isotope ratio

distributed by sampling sites...........................................................................................71

xiii

Figure 4. Distribution of elemental compositions from adult Cichla temensis peacock

bass’ otolith core………………………………………………………………………..72

Figure 5. Cichla temensis peacock bass’ movement history from their birth to the

locations where they were caught as adult individuals, based on variation in 87Sr/86Sr

isotope ratio values on the otolith transect……………………………………………..73

Figure 6. Transect lines displaying Cichla temensis peacock bass’ movement histories

from their birth to catch locations………………………………………………………76

Figure 7. Organizational chart with C. temensis movements…………………………..79

Figure 8. The organogram illustrates C. temensis movement behaviour among different

water types in relation to differences in otoliths’ geochemical composition (87Sr/86Sr

isotopes)………………………………………………………………………………...83

Figure 9. Theoretical spatial movement distribution of Cichla temensis in the middle

Negro River basin………………………………………………………………………84

1

Introdução geral

A complexidade da pesca na Amazônia dificulta o pleno entendimento de alguns

pressupostos de modelos tradicionais para avaliação dos estoques pesqueiros, aumentando o

grau de incerteza dos resultados obtidos. Como regra geral, a aplicação dos modelos analíticos

é embasada em parâmetros de dinâmica populacional e ainda existem poucos estudos

conclusivos sobre a identificação das populações de peixes que habitam os ambientes

aquáticos amazônicos. Barthem e Petrere Jr. (1996) postularam, por meio de estudos de

crescimento, a existência de uma única população de piramutaba Brachyplatystoma vailantii

(Valenciennes, 1840) desde os trechos superiores dos tributários de águas brancas da calha do

rio Solimões/Amazonas até o estuário. No entanto, a maioria das espécies importantes para a

pesca comercial ainda são pouco estudadas, e não temos informações suficientes para afirmar

que estes indivíduos pertencem a uma única ou a várias populações (Bailey e Petrere, 1989;

Batista, 2001). Sendo assim, a falta de informações precisas sobre os comportamentos

migratórios das espécies se torna uma das principais limitações para a sua conservação, o que

precisa ser corrigido com mais pesquisas nessa área.

A descrição dos padrões de movimentos migratórios tem sido uma questão central na

biologia e manejo de peixes de água doce. As migrações entre os ambientes marinhos e de

água doce, realizadas por peixes anádromos e catádromos, são relativamente bem

conhecidas (Groot e Margolis, 1991, Walter e Thorrold, 2006), mas o mesmo não ocorre

com os movimentos migratórios de peixes de áreas continentais (Gowan et al., 1994;

Northcote, 1997).

Os processos de migração e dispersão dos peixes são considerados fatores

fundamentais na definição de populações e metapopulações (Hanski e Gilpin, 1997; Rieman

e Dunham, 2000). No entanto, para rastrear esses indivíduos entre ambientes distintos é

necessário o uso de alguns artifícios e metodologias. Dentre as metodologias mais utilizadas

se destacam a marcação e recaptura (Hoeinghaus et al., 2003), a telemetria (Semmens et al.,

2006) e, atualmente, o uso de marcadores ambientais (isótopos e elementos traços)

encontrados em otólitos e outras estruturas rígidas em peixes (Thorrold et al., 2001; Walther

et al., 2008).

2

Análises de isótopos e de elementos traços em otólitos têm apresentado resultados

mais acurados e completos na descrição retrospectiva da história dos movimentos de

dispersão de indivíduos e de grupos de peixes (Wells et al., 2003; Rich et al., 2004; Walther

et al., 2008; Walther e Thorrold, 2010), fornecendo informações robustas e confiáveis sobre

a dinâmica espacial de populações (Rieman et al. 1994; Humston & Harbor, 2006).

No entanto, a maioria dos estudos realizados com essa metodologia até o presente

momento, tem contemplado espécies de peixes que habitam ambientes marinhos ou

estuarinos (Gillanders 2002; Walther e Thorrold, 2010), onde é esperada uma grande

variação na concentração de solutos nos diferentes ambientes. Recentemente, alguns estudos

realizados em ambientes de águas interiores também têm mostrado bons resultados em

monitorar movimentos de peixes entre ambientes quimicamente distintos (Rich et al., 2004;

Humston et al., 2010; Johnson et al., 2012).

Na região amazônica, vários estudos sobre os processos migratórios de peixes foram

realizados (Ribeiro e Petrere Jr. 1990; Barthen et al., 1991; Freitas e Garcez, 2004).

Entretanto, devido às dificuldades em monitorar indivíduos pequenos (ou jovens) com os

modelos de marcação tradicionais, somados às adversidades encontradas em recapturar ou

acompanhar indivíduos em áreas remotas, a maioria desses estudos gerou resultados não

conclusivos. Assim, os isótopos de elementos traços detectados na composição de otólitos

de peixes, pode ser utilizados como marcadores ambientais naturais, para rastrear

movimentos migratórios de indivíduos que transitam entre ambientes quimicamente

diferentes (Johnson et al., 2012), e solucionar essa deficiência metodológica.

Nesse contexto, a análise química de otólitos de peixes pode contribuir para esclarecer

dúvidas relacionadas à existência de migração de indivíduos entre populações, melhorando o

entendimento dos parâmetros da dinâmica populacional de peixes. Isso poderá propiciar o

aprimoramento das estratégias atuais de manejo pesqueiro utilizadas na Região Amazônica,

principalmente nas áreas onde atuam várias modalidades de pesca sobre o mesmo recurso.

Na região do Rio Negro ocorrem quatro modalidades de pesca: a comercial, a

esportiva, a ornamental e a de subsistência (Sobreiro et al., 2010). Dentre estas, a que mais

vem crescendo nessa região é a pesca esportiva, com a modalidade pesque e solte (Holey et

al., 2008), a qual atua principalmente sobre as espécies de ciclídeos, com destaque para o

3

tucunaré-açu C. temensis, por apresentar maior tamanho e voracidade em relação aos seus

congêneres (Taphorn e Barbarino-Duque, 1993).

Alguns estudos sobre a distribuição espacial de espécies do gênero Cichla foram

realizadas na bacia do Rio Amazonas (Macrander 2010; Willis et al., 2007; 2010). No

entanto, os resultados apresentados por esses estudos não mostram claramente a existência

de movimentos de dispersão ou processos migratórios entre os diferentes sistemas aquáticos,

o que dificulta a compreensão adequada da dinâmica de movimentos dessas espécies na

região. Desse modo, também pouco se sabe sobre a distribuição espacial do tucunaré-açu C.

temensis na região do Médio Rio Negro, área onde a atividade da pesca esportiva ocorre

com maior intensidade.

Pelo exposto, e visando compreender a dinâmica de dispersão dos tucunarés-açu, e, ao

mesmo tempo, subsidiar informações que possam contribuir com o entendimento da

distribuição espacial dessa espécie na região do Médio Rio Negro, a presente pesquisa

analisou os isótopos de 87Sr/86Sr e a concentração dos elementos traços Sr/Ca e Ba/Ca em

otólitos de tucunaré-açu Cichla temensis, no intuito de identificar o local de nascimento de

juvenis menores de um ano e adultos. Este estudo verificou ainda, através das diferenças

químicas existentes nos otólitos dos tucunarés, variações indicando movimentos de

dispersão entre ambientes quimicamente distintos, o que, posteriormente, foi utilizado para

avaliar a existência ou não de uma estrutura de metapopulação de Cichla temensis na região.

Biologia do tucunaré-açu Cichla temensis

O tucunaré pertence à família Cichlidae, é peixe adaptado principalmente a ambientes

lênticos e ocorre naturalmente na América do Sul, em rios de água preta, clara ou branca

(Winemiller et al., 2008). Pertence ao gênero Cichla, que abriga 14 espécies válidas

(Kullander e Ferreira, 2006). Na região amazônica as espécies de tucunarés utilizadas na

pesca são: Cichla temensis Humbolt, 1821, C. ocellaris Bloch e Schneider, 1801, C.

orinocencis Humbolt, 1821, C. intermedia Machado-Allison, 1971 e C. monoculus Spix e

Agassiz, 1831.

4

O tucunaré-paca ou tucunaré-açu Cichla temensis (Figura 1) é um importante predador

de topo de cadeia alimentar em diversos ambientes aquáticos (Barros, 1980). Segundo Graef

(1995) o C. temensis atingem a idade adulta por volta dos dois anos, em ambiente natural. A

espécie possui desova parcelada, a qual ocorre durante todo o ano e se intensifica na época

da seca (Fontenele, 1950; Gomiero et al., 2009). É um peixe popular na pesca esportiva, mas

também tem alto valor na pesca comercial e de subsistência (Corrêa, 1998; Jepsen et al.,

1999). Por este motivo, conflitos são frequentes entre os diferentes usuários do recurso,

quando estes atuam nas mesmas áreas de exploração pesqueira (Sobreiro et al., 2010).

Figura 1. Exemplar de tucunaré-açu Cichla temensis, capturado na região do Rio Padauari em

2012.

Mobilidade de dispersão de C. temensis

Estudos indicam que o tucunaré Cichla temensis é uma espécie sedentária e

territorialista (Barros, 1980, Santos et al., 1984, Jepsen et al., 1999), por outro lado, alguns

estudos afirmam a existência de movimentos de dispersão desses indivíduos (Hoeinghaus et

al., 2003; Holley et al., 2008). Essa controvérsia nas informações causam incertezas quanto a

distribuição desses indivíduos dentro dos sistemas aquáticos amazônicos, e tampouco se estes

realizam movimentos territoriais entre populações distintas. Essa lacuna de informações

também se estende à maioria das espécies importantes para a pesca comercial no Amazonas

(Bailey e Petrere, 1989; Batista, 2001).

5

Pesquisas têm sido realizadas no intuito de compreender os mecanismos que regulam

o recrutamento e a ecologia espacial do tucunaré, particularmente em sistemas de rios em

áreas abertas de bacias hidrográficas brasileiras (Smith et al., 2005). A variabilidade de

recrutamento em estoques é geralmente alta, tornando-se visível a correlação com fatores

abióticos, particularmente descargas d'água e temperaturas (Smith et al., 2005).

Em recentes pesquisas realizadas na bacia amazônica sobre a dispersão gênica de

tucunarés, foi observada a separação de populações ao longo dos diversos tributários nessa

região (Macrander 2010; Willis et al., 2007; 2010). Estudos sobre marcação e recaptura

realizados com Cichla temensis, C. orinocensis e C. intermedia em rios da Venezuela

demonstraram que a maioria dos peixes recapturados estava dentro de um raio de 1 km, a

partir do ponto de sua marcação (Hoeinghaus et al., 2003). Por outro lado, indivíduos de C.

temensis marcados nesse mesmo estudo, foram recapturados com distâncias entre 17 a 21 km

(o tempo entre o período de marcação e a recaptura dos exemplares de C. temensis, não foi

divulgado no estudo). Holley et al. (2008) utilizaram a mesma metodologia de marcação e

recaptura com indivíduos de C. temensis na região do Médio rio Negro, e relataram que um

indivíduo dessa espécie foi recapturado a 40 km de distância de seu local de origem, após um

ano de sua marcação.

No entanto, estes esforços não foram suficientes para elucidar completamente o

processo de dispersão desses ciclídeos, o que torna necessária a realização de investigações

acerca dos padrões migratórios e/ou de movimentos de dispersão desses indivíduos entre

regiões distintas. Tais estudos poderão contribuir para um entendimento mais aprofundado

sobre a distribuição espacial de (sub)populações de tucunarés na região do Médio Rio Negro.

Otólitos

Os peixes teleósteos têm três pares de estruturas inorgânicas em seu sistema auditivo,

conhecidas como otólitos (lapillus, sagitta e asteriscus), que estão localizados atrás dos seus

globos oculares (Campana, 1999). Estas estruturas são formadas basicamente por carbonato

de cálcio (aragonita) que é sedimentado nos otólitos diretamente da química do ambiente

aquático onde vivem (Campana 1999; Bath et al., 2000; Campana e Thorrold, 2001; Walther

6

e Thorrold 2006). Os otólitos são metabolicamente inertes e crescem continuamente através

da sedimentação de finas camadas diárias (2 a 5 µm), que formam anéis concêntricos durante

toda a vida do peixe (Pannella, 1971; Gauldie, 1993; Lord et al., 2011).

Em muitos peixes, incluindo os ciclídeos, os otólitos sagitta são os maiores (Gaemers,

1984) e por essa razão são os preferidos em estudos de análises de anéis de crescimento e

microquímica (Pannella, 1980; Gomiero e Braga, 2007) (Figura 2).

Figura 2. Estrutura de um otólito Sagitta, retirado de um tucunaré-açu adulto Cichla temensis.

A= vista superior, B= vista da parte inferior e C= vista lateral esquerda. (Modificado de

Gomiero e Braga, 2007).

Os otólitos de peixes com idade de até duas semanas apresentam composição química

bastante específica, relacionada ao local de seu nascimento (Lyons e Kanehl, 2002). A

composição química destas estruturas sofre mudanças graduais durante a vida do indivíduo,

recebendo influências de outras áreas exploradas pelo peixe (Campana, 1999). Em função

disso, a composição química dos otólitos de indivíduos jovens pode ser utilizada como

registro do local de seu nascimento. Já o perfil dos otólitos dos peixes adultos, iniciado

próximo do centro do otólito até sua borda, pode ser utilizado para identificar os possíveis

ambientes por onde o peixe percorreu, e, desse modo, reconstruir as possíveis rotas

migratórias realizadas por indivíduos separadamente (Walter et al., 2008).

7

Migração e microquímica de otólitos

Estudos recentes sugerem que a heterogeneidade na geologia do canal principal de um

rio pode apresentar diferenças detectáveis na química dos otólitos dos peixes, se estes

percorrerem diferentes seções de um único rio ao longo de seu crescimento e vida adulta

(Wells et al., 2003). Do mesmo modo, a variação química entre os sistemas aquáticos pode

contribuir para a distinção entre os locais de nascimento de peixes entre rios com diferentes

características químicas da água (Campana, 1999).

As análises microquímicas de otólitos mostram que é possível correlacionar tais

características com diferenças limnológicas entre ambientes aquáticos, através dos registros

dos isótopos e elementos traços sedimentados nessa estrutura ao longo da vida do peixe

(Campana e Thorrold, 2001). Essas análises podem ser utilizadas para gerenciamento

estratégico de uma determinada espécie que apresente padrões migratórios ou de dispersão,

como troca de indivíduos entre duas ou mais áreas ou populações, os quais podem afetar o

resultado do recrutamento de indivíduos de uma determinada região (Bilby et al., 2001) e sua

distribuição espacial (Ridgeway et al., 2002).

O presente estudo teve como objetivo analisar populações de tucunaré-açu Cichla

temensis da região do médio Rio Negro, por meio da aplicação da metodologia de análise de

isótopos e elementos traços ambientais encontrados em otólitos de peixes, procurando

contribuir com dados mais precisos sobre os movimentos inter-habitats para essa espécie. A

detecção desses movimentos é possível caso existam diferenças químicas significativas entre

os ambientes percorridos pelo peixe (Wells et al., 2003), uma vez que a química dos otólitos

registra com precisão os movimentos de saída e/ou de retorno desses indivíduos, desde o seu

local de nascimento/origem até o local de sua captura (Guido et al., 2006).

Distribuição da razão isotópica de 87Sr/86Sr no ambiente

Estrôncio (Sr) é um elemento traço encontrado na maioria das rochas ígneas,

metamórficas e sedimentares. Está distribuído em vários tipos de matérias e ambientes, como

8

na água, no solo, nas plantas e em animais (Slovak e Paytan, 2011). Naturalmente o Estrôncio

ocorre distribuído em quatro formas estáveis (84Sr, 86Sr, 87Sr, e 88Sr), das quais três não são

radioativas: 84Sr (0,56 %), 86Sr (9,87%) e 88Sr (82,53%). O isótopo 87Sr (7,04%) é radiogênico

(radioativo) e é formado pela queda da radioatividade do rubídio 87Rb (Faure e Mensing,

2005). Devido à seus valores proporcionais no ambiente terrestre o 87Sr e o 86Sr podem ser

correlacionados, para normalizar a distribuição do 87Sr em substituição ao rubídio,

representado na forma do isótopo de 87Sr/86Sr.

Faure (2001) relata que a proporção média do isótopo de 87Sr/86Sr na crosta

continental é de 0,70916. Entretanto, estudos recentes encontraram valores isotópicos de

87Sr/86Sr originários de duas medidas de referência: a Eimer e Amend (E&A) com valor de

87Sr/86Sr de 0,70800, e a U.S. National Bureau of Standards-NBS987, apresentando valor de

0,71025. Os mais altos valores de 87Sr/86Sr foram encontrados em rochas muitos antigas, tais

como granitos, que geralmente apresentam altos valores de Rb/Sr com valores de 87Sr/86Sr

acima de 0,710 (Faure 2001). Ao contrário, rochas jovens apresentam baixos valores de Rb/Sr

e também valores de 87Sr/86Sr menores que 0,704 (Faure 1977; Capo et al. 1998; Slovak e

Paytan 2011).

Uma vez que os valores de 87Sr/86Sr variam muito pouco quando passam das rochas

lixiviadas indo para o solo, de onde são absorvidos pela vegetação e posteriormente pela

cadeia alimentar (Graustein e Armstrong, 1983; Miller et al., 1993), seus valores não se

alteram significativamente nesse processo, e, consequentemente, preservam os valores

originais de suas rochas matrizes (Hurst e Davis 1981; Faure, 1986; Åberg et al., 1995;

Wakabayashi et al., 2007; Ruggeberg et al., 2008). Desse modo, o isótopo de 87Sr/86Sr pode

ser utilizado como marcador ambiental em estudos de movimentos de dispersão de animais

terrestres e/ou aquáticos que tenham incorporado esses elementos em suas estruturas rígidas

(Capo et al., 1998; Elsdon et al., 2008; Turner e Limburg, 2012).

9

Problema enfocado no estudo

Estudos como os de Hoeinghaus et al. (2003) e Holley et al. (2008), mostraram que os

ciclídeos, principalmente o C. temensis, realizam movimentos de dispersão consideráveis

entre os tributários de grandes rios, como o Rio Cinaruco (Venezuela) e o Rio Negro (Brasil).

Por outro lado, diversas pesquisas apontam o tununaré-açu como uma espécie sedentária

(Winemiller, 2001; Granado-Lorencio et al., 2005). Essa polêmica sobre o tucunaré-açu ser

ou não ser um peixe sedentário, é reforçada quando Macrander (2010) sugere a existência de

barreiras hidroquímicas que impedem o fluxo de indivíduos de C. temensis entre as bacias dos

rios Orinoco (Venezuela) e Rio Negro. Será que os indivíduos de tucunarés que não realizam

seus movimentos de dispersão entre os ambientes ondem residem é devido as barreiras

hidroquímicas ou porque são de fato sedentários? Ainda, se realizam movimentos de

dispersão entre ambientes distintos, formam estruturas metapopulacionais?

Procurando entender a dinâmica de dispersão do tucunaré-açu Cichla temensis, a

presente pesquisa, se propôs a estudar os movimentos de dispersão da espécie C. temensis na

bacia do Médio Rio Negro. Por haver nessa região vários tributários do Rio Negro, com tipos

distintos de água, onde habitam a espécie em questão. Alguns tributários desta região, foram

selecionados para a presente pesquisa por formarem pares de rios conectados entre si, mas

serem compostos por diferentes tipos de água. Por exemplo, o Rio Aracá (água preta) e o

Demeni (água branca), o Rio Preto (água preta) e o Padauari (água branca). A exceção foi o

Rio Cuiuni (água preta) que é diretamente conectado com o Rio Negro (água preta). No

entanto o Rio Cuiuni, apresenta sua bacia de drenagem situada em região geologicamente

diferenciada do leito do canal principal do Rio Negro. Outro fator importante dos tributários

dessa região é que eles drenam bacias com geologias distintas, o que esperamos ser visível na

microquímica dos otólitos dos exemplares de tucunaré-açu ali coletados.

Além disso, o presente estudo verifica se as diferenças microquímicas dos ambientes

registradas nos otólitos, podem indicar a formação de barreiras hidroquímicas entre os

tributários com diferentes tipos de água (água preta vs água branca), o que poderia indicar a

existências de populações isoladas localmente ou de metapopulações, quando conectadas pelo

fluxo de entrada e saída de indivíduos procedentes de outros tributários. Com esse panorama

ambiental surgiu a pergunta chave para o presente trabalho. Existem movimentos de dispersão

10

de tucunarés-açu Cichla temensis que mostram padrões de metapopulações entre o Rio Negro

e seus tributários (rios Cuiuni, Aracá, Demeni, Preto e Padauari)? É possível verificar esses

movimentos de dispersão comparando as diferenças microquímicas dos otólitos de tucunaré-

açu com os tipos de geologia das áreas por eles habitadas?

Sabe-se que os ciclídeos fazem pequenos deslocamentos entre ambientes, entretanto

ainda pouco se conhece sobre os padrões de movimentos de dispersão entre essas populações.

No entanto, estudos realizados sobre espécies de peixes que habitam ambientes de água doce

(Bataille & Bowen 2012; Hegg et al. 2013), mostram que é possível a verificação da origem

dessas populações de peixes através de registros químicos existentes em otólitos relacionados

aos ambientes em que estas espécies vivem ou viveram. Informações sobre a dinâmica de

dispersão de indivíduos de uma mesma população e implicações da existência de estrutura

metapopulacional para espécies de peixes de interesse múltiplos, como o C. temensis pode

contribuir para a criação de estratégias de manejo para essa espécie. Hoje em dia, existem

algumas tentativas de manejo participativo entre os usuários do recurso na região de Barcelos

no Amazonas, comunidades ribeirinhas, pescadores comerciais e pescadores esportivos. No

entanto, essas tentativas de acordo de pesca não tiveram êxito (Sobreiro et al., 2010). Nesse

sentido, se faz necessário produzir informações sobre a distribuição espacial do tucunaré-açu

que possa auxiliar no manejo efetivo das pescarias dessa espécie na região do Médio Rio

Negro.

Hipóteses

H0 = A composição química de otólitos não é um marcador natural do local de

nascimento de tucunaré-acú jovens e adultos;

H1 = A composição química de otólitos é um marcador natural do local de nascimento

de tucunaré-acú jovens e adultos;

11

H0 = As populações de tucunaré-açu Cichla temensis dos rios Cuiuni, Aracá, Demeni,

Preto, Padauari e Negro não são conectadas entre si através de movimentos de dispersão entre

diferentes ambientes;

H1 = As populações de tucunarés Cichla temensis dos rios Cuiuni, Aracá, Demeni,

Preto, Padauari e Negro, são conectadas entre si através de movimentos de dispersão entre

diferentes ambientes.

Objetivo geral

Verificar se a composição microquímica de otólitos atua como marcador ambiental do

local de nascimento de tucunaré-acú jovens e adultos e se existem populações diferentes dessa

espécie para os rios Cuiuni, Aracá, Demeni, Preto, Padauari e Negro.

Nesse contexto, esta pesquisa propõe:

1) Apresentar informações que indiquem a região de nascimento dos indivíduos de

tucunaré-açu e a distribuição espacial de suas populações, dentro do sistema aquático

estudado, utilizando isótopos e elementos traços acumulados temporalmente nos otólitos dos

peixes; e

2) Analisar a aplicação dos resultados dessa pesquisa para o estudo da dispersão e o

intercâmbio dos tucunaré-açu adultos entre os rios Cuiuni, Aracá, Demeni, Preto, Padauari e

Negro.

12

Objetivos específicos

a) Investigar a geoquímica de otólitos como potencial marcador natural do local

de nascimento de peixes jovens; ao longo dos rios Cuiuni, Aracá, Demeni, Preto, Padauari e

Negro na região do Médio Rio Negro;

b) Identificar o local de nascimento dos tucunarés com menos de um ano e

também de tucunarés adultos, coletados nesses diferentes locais, analisando a microquímica

da parte central dos otólitos;

c) Avaliar os padrões de movimentos de dispersão de indivíduos de tucunaré-açu

entre os ambientes de águas preta ou branca, através da variação microquímica do centro até a

borda dos otólitos, que possam indicar a ocorrência de metapopulação dessa espécie na

região; e

d) Sugerir estratégias de gestão do(s) estoque(s) de tucunaré-açu, incorporando

informações sobre a estrutura espacial e padrões de movimentos de dispersão entre os rios

acima citados.

Organização da Tese

O Capítulo I apresenta um estudo sobre a distribuição espacial dos berçários de

tucunarés-açu jovens, com menos de um ano, em diferentes tributários de águas brancas e

pretas na região do Médio Rio Negro. Esse estudo foi desenvolvido utilizando informações de

mapas geológicos relacionados aos tipos de sedimentos encontrados na região do Médio Rio

Negro. Os dados sobre a geologia da área do estudo foram posteriormente utilizados para

identificar a distribuição espacial dos isótopos de 87Sr/86Sr e dos elementos traços Sr/Ca e

Ba/Ca, encontrados na composição dos otólitos dos tucunarés-açu jovens.

No Capítulo II foram analisadas as razões isotópicas de 87Sr/86Sr e dos elementos

traços Sr/Ca e Ba/Ca, mensuradas na parte central dos otólitos de tucunarés adultos, para

13

determinar a origem de cada peixe. Foram comparadas as características microquímicas do

centro dos otólitos desses peixes adultos com a composição química dos otólitos dos peixes

jovens de origem conhecida. Ainda, foi examinado as diferenças microquímicas existentes na

parte central até a borda dos otólitos dos peixes adultos para verificar se esses indivíduos

realizavam movimentos de dispersão deixando os seus locais de nascimento e se retornavam

posteriormente aos seus locais de origem.

Uma seção de conclusões finais apresenta um sumário sobre os resultados obtidos em

ambos os capítulos. Os capítulos desta Tese encontram-se em formato de manuscritos,

conforme recomendação do Programa de Pós Graduação em Biologia de Água Doce e Pesca

Interior do INPA. Esses, por sua vez, estão formatados segundo as normas das revistas às

quais serão submetidos para publicação.

Metodologia geral

Área de estudo

A bacia do Rio Negro tem cerca de 715.000 km2 (Latrubesse e Franzinelli, 2005),

formando uma área que ultrapassa as fronteiras do nosso país (Franzinelli e Igreja, 2002). O

Rio Negro tem aproximadamente 1.700 km de extensão da nascente ao seu delta (Leon et al.,

2006), se tornando o maior rio de água preta do globo terrestre (Latrubesse e Franzinelli,

2005).

A bacia de drenagem desse rio banha regiões com rochas antigas e jovens (Horbe e

Santos, 2009), as quais, consequentemente, influenciam na coloração da água de seus

tributários (Sioli, 1984). A água preta é a que predomina na bacia do Rio Negro (Junk et

al.,1997; Latrubesse e Franzinelli, 2005), embora existam vários tributários de água branca ou

clara. Essa diversidade da geologia e os diferentes tipos de água de seus tributários foram de

crucial importância para o desenho amostral desta pesquisa (Figura 3).

14

Figura 3. Localização dos locais amostrais na região do Médio Rio Negro, município de Barcelos-Amazonas. Pt01 = Rio Cuiuni (água preta),

Pt02 = Confluência do Rio Demeni com o Rio Negro (água preta), Pt03 = confluência do Rio Aracá com o Rio Demeni (água mista), Pt04 = Rio

Aracá (água preta), Pt05 = Rio Demeni (água branca); Pt06 = confluência do Rio Padauari com o Rio Negro (água preta), Pt07 = confluência do

Rio Preto com o Rio Padauari (água mista), Pt08 = Rio Preto (água preta) e Pt09 = Rio Padauari (água branca).

15

Coleta e análise dos dados

Foram coletados 142 exemplares de Cichla temensis, sendo 99 de indivíduos jovens

com menos de um ano e 43 adultos. As coletas foram realizadas nos períodos de seca, em

janeiro de 2011 e de março 2012 (Tabela 1).

Tabela 1. Locais amostrais e comprimento padrão (mm) dos tucunarés-açu C. temensis

coletados na região do Médio Rio Negro – Amazonas.

Período das coletas

Locais amostrais

2011 2012

Juvenis

N (*Min-Máx)

Adultos

N (*Min-Máx)

Juvenis

N (*Min-Máx)

Adultos

N (*Min-Máx)

Pt 01 14 (10.5 – 180)

5 (245 -385)

Pt 02 - 4 (280-460) 15 (11-15) -

Pt 03 - - 15 (72-92) 6 (290-540)

Pt 04 - 1 (765) 11 (14-18) 5 (225-260)

Pt 05 - 2 (325-465) 15 (65-93) 4 (245-425)

Pt 06 - - 14 (40-53) 1 (240)

Pt 07 - 1 (570) - 4 (240-510)

Pt 08 - - 15 (13-22) 6 (465-625)

Pt 09 - 1 (500) - 5 (210-465)

Total 14 8 85 35

N = número de indivíduos (* Comprimento padrão em milímetros)

Os indivíduos foram submetidos a choque térmico em gelo logo após sua captura,

embalados, etiquetados e acondicionados em caixas térmicas. Os peixes adultos (maiores que

24 cm) foram submetidos a um processo de dissecação para a retirada dos otólitos ainda em

campo. Os otólitos dos indivíduos jovens foram extraídos em laboratório, com o auxílio de

equipamentos especializados (Figura 4).

16

Figura 4. A = Biometria dos peixes adultos, B = Extração dos otólitos dos peixes adultos, C =

Biometria dos peixes jovens e D = Extração dos otólitos dos peixes jovens.

Os otólitos extraídos foram limpos com água destilada, secos, identificados e

armazenados em tubos eppendorf, para posterior montagem. Em laboratório, cada otólito foi

colado sobre uma lamínula de vidro para microscopia com a sua superfície côncava voltada

para cima (Figura 5).

A B

C D

17

Figura 5. A = Material utilizado para a separação e montagem dos otólitos, B = Otólito fixado

em lamínulas.

Posteriormente cada otólito foi lixado com lixas diamantadas com textura entre 30 a

200 µm, até alcançar a visualização (sob microscópio óptico) dos anéis de crescimento com

distância aproximada de 30 micrometros (µm) do núcleo do otólito (Figura 6).

Figura 6. A = Lixamento dos otólitos, B = Microscópio óptico utilizado na visualização da

distância entre a superfície do otólito e sua parte central (µm).

Depois de lixados os otólitos foram submetidos a uma limpeza com água destilada em

banheira ultrassônica (Branson, 200), por dois minutos. Em seguida foram acomodados para

A B

A B

18

secagem. Todo o processo de limpeza e secagem foi realizado em uma câmara ultralimpa

(AirClean 600 PCR workstation) (Figura 7).

Figura 7. A = Banheira ultrassônica para limpeza dos otólitos, B = Câmara ultralimpa para

limpeza e secagem dos otólitos.

Após a limpeza e secagem dos otólitos, eles foram armazenados em caixas de

lamínulas limpas, e essas acondicionadas em sacolas plásticas do tipo zip lock (Figura 8).

Figura 8. A = Acondicionamento dos otólitos em sacolas e caixas esterilizadas, B =

Acondicionamento dos otólitos para transporte.

A B

A B

19

No Laboratório de Espectrometria de Massa do Instituto Oceanográfico Woods Hole

(WHOI) em Massachusetts nos Estados Unidos da América, os otólitos foram selecionados

para as análises isotópicas e de elementos traços, realizadas com queima a laser de parte da

superfície do otólito, com uso de equipamento óptico associado a um coletor múltiplo de

espectrometria de massa por plasma indutivamente acoplados (LA-MC-ICP-MS) (Figura 9).

Figura 9. A = Coletor Múltiplo de Espectrometria de Massas por Plasma Indutivamente

Acoplado, B = Imagem de um otólito com a região central mapeada (amarelo) e o transecto

estabelecido para queima (verde), prontos para a análise química a laser.

Cada otólito submetido à análise microquímica teve seus valores químicos

visualizados automaticamente através de uma tela de computador (Figura 10).

Figura 10. A = Queima a laser da superfície do otólito, B = Resultados das análises

microquímicas em computador.

A B

A B

20

Os dados coletados foram tabulados e posteriormente analisados estatisticamente

empregando-se os programas SPSS (SPSS, Inc) e Statistica 7.0 (StatSoft, Inc). Os resultados

obtidos com as análises foram utilizados na elaboração dos Capítulos I e II desta Tese.

21

Capítulo I

Garcez, R.C.S.; Humston, R.; Harbor, D. & Freitas, C.E.C.

Otolith geochemistry in young-of-the-year peacock bass

Cichla temensis for investigating natal dispersal in the Rio

Negro (Amazon - Brazil) river system. Submetido e aceito

na revista Ecology of Freshwater Fish, em 22 Março de

2014.

22

Otolith geochemistry in young-of-the-year peacock bass Cichla temensis for

investigating natal dispersal in the Rio Negro (Amazon - Brazil) river

system

R. C. S. Garcez1, R. Humston 2, D. Harbor3, and C. E. C. Freitas4

1 Programa de Pós-Graduação em Biologia de Água Doce e pesca Interior, Instituto Nacional

de pesquisas da Amazônia, Av. André Araújo, 2936. Aleixo, Manaus, AM 69.060-001,

Brazil.

2Department of Biology, Washington and Lee University, Lexington, VA 24450,

U.S.A.

3Department of Geology, Washington and Lee University, Lexington, VA 24450,

U.S.A.

4Departamento de Ciências Pesqueiras. Universidade Federal do Amazonas, Avenida General

Rodrigo Otávio Jordão Ramos, 3000. Manaus, AM 69.070-000, Brazil.

Running head: Otolith geochemistry of peacock bass C. temensis

23

Abstract

This study examined otolith geochemistry as a natural marker of natal origins in young-of-

the-year (YOY) C. temensis in the Negro River basin of Brazil. We analysed trace element

and isotopic composition of otoliths of YOY collected off spawning nests from the main stem

and major tributaries. These were compared to regional bedrock geologic composition to

explore underlying mechanisms of differences in otolith geochemistry. Our results suggest

that spatial differences in otolith geochemistry can be used to distinguish natal origins based

on 87Sr/86Sr, Sr/Ca, and Ba/Ca ratios. This approach allowed us to correctly classify 99% of

juvenile fish to their natal streams using cross-validation in a linear discriminant function

analysis (LDFA). Patterns of otolith isotopic composition correspond with patterns in regional

geology as expected based on previously demonstrated correlations, though some fine-scale

spatial differences cannot be accounted for by available geologic information. These results

demonstrate that otolith chemistry is valuable as a natural marker of natal origins in this

system and suggest that inferences from geologic maps may be useful for interpreting

movements based on otolith geochemical signatures. This information provides the basis for

future work to investigate the early life history and spatial ecology of this important cichlid.

Key words: Amazon Basin; Fish movement; Fresh water; Geochemistry; Strontium isotope;

Trace elements.

24

Introduction

The fish species of the Amazon Basin exhibit an incredible diversity of life histories,

and greater scientific understanding of fisheries ecology in the area is crucial to the

development of feasible and realistic strategies of conservation. Species of the genus Cichla

support economically important fisheries for small-scale commercial harvest as well as

recreational angling and associated ecotourism. The biology of Cichla species has been fairly

well documented, including population structure (Winemiller et al. 1997); age and growth

(Jepsen et al. 1997); abundance (Taphorn & Barbarino Duque 1993); feeding habits (Novaes

et al. 2004); spawning (Chellappa et al. 2003); and dispersal movement (Hoeinghaus et al.

2003). In general, reports about dispersal movements of Cichla spp. in the Amazon Basin

come from research using capture–mark–recapture technique (Taphorn & Barbarino Duque

1993; Hoeinghaus et al. 2003; Holley et al. 2008). However, fish must be large enough to bear

physical tags; therefore, these methods are less useful for tracking movement early in a fish’s

life history (Holley et al. 2008). Dispersal in the early life stages is important for

understanding spatial population dynamics and recruitment patterns across the river tributary

network (Humston et al. 2010) and therefore represents a considerable knowledge gap for

managing these fisheries.

Like many South American rivers, the Negro River near the equator in Brazil supports

a popular recreational sport fishery that focuses primarily on Cichla species (Hoeinghaus et

al. 2003; Holley et al. 2008). In this region, Cichla are abundant and colonise the main

channel of the Negro River and the majority of its white and black water tributaries (Jepsen et

al. 1997). Sport fishing occurs primarily during the receding water period between September

and April and brings approximately 1800 anglers to the Negro River Basin during this period

(Holley et al. 2008). Cichla reproduction takes place at the same time, following the receding

water period (Zaret 1980). These cichlids tend to prefer lentic habitats (Winemiller 2001),

make their nests on the river bottom (Jepsen et al. 1997) and demonstrate parental care

behaviour (Winemiller & Jepsen 1998). In dispersal following this period of parental care,

young-of-year could redistribute widely throughout the extensive network of rivers and

tributaries, particularly during the high flood season flows that follow in May–August.

Emigration and recruitment from spawning areas could therefore support distant fisheries,

obscuring the effects of harvest on local populations and/or creating source-sink dynamics.

25

Finding a way to effectively study movement across the full range of ontogeny is therefore

important for identifying such dynamics.

Isotopic and trace elemental composition of hard parts of animals has been

demonstrated to be useful as a natural fingerprint of past residency in studies of movement

ecology (Price et al. 2000; Walther et al. 2008; Walther & Limburg 2012; Wolff et al. 2012).

The chemistry of fish otoliths in particular has been demonstrated to be an effective tool for

tracking fish movement, as this structure readily incorporates the chemical signatures of

specific water bodies as influenced by their lithospheric geochemistry (Thorrold et al. 2001;

Humston & Harbor 2006; Walther et al. 2008; Nowling et al. 2011). The use of otolith

microchemistry as an alternative method of estimating natal origin and fish movement rates

could circumvent existing limitations to the use of physical tags in the study of movement by

Cichla; however, no study of Cichla species in the Amazon Basin has investigated this

possibility. In particular, isotopic chemistry (e.g., 87Sr/86Sr) holds potential as a distinct

marker in these rivers as it correlates with age and composition of underlying geology

(Barnett-Johnson et al. 2008; Bataille & Bowen 2012; Hegg et al. 2013), both of which vary

substantially across this system. In this study, we sought to determine whether otolith

geochemistry could provide a useful indicator of provenance of Cichla temensis in the Rio

Negro system. Correspondingly, we qualitatively explored whether regional differences in

otolith chemistry correspond with variation in underlying lithology based on available data.

Taken together, these sources of inference could provide a foundation for reconstructing

movement patterns across the full range of ontogeny in Cichla species in the Amazon Basin

based on retrospective analysis afforded by otolith chemical composition.

Materials and Methods

Study Area

The Negro River Basin (NRB) comprises over 715,000 km2 that extend from latitude

3°140S to 5°80N and from longitude 72°570W to 58°160W (Latrubesse & Franzinelli 2005)

26

and includes large, remote areas with minimal exploration or study. The NRB is a

transboundary basin, with its area divided among Brazil, Colombia, Venezuela and Guiana

(Franzinelli & Igreja 2002). The Negro River forms from the confluence of the Guiana River

and the Casiquiare Channel and flows roughly 1700 km to its terminus at the Solimões

(Amazon) River. It contributes a mean annual discharge of over 50,000 m3∙s-1 (Leon et al.

2006) to the system making it the second largest Amazon River tributary in this respect

(Latrubesse & Franzinelli 2005).

The Negro River catchment incorporates three distinct upper, middle and lower

regions: (i) the upper region beginning in the Guyana Shield, where the Negro River

headwaters are located, and ends in Santa Isabel do Rio Negro (Franzinelli 2011); (ii) the

middle region extends to the confluence with the Branco River and exhibits Cenozoic

sediments derived from Precambrian crystalline basement or Palaeozoic and Mesozoic

sedimentary rocks (Latrubesse & Franzinelli 2005); and (iii) the lower region begins at the

confluence with the Branco River, occurs nearly entirely in the Cenozoic floodplain, passing

across the Archipelago of Anavilhanas and ending at the confluence with the Solimões River

(Franzinelli & Igreja 2002; Fig. 1). The chemical composition of the water in the Negro River

and its tributaries is highly variable and strongly influenced by forest characteristics and

geological location (Horbe & Santos 2009). Within the NRB, black water with high acidity is

predominant (Stallard & Edmond 1987).

27

Fig.1. Location and geological conditions of the study area: (a) location of the sample

sites with respect to the major tributaries of the Negro River, (b) ages of the geological units

from Ferreira et al. (2005) and dashed outlines of the basins upstream of the sample sites, and

(c) geologic units as potential sources of isotopic differentiation.

Geologic age corresponds roughly with 87Sr/86Sr ratios due to radiogenic Sr

accumulation (Faure 2001), and geologic composition determines how geochemistry of

bedrock and corresponding weathering can contribute to Sr abundance and isotopic

composition in surface waters (Barnett-Johnson et al. 2008; Bataille & Bowen 2012; Hegg et

28

al. 2013). The NRB and the majority of its northern tributaries are primarily underlain by

silicate rocks rich in Sr with high 87Sr/86Sr ratios (Edmond et al. 1995). Conversely, the

southern area is formed by younger Quaternary sediments with low radiogenic 87Sr/86Sr ratios

(Allégre et al. 1996). We would expect to see this regional variation reflected in otolith Sr

ratios as well.

We focused our sampling effort in the geologically diverse ‘middle’ section of the

Negro River. Fish were collected from seven sites separated by at least 20 river kilometer

(rkm) in two general areas area we termed upper river and lower river areas, which are

separated by at a distance of approximately 150 rkm (Table 1; Fig. 1a).

Table 1. Geographic information on sample locations

Location names Code Types of

water

Latitude Longitude

Lower Rivers

Cuiuni River Pt01 BW -00 47' 32,90280'' -63 15' 23,38560"

NRMC Pt02 BW -00 46' 26,60520'' -62 56' 26,84760"

ADC Pt03 MW -00 26' 25,63440'' -62 54' 27,25560"

Aracá River Pt04 BW -00 16' 48,77760'' -63 00' 44,67600"

Demini River Pt05 WW -00 18' 03,43800'' -62 46' 26,85360"

Upper Rivers

NRMC Pt06 BW -00 15' 04,26240'' -64 04' 33,52080"

Preto River Pt07 BW -00 05' 31,06320'' -64 09' 24,28920"

Six of our seven sampling sites were in Negro River tributaries entering from the

northern side of the main stem, situated in large regions of Paleoproterozoic accreted craton,

added to by Mesoproterozoic tectonic belts and igneous intrusions (Gradstein et al. 2012).

The exception is Pt01 located in the Cuiuni River, a tributary whose catchment basin lies to

the south of the Negro River in a Cenozoic basin formed in unconsolidated Neogene sediment

(Fig. 1b). We refer to these locations by their numeric codes to make spatial arrangement

clearer for the reader; numbering is ordered roughly proceeding from downstream to upstream

sites.

29

Sample collection

Fish were collected at two separate times, with the first collection occurring in March

2011; only one site was sampled (Pt01) in this period due to poor conditions. A second

collection effort was organised in January 2012 to increase sample size and spatial coverage.

Collections were conducted during these months to correspond with spawning and so that

young-of-the-year (YOY) C. temensis could be collected near their natal nests in the shallow

floodplain streams and lake margins while still under parental care. Zaret (1980) reported that

YOY Cichla spp. Do not disperse from their nursery ground until they are at least 2 months in

age and 60 mm standard; therefore, we only collected YOY well below this size. Captured

fish were euthanized via thermal shock, covered with ice and stored in sealed plastic bags.

They were labelled and transported to the fish biology laboratory facility at the Federal

Amazon University. In the laboratory, the standard length (SL) of each YOY was measured to

the nearest 1 mm (±1), and the fish were weighed to the nearest 0.01 g before otolith

extraction.

Otolith preparation and analysis

Sagittal otoliths were removed under a dissecting microscope, triple-washed in

ultrapure deionised water to remove attached fleshy tissue, dried and stored in a 2 ml

Eppendorf tube for further analysis. Then, they were transported (IBAMA licence number

110752) to Washington and Lee University (WLU - Virginia, USA) for preparation. The right

otolith from each individual fish was selected for chemical analysis, and the left otolith was

saved as a potential replacement. Otoliths were mounted with the convex surface up (i.e.,

sulcus up) on microscope slides using gel-consistency cyanoacrylate. Otoliths were then

hand-polished on the saggital plane using 30 µm and 3 µm diamond lapping film to a distance

of approximately 50 µm above the core.

All mounted and polished otoliths were cleaned in ultrapure (Milli-Q, EMD Millipore,

Billerica, MA, USA) water for two minutes using an ultrasonic bath (Bransonic 200) under

30

class 100 clean conditions. Afterwards, the otoliths were dried in a laminar flow hood

(approximately 2 h) and stored for transport to the Woods Hole Oceanographic Institute

(WHOI).

Otoliths with a diameter of length and width of more than 250 µm were selected for

chemical analyses, to ensure enough area and mass for laser ablation. An exception was made

for the otoliths from Pt04, which were approximately 200 µm in length and 100 µm in width.

The analytical procedure was performed using a Neptune multicollector ICP-MS (Thermo

Scientific, Waltham, MA, USA) attached to a 193 nm laser ablation system (New Wave

Research, ESI Inc., Portland, OR, USA), which was used to ablate a raster centred on the core

of the otolith. The laser ablated a 100-µm-diameter spot per cycle while running at 100%

power, 10 Hz repetition rate and 6 µm∙s-1 scan speed. We were able to simultaneously

measure 87Sr/86Sr, Sr/Ca and Ba/Ca ratios from a single raster following procedures outlined

in Walther and Thorrold (2010). Blank samples and two different standard reference materials

were analysed between every 12 otolith samples. Reference materials included a strontium

carbonate isotopic standard (SRM 987) and a dissolved otolith certified reference (FEBS-01;

Sturgeon et al. 2005). Blank, mass bias and interference corrections were applied as outlined

in Walther et al. (2008) and Walther and Thorrold (2010). Mean 87Sr/86Sr in SRM 987

standard measurements (N = 17) was 0.7102502 with a standard deviation (SD) of 0.0000218,

which is within 1 SD of the known value of 0.71024 for SRM 987.

Statistical analysis

We initially employed analyses of variance – ANOVA (Zar 1999) to examine spatial

differences in otolith 87Sr/86Sr, Sr/Ca and Ba/Ca composition individually. Tukey’s HSD test

(Zar 1999) was employed to identify pairwise differences when significant differences were

identified among sampling locations. This allowed us to elucidate how each variable varied

among locations and corresponded to geologic variation. We then used linear discriminant

function analysis (LDFA) to determine the degree to which multivariate geochemical

signatures in otoliths could be used to distinguish origins of individuals from our sample.

Variables were entered into the model using a stepwise forward method, using Wilk’s

31

Lambda and F-statistic probabilities to determine sequence of variable addition and evaluate

model improvement, respectively (Huberty 1994). A serial deletion cross validation procedure

was used to quantify classification accuracy and error rates. All statistical analyses were

conducted using SPSS software (SPSS, Inc., IBM Corporation, Armonk, NY, USA).

Geologic analyses

Due to the sheer number of tributaries and the vast size and remoteness of this area, it

is not feasible to collect a ‘catalog’ of reference data on otolith chemical composition from all

possible natal sources in this system. Therefore, we investigated correspondence between

geologic composition of watersheds and chemical composition of otoliths collected from

sampling locations. We focused on 87Sr/86Sr in particular in this analysis, as previous

examples demonstrate that this can be correlated with geologic data (Barnett- Johnson et al.

2008; Bataille & Bowen 2012; Hegg et al. 2013) and that it is often less variable than trace

element chemistry in surface waters (with notable exceptions, e.g., Clow et al. 1997; Aubert et

al. 2002; Voss et al. 2014). Following Barnett-Johnson et al. (2008) and Bataille and Bowen

(2012), we used available data to examine the age of geologic features in watersheds as well

as their relative composition. Although limited data were available in this regard, we

attempted to infer the degree to which particular types of rocks in the study area would

contribute radiogenic strontium and followed data reported in Bataille and Bowen (2012) for

guidance. We divided the map units of middle Negro River region following Ferreira et al.

(2005) into the following broad units: (i) Paleoproterozoic basement of the Santa Izabel do

Rio Negro unit, which comprises metasedimentary, metamorphosed felsic volcanic and

plutonic rocks including granodiorite, quartz diorite, migmatites and monzogranites, and

gneiss in the northwest corner; (ii) Paleoproterozoic granite intrusions (syeno- and

monzogranite); (iii) Mesoproterozoic granite intrusions (syeno- and monzogranite, 93%) and

minor mafic and ultramafic intrusions (< 7%); (iv) Mesoproterozoic quartzite; (v) Cenozoic

sediments including river floodplains, Quaternary terraces and aeolian deposits. We then

quantified the relative composition of watersheds in these different geologic categories and

compared these with otolith 87Sr/86Sr composition from sampling locations in those

watersheds. Finally, we examined scatter plots of per cent rock type against mean 87Sr/86Sr

32

observed in collections and performed simple linear regressions to explore the degree to

which watershed geologic composition could be used to predict stream isotopic chemistry.

Results

We collected a total of 175 YOY C. temensis from seven experimental reaches in two

general areas, designated as upper river and lower river areas in this study (Table 2). The

sampled fish ranged in weight from 0.02 to 69.25 g (±25.30) and in standard length (SL) from

1.62 to 8.98 cm (±2.95). This size range suggests these fish were still in close proximity to

their spawning site at the time of collection. Their mean age in months was 2.41 ± 1.39. Of

these, a total of 99 otoliths were of sufficient size for laser ablation and chemical analyses.

Table 2. Strontium 87Sr/86Sr isotope and trace element ratio values (mean ± SD) from

the otoliths of young-of-the-year C. temensis (sampling site names are listed in Table 1).

Area Location

Code

N 87Sr/86Sr Sr/Ca (mmol/mol-1) Ba/Ca (mmol/mol-1)

Lower rivers

Pt01 14 0.71314 ± 0.00066 0.01458 ± 0.00303 0.00120 ± 0.00057

Pt02 15 0.74097 ± 0.00147 0.00624 ± 0.00098 0.00041 ± 0.00017

Pt03 15 0.78554 ± 0.00104 0.00945 ± 0.00155 0.00081 ± 0.00024

Pt04 11 0.78292 ± 0.00101 0.00621 ± 0.00130 0.00127 ± 0.00010

Pt05 15 0.73494 ± 0.00016 0.00480 ± 0.00034 0.00026 ± 0.00006

Upper rivers Pt06 14 0.74230 ± 0.00086 0.00847 ± 0.00051 0.00074 ± 0.00010

Pt07 15 0.74211 ± 0.00100 0.00552 ± 0.00236 0.00135 ± 0.00025

Results of univariate analyses (ANOVA) indicated significant differences among

sample sites in otolith 87Sr/86Sr (F6, 92 = 9919.871, P < 0.0001), Sr/Ca (F6, 92 = 55.323, P <

0.0001) and Ba/Ca ratios (F6, 92 = 35.250, P < 0.0001). Otolith strontium isotopic ratios

varied widely among locations, with location means ranging from 0.71314 (Pt01) to 0.78554

(Pt03) with low variance within sites. Tukey’s HSD tests revealed pairwise differences among

all locations except the two most upstream sampling sites (Pt06 and Pt07). Differences

between the two mainstem Negro River sites (Pt02 and Pt06) were subtle, with the

downstream location (Pt02) slightly lower than upstream. In the lower sampled section, the

southern tributary had the lowest 87Sr/86Sr by far. An interesting pattern emerged among the

33

northern tributaries of the lower section, which includes three sites comprising two smaller

tributaries (Pt05 and Pt04) and one larger tributary (Pt03) formed by their confluence (Fig.

2a). Otoliths sampled from the western tributary (Pt04) were very high in radiogenic

strontium, while otoliths collected from the eastern tributary (Pt05) were much lower.

Although we would expect the area below the confluence of these rivers (Pt04) to produce

otoliths with 87Sr/86Sr somewhere between these two, we actually observed the highest overall

levels of radiogenic strontium in otoliths from this location.

Analyses suggested greater separation among sites based on Sr/Ca ratios. Locations

Pt02, Pt04 and Pt07 were indistinguishable with respect to Sr/Ca, but all other remaining

locations separated out individually (Fig. 2b). Uniquely high Sr/Ca was observed in the

southern tributary sampled at site Pt01. By contrast, univariate screening suggested that

otolith Ba/Ca appeared to hold the lowest potential as a discriminatory variable, with

sampling sites roughly separating into two groups: locations Pt02 and Pt05 form one group

with relatively low Ba/Ca, while all other locations had similarly higher Ba/Ca ratios (Fig.

2c). Variance within locations was high leading to substantial overlap in observed ranges

among sites.

36

Fig. 2. Mean isotopic and trace element concentration ratios in the otoliths of YOY

fish groups. (a) 87Sr/86Sr ratio, (b) Sr/Ca ratio and (c) Ba/Ca ratio. Means sharing a lowercase

letter are not significantly different (Tukey’s HSD pairwise mean comparisons, α = 0.05).

In LDFA, 99% of YOY C. temensis were classified accurately with respect to their

natal origin in cross-validation based on all three isotopic and trace element variables. As

expected from results of univariate analyses, 87Sr/86Sr was the most influential variable in

discriminant functions, with an eigenvalue of 894.476 (98.5% of variance) followed by Sr/Ca

with 11.835 (1.3% of variance) and by Ba/Ca 1.987 (0.2% of variance). All three variables

were retained in stepwise model construction (P < 0.001 for each variable addition). The only

error committed in crossvalidation was a single fish from location Pt02, which was predicted

to be from location Pt06. This individual fish is 12.0 mm and 0.0092 g, indicating that it was

still under parental care (Winemiller et al. 1997) and unlikely to have dispersed from its natal

site, especially over this great a distance (~150 rkm). Bivariate plots (Fig. 3a-c) demonstrate

overlap occurs between samples from these locations in all bivariate combinations, providing

a simpler, statistical explanation for this misclassification.

39

Fig. 3. Microchemical isotopic and trace element ratios from the otoliths of YOY C.

temensis distributed within groups and between areas. Sr/Ca versus 87Sr/86Sr ratios (a), Ba/Ca

versus 87Sr/86Sr ratios (b) and Ba/Ca versus Sr/Ca ratios (c). The sample location codes are

listed in Table 1. YOY, young-of-the-year.

Spatial patterns and correlations with geology

The most significant geological differentiation of the sample locations identified in our

analysis is the southern versus northern tributaries (Table 3). The southern watersheds

comprise depositional lowlands of the Solimões-Negro divide, which includes extraregional

sediments of the Amazon Basin derived in part from the Andean highlands. The northern

watersheds include Proterozoic igneous and metamorphic highlands that have shed sediments

into the basin during the Cenozoic. As would be predicted, 87Sr/86Sr was lowest in the

southern tributary sampled (Pt01), reflecting the dominance of unlithified Neogene sediments

40

in that watershed compared with the older cratonic composition of the northern tributary

watersheds (Fig. 1b, c).

Table 3. Percentage of major rock groups in watersheds above sample locations

Pt01 Pt02 Pt03 Pt04 Pt05 Pt06 Pt07

Cenozoic sediments 100.0 46.5 46.1 75.5 22.6 48.5 63.3

Mesoproterozoic quartzite 0.0 1.5 1.5 3.3 0.2 0.8 1.6

Mesoproterozoic quartzite 0.0 7.0 7.0 4.5 9.2 2.5 1.3

Paleoproterozoic intrusions 0.0 11.6 11.7 8.1 14.8 21.7 19.9

Paleoproterozoic basement 0.0 33.2 33.6 8.6 53.2 26.5 13.9

Per cent of hardrock only

Mesoproterozoic quartzite - 2.7 2.7 13.6 0.2 1.5 4.4

Mesoproterozoic intrusions - 13.1 13.1 18.2 11.9 4.9 3.5

Paleoproterozoic intrusions - 21.8 21.8 33.1 19.2 42.2 54.1

Paleoproterozoic basement - 62.4 62.4 35.1 68.7 51.4 38.0

The per cent of total Paleoproterozoic hard rock in a watershed showed no correlation

with 87Sr/86Sr (P = 0.3); however, per cent Mesoproterozoic hard rock was significantly

correlated with otolith 87Sr/86Sr (r2 = 0.64, P = 0.02). Quartzite can contribute radiogenic Sr to

a great degree and may be the driver behind this correlation. However, our sample size (N =

7) is insufficient for further exploration of how these and other potential drivers of basin

87Sr/86Sr (e.g., geologic heterogeneity; see Hegg et al. 2013) may be used to predict river

87Sr/86Sr.

Differentiation of the headwater basins for the northern watersheds is complicated by a

lack of knowledge about the source of Cenozoic sediments and the lack of detailed

discrimination of rock units that could be used to constrain strontium ratios following the

method of Bataille & Bowen (2012). In the upper sampled section of the river, otolith isotopic

ratios were relatively consistent and in the middle range of observed values. The floodplain

habitat sampled at Pt06 was adjacent to the northern shore of the mainstem Negro River, and

therefore, its high 87Sr/86Sr likely reflects inputs from the older Paleoproterozoic and

Mesoproterozoic exposures to the north with little influence from southern watersheds. This

explains the similarity between Pt06 and Pt07. In the lower sampling area, the high strontium

ratios in otoliths from Pt04 may be attributed to the increased percentage of Mesoproterozoic

41

quartzite or the higher percentage of Mesoproterozoic granite intrusions in the exposed rocks

of the highlands (Fig. 1b,c and Table 3). In addition, aeolian sediments are only exposed in

basin 4, where they comprise over 5% of the basin (Fig. 1c). The source of these late

Quaternary sediments (Tatumi et al. 2002) is unknown but may include winnowing of

sediments from the igneous and metamorphic highlands and therefore could be a source of the

isotopic difference. Our geologic analyses do not shed light on why otoliths from fish

collected at Pt03 would be enriched in radiogenic strontium compared with its adjacent

tributary (Pt04).

Discussion

Chemical composition of YOY otoliths from C. temensis showed strong potential for

differentiating natal origins, particularly for distinguishing between fish emerging from

southern versus northern tributaries based on Sr isotopic and elemental composition. The

former is likely the most reliable indicator of provenance in this respect, as it is less

susceptible to temporal variation in stream water chemistry. Given the large differences in

87Sr/86Sr we observed between northern and southern tributaries and its strong correspondence

with regional geology, this marker is likely also useful for reconstructing movement patterns

between northern and southern tributaries over entire life histories from adult otoliths

(Walther & Limburg 2012). Differentiating movement between tributaries from upstream

versus downstream reaches of the Negro River (e.g., between upper and lower areas in our

study) based solely on isotopic ratios is less straightforward. As spawning takes place in

floodplain lakes adjacent to the rivers, it is possible that fine-scale variation in geology

influencing the water chemistry of lake inputs from groundwater or small feeder streams is

reflected in otolith chemistry of YOY from these habitats. Recent examples demonstrate

methods for characterising ‘isoscapes’ of spatial 87Sr/86Sr variation based on geologic

composition and weathering dynamics (Bataille & Bowen 2012; Hegg et al. 2013), and this

approach could be useful for explaining observed fine-scale variation and predicting tributary

87Sr/86Sr. However, the lack of sufficient data differentiating regional geology to the same

degree precludes applying these same methods in this study area.

42

Although temporal variation in trace element-to-calcium ratios in rivers can reduce

their reliability as a marker of provenance in a river tributary network, there is some

indication in the geology of the region that Sr/Ca could provide reasonable information for

retrospective analysis of movement from otoliths. Our limited sample was not sufficient to

explore this correlation at length; however, recent examples (Hegg et al. 2013) demonstrate

that this is a rich area for further consideration. Although geologic data can provide inference

on relative spatial variation in otolith trace element composition (Humston & Harbor 2006),

predictions of trace elemental composition of stream water (and hence otolith chemistry) from

geology are not possible to the same extent as for isotopic composition. Likewise, these

methods cannot account for differences in trace element incorporation in otoliths attributable

to changes in fish physiology. We therefore suggest that strontium isotopes provide the most

reliable ‘natural tag’ for reconstructing fish movement in this system from otolith chemistry

chronologies.

This research confirms that otolith geochemical signatures in YOY C. temensis are

distinct and specific to their river of origin. This method can therefore be applied to examine

the number of spawning sources contributing to a regional stock, and the relative contribution

of rivers differentiated by unique isotopic ‘signatures’ (e.g., northern vs. southern tributaries)

can be elucidated as well. Isotopic ratios show potential for retrospective analysis of adult

movement patterns; however, differentiating movement among northern tributaries may be

complicated by similarity of isotopic ratios among rivers. Applicability of these methods for

studying movement of Cichla and other fish species in the Negro River system can be

measurably enhanced by further characterization of bedrock and stream water geochemistry in

this region.

Acknowledgments

We thank B. A. Harris for his helpful review of previous drafts and Mr. J. A. D.

Siqueira for his assistance in the field collecting the samples. Financial support of this study

was provided by CAPES, CNPq, FAPEAM and INCT-Adapta. Laboratory, and logistic

support also was provided by UFAM, INPA, I-PIATAM and WLU.

43

Capítulo II

Garcez, R.C.S.; Humston, R. & Freitas, C.E.C. Philopatry

and metapopulation patterns for the peacock bass Cichla

temensis of the middle Negro River Basin (Amazonas -

Brazil): an otolith chemical analysis. Manuscrito

formatado para a Ecology of Freshwater Fish.

44

Philopatry and metapopulation patterns for the peacock bass Cichla

temensis of the middle Negro River Basin (Amazonas - Brazil): an otolith

chemical analysis.

Raniere C. S. Garcez1, R. Humston 2, and C. E. C. Freitas3

1 Programa de Pós-Graduação em Biologia de Água Doce e pesca Interior, Instituto Nacional

de pesquisas da Amazônia, Av. André Araújo, 2936. Aleixo, Manaus, AM 69.060-001,

Brazil.

2Department of Biology, Washington and Lee University, Lexington, VA 24450,

U.S.A.

3 Department of Fisheries Sciences. Federal University of the Amazonas, Avenida

General Rodrigo Otávio Jordão Ramos, 3000. Manaus, AM 69.070-000, Brazil.

Running head: Otolith geochemistry and philopatry of peacock bass C. temensis

45

Abstract

Strontium isotope and trace elements from otolith of peacock bass Cichla temensis

(Humboldt, 1833) indicated that it is possible to track movements of young-of-year (YOY)

and adult fish in the Negro River and its tributaries. A canonical discriminant function

analysis (CDFA) shows in a cross table that 93.9% of YOY and 41.9% of adult peacock bass

otolith core fingerprints matched correctly with their nursery areas. Variation in 87Sr/86Sr

isotopes ratio from a transect line on the otolith surface indicated the existence of isolated and

mixed populations for adult fish in the study area. Our results also confirm that adult peacock

bass present a meta-population structure and disperse up and down river with philopatric

behaviour. Therefore, the present report offers new information about spatial distribution of

C. temensis in their natural environment, which could be used to support effective strategies

for stocks and fisheries management and conservation.

Keywords: Fish management; geochemistry; homing, otolith fingerprints; trace elements.

46

Introduction

The Negro River basin is a vast and complex aquatic ecosystem that supports large

Cichla populations (Hoeinghaus et al., 2003). The main Cichla species in this huge aquatic

environment is the peacock bass Cichla temensis (Humboldt, 1833), which is the largest

representative of the family Cichlidae. It attains large sizes of approximately 81.5 cm

Standard Length (SL) (Taphorn and Barbarino-Duque, 1993) and more than 12kg (Montana et

al., 2006; Holley et al., 2008).

In this region three Cichla species, C. monoculus, C. temensis, and C. orinocensis,

(Kullander and Ferreira, 2006; Willis et al., 2010) co-exist, but among these species C.

temensis reportedly occurs in a greater variety of aquatic habitats than its congeners (Jepsen et

al., 1997; Hoeighaus et al., 2003). Other Cichla species naturally occupy black and white

waters in the Orinoco River (Colombia and Venezuela) and the Negro River in Brazil

(Winemiller et al., 1997; Brinn et al., 2004; Kullander and Ferreira, 2006).

The abundance and voracity of this large-sized predator (Winemiller 2001) gained the

attention of the catch-and-release sport-fishing industry (Macrander, 2010) that has emerged

in the Negro River, attracting over 2000 anglers to the region annually (Holley et al., 2008).

Despite the importance of the Cichla species, both as a food resource and sport fishing target

(Hoeinghaus et al., 2003; Willis et al., 2007), makes different users (subsistence, commercial

and sport fishing) competing for the same resource, that brought the necessity to increase

efforts to study the distribution of Cichla species populations in the Negro River Basin, in

order to develop an effective fisheries management strategy.

The knowledg about fish movements dispersiton among locations is the first step to

understand the fish population dynamics. For that is crucial to understand the concept of

metapopulations (Levins, 1970), that consicts in a conjunct of separated populations that are

conected by the emigration and immigrations of individuals in those aquatics system (Kritzer

and Sale, 2004).

The metapopulation is a potential approach to understand the spatial distribution of

animal populations (Levins, 1970) in a variety of aquatic and terrestrial systems (Hanski and

Simberloff, 1997; Kritzer and Sale, 2004). One way to better understand this idea for systems,

such as large rivers with adjacent floodplains, would be to assume that lakes and rivers are

47

patches of aquatic habitats for resident fish. The fish movements between the patches of these

open and connected systems could be interpreted in terms of metapopulational dynamics.

Mapping fish population connectivity through adults’ dispersal and their emigration or

immigration movements among different geological natal patches is the basis of

understanding this theory (Thorrold et al., 2001).

Adult peacock bass are presumed to be sedentary fish (Granado-Lorencio et al., 2005),

and have been classified as an equilibrium species (Winemiller, 2001). Meanwhile, some

studies have reported that the Cichla species realizes small-scale dispersal movements

between main channels and lakes (Hoeinghaus et al., 2003). On the other hand, this

supposition has basically been tested using conventional approaches such as field

observations (Lowe-McConnell, 1969), tagging (Taphorn and Barbarino-Duque 1993;

Hoeinghaus et al., 2003; Holley et al., 2008; Willis et al., 2010), radio telemetry (Thorstad et

al., 2001), and genetic procedures (Macrander, 2010). The information collected, however, is

inconclusive and leaves many lacunas, making it difficult to understand the movements of C.

temensis and its distribution pattern, which may be attributed to problems with these

methodologies.

Previous studies using fish’s otolith geochemical signatures (Brazner et al., 2004;

Walter and Thorrold, 2006; 2010; 2013) have shown accurate results tracking small and large

fish in marine (Humphreys Jr. et al., 2005) and fresh water habitats (Humston et al., 2010).

These findings confirm that this technique is useful as a natural marker approach (Thorrold et

al., 2001), and an effective apparatus to resolve the discrepancy regarding frequency of large

scale movements (between rivers with different geological composition) by drawing a

complete route history for each fish individually among river habitats.

It is possible to identify every different geochemical locations through which a fish

passes during its lifetime because fish otoliths are calcareous structures formed by the

sequential addition of inert layers of calcium carbonate (Campana 1997; 1999). The calcium

carbonate is derived from surrounding aquatic environment, and shapes a geochemical

fingerprint (Walther and Thorrold, 2006). Even if the chemical changes are minuscule, we can

track the fish movements across different geologic locations (Humston and Harbour, 2006).

Based on this premise, this study intends to verify the peacock bass spatial distribution

in the Negro River basin in Brazil, by investigating if the young-of-year C. temensis otoliths’

48

core microchemical signatures differ enough between different geological sampling sites to be

used as an effective reference for tracking back its birth place. If so, it seeks to verify the

otolith core isotope and elemental composition for the existence of similarities between YOY

and adult chemical fingerprints that could be used to predict adult fish origins (i.e. birth

place).

Also, in order to verify the existence of adult C. temensis’ spatial distribution and

movements down or upstream, this study analyses variation in 87Sr/86Sr isotopes in a laser

ablated transect from the otoliths surfaces of adults fish for the purpose of identifying some

pattern of philopatry or metapopulation structure. In this way, this study intends to test the

following null hypotheses:

i) There is no difference in the chemistry between otolith core of the YOY and adult

peacock bass; ii) Adult peacock bass’ otolith core isotope and elemental composition do not

vary among tributaries of the Negro River with different water types; iii) There are no

chemical differences in the values of 87Sr/86Sr isotopes from the adult otoliths expressed in a

transect line (from core to the edge) that could indicate fish movements among sampling sites;

and iv) Adult C. temensis do not return to their nursery areas after leaving it.

Material and Methods

Study area

The study area is located at the Middle Negro River region, about 500 kilometers

upstream from Manaus, the capital of Amazonas State, Brazil (Holley et al., 2008) (Figure 1).

The Negro River is the largest black water river in the globe (Latrubesse & Franzinelli, 2005),

and it is formed of a complex network of tributaries, which differ greatly in their geologic

basement and water types (Gaillardet et al., 1997; Queiroz et al., 2009).

49

Figure 1. Study area and geographic distribution of sampling sites in the middle Negro River, Barcelos Municipality. Different colors

in the basin rivers delineated its areas.

B

A

50

Sample collections and preparations

A total of 142 individuals of Cichla temensis were collected (43 adults and 99 young-

of-year) during two ocasions, March 2011 and January 2012 from nine sampling sites in the

Middle Negro River Basin. Data values from sampling sites Pt07 and Pt09 related to YOY

were absent. Consequently, to get data to represent adults birth from this two locations, the

otolith core chemical fingerprints from fish coded as G2 and ID1 were used to represent Pt07

and Pt09, respectively. The distance between samples location was about 25 rkm (river

kilometer), this procedure was made once C. temensis as reported by Hoeinghaus et al. (2003)

made movements dispersion from their targed locatins in a distance around 21 Km. The

sample locations were choose in trybutaries located in diferente geological formation and also

with diferente water types (black or white). Peacock bass were caught with rod and reel, dip

net, and line and hook. Standard length (SL) was measured for both adults and YOY to the

nearest centimeter and millimeter, respectively, and weight was recorded to the nearest gram.

To ensure that YOY individuals were collected from their natal rivers, they were sampled

while in their nests or close to them. Further, all specimens were subsequently euthanized

with ice, saved in plastic bags with identification labels, and transported in insulated boxes

with ice to the laboratory.

In the laboratory Sagittae otoliths were removed from each fish under dissecting

microscopes, rinsed in ultrapure deionizer water to facilitate the removal of remaining tissue,

and stored in numbered two-milliliter Eppendorf microcentrifuge tubes for subsequent

preparation. Afterward, one otolith of each pair was mounted with the convex surface up on

petrographic glass slides using ultra gel super glue. Otoliths were then hand-polished in the

horizontal plane using a 30μm and 3μm Al2O3 lapping film at an approximate 50µm distance

from the core (Jones and Chen, 2003). Once ground, the otolith was cleaned for two minutes

in ultraclean water in an ultrasonic bath machine (Bransonic 200®), and air-dried under a

laminar flow hood for two hours. All cleaning took place in an air-clean system (600 PCR

workstation). Next, the otoliths were saved in a slide box in Ziplock plastic bags.

51

Strontium isotope and elemental analysis

Strontium isotopes and elemental compositions of Ba/Ca and Sr/Ca ratios from

individual peacock bass otoliths were analyzed at the Wood Hole Oceanographic Institution

(WHOI), by means of a Thermo Finnigan Neptune multiple collector and an inductively

coupled plasma mass spectrometer (LA-MC-ICP-MS) in conjunction with UP-193 laser

ablation systems were used. Primarily, the otolith samples of YOY and adult peacock bass

were organized in a chamber. Subsequently the best annuli position in the YOY and adult fish

otoliths was determined using a microscope attached to a video camera and computer system.

Once the otolith annuli was found, a spot raster was drawn on the otolith surface of the YOY,

and a raster and transect line (from the core moving to the otolith edge) were drawn on the

otolith surface of adults. These procedures were programmed and saved for ablation.

This methodology was used to ablate a 250 µm x 250 µm spot raster centered on the

nucleus of each otolith in order to obtain the natal origin concentrations of trace elements and

87Sr/86Sr isotope ratios from adult and YOY peacock bass. A laser ablated a spot with a 100

µm diameter per blast on the adults otolith surface along the transect line while running at

100% power, or 10 Hz repetition rate and 6 µm·s-1scan speed. The methods used included

blank and mass bias corrections as outlined by Walther et al. (2008) and Walther and

Thorrold (2010). Thus, ratios of Sr/Ca, Ba/Ca and 87Sr/86Sr isotopes were quantified with a

single ablated spot raster or on the transect line through the microscope’s objective lens for

each YOY and adult peacock bass.

Water data collection and analysis

Water quality parameters were measured during the experimental fisheries, using a HI-

9828 Multi-Parameter Water Quality Portable Meter, in order to get data about hydrogenionic

potential (pH); temperature, TEMP (oC); conductivity, COND (µS·cm-¹); and Dissolved

oxygen, DO (mg·l-¹) from each selected tributaries in Negro River basin. To test water

parameters difference among sample locations were used analysis of variance (ANOVA)

52

applied separately for each parameter. When differences was confirmed were used a Tukey

HSD test to verify where the differences occurred within water locations.

Otoliths statistical analyses

To test the first hypothesis, the correlation between YOY and adult otolith core

chemical composition was analyzed by applying a Pearson's correlation analysis on the data

set for fish from the same place. Subsequently, a canonical discriminant function analysis

(CDFA) with a Wilks’ lambda (λ) test was performed to verify which variable (87Sr/86Sr,

Sr/Ca and Ba/Ca) better reflect the distribution of the data groups (Cruz-Castillo et al., 1994;

Nowling et al., 2011) through the difference of distance among canonical variables. A cross-

validation table procedure was also used to verify the probability of adult fish to be accurately

predicted in their nursery areas.

The second hypothesis was tested using an analysis of variance (ANOVA) applied

individually for each variable (87Sr/86Sr isotope, Sr/Ca and Ba/Ca) to verify significant

differences among groups of adults’ otolith core chemical fingerprint sampled in tributaries

with different water types. If this hypothesis was rejected, a post hoc Tukey’s HSD (honestly

significant difference) test was used to identify where these differences occur between

tributaries.

The third hypothesis was tested by direct observations of 87Sr/86Sr values from adult

fish’s otolith transects plotted in a scatter-plot in order to verify differences in 87Sr/86Sr values

that could imply fish movement among sampling sites.

Finally, the values of 87Sr/86Sr from adults fish's transect line (navigated route) were

plotted in a scatter-plot (x, y), to verify any correlation between the otolith microchemistry

from their nursery grounds to their caught location, in order to verify possible indications of

homing back movements.

The Pearson, ANOVA and Tukey tests were performed using Statistica 7.0 (StatSoft,

Inc), whereas CDFA analysis was executed with SPSS (SPSS, Inc). All data analysis were

carried out using probability values of 0.05 as the level of statistical significance.

53

Results

Young-of-year (YOY) and adult peacock bass otolith core microchemical analyses

A Pearson's analysis was applied in the Sr isotopes and elemental composition data

from YOY and adults’ otoliths core chemical compositions and showed positive correlation,

which were mainly explained by 87Sr/86Sr with r2= 0.8903 and by Sr/Ca ratios’ composition

with r2= 0.5512. These results indicated a strong correlation among the groups of variables

analyzed.

The CDFA analysis revealed function values of 87Sr/86Sr (Wilk's λ = 0.0001, χ2 =

971.26, df=18, p<0.0001), Sr/Ca (Wilk's λ = 0.026, χ2 = 339.11, df = 10, p<0.0001), and of

Ba/Ca (Wilk's λ = 0.335, χ2=101.76, df = 4, p<0.0001) presenting significant differences

among canonical variable groups. The distribution of canonical variables was largely driven

by variations in 87Sr/86Sr values emphasized in function 1, explained by the Eigenvalue of

98.5% and canonical correlation of 0.999.

The canonical scores of YOY and adult peacock bass were distributed according to

their influence to plot the data set in the canonical bidimentional space, resulting in similar

values for both YOYs and adults, which matched positively. This finding suggests that the

confidence with which adults were classified to a particular YOY birth area was based on the

homologous geochemical signatures. There were two adult fish that did not match properly

with YOY sampling sites: one fish from Pt03 and another from Pt04 (Figure 2).

54

Figure 2. A scatter-plot displaying the distribution of Cichla temensis’ YOY and adult

otolith core chemical fingerprints. The diamond symbols within the colored ellipses are the

YOY otolith fingerprints, and all of the other symbols correspond to the different adult fish

sampling sites. The same colors for the ellipses and for the solid symbols indicate possible

relationships between YOY and adult fish.

The CDFA joined the YOY and adults together according to similarities in their

microchemical signatures. This tendency was clearly observed in function 1, with the

distribution of adult peacock bass from sampling sites Pt01, Pt03, Pt04 and Pt05; the other

sampling sites were grouped together, which made interpretation more difficult.

These results were also confirmed using a cross-validation table procedure that shows

classification accuracies ranging from 14.3% to 100% and from 16.7% to 100% for both

YOY and adult fish, respectively. These chemical distribution signatures among fish groups

and sampling sites were predicted as follows:

Adult peacock bass caught at the Cuiuni (Pt01) and Demeni (Pt05) Rivers were

correctly matched to their birth locations with an accuracy of 100%. One fish from location

Pt06 was predicted to have originated from the Demeni River (Pt05) with 100% accuracy.

The fish caught at the Aracá River (Pt04) were classified as coming from the Pt03 with 83.3%

55

accuracy, and one fish was predicted to have come from location Pt06 with an accuracy of

16.7%. Also, individuals collected at Pt03 were correctly classified as from this location with

83.3% accuracy, while 16.7% were predicted to come from location Pt06. The otolith of fish

collected at the Pt02 were correctly predicted with 25% accuracy, while the other 25% and

50% were misclassified as having come from the Demeni River (Pt05) and location Pt06,

respectively.

Finally, the fish caught at the Preto River (Pt08) were classified with 16.7% accuracy

as originating from this location, while 66.7% were predicted to have come from location

Pt06, followed by 16.7% classified as from location Pt02. All of the adult otolith core

chemical signatures were correctly classified at the YOY sampling sites with an average of

41.9% accuracy (Table 1).

Table 1. Summary of linear discriminant functions analysis in matching adults’

unknown origin to known YOY sampling sites. Outside the parentheses the fish classification

accuracy is noted in percentiles, and the correlated number of individuals is located inside.

The symbol “*” means not YOY or adult fish used in the analysis.

Classification Resultsa,b

Predicted birth locations for YOY and adult fish

Pt01 Pt02 Pt03 Pt04 Pt05 Pt06 Pt07 Pt08 Pt09 (N)

Cases

Selected

Cases not

Selected

Birth

location

of YOY

Pt01 100.0 14

Pt02 73.3(11) 26.7(4) 15

Pt03 100.0 15

Pt04 100.0 11

Pt05 100.0 15

Pt06 14.3(2) 85.7(12) 14

Pt07 * 00

Pt08 100.0 15

Pt09 * 00

Caught

location

of Adult

Pt01 100.0 5

Pt02 25.0(1) 25.0(1) 50.0(2) 4

Pt03 83.3(5) 16.7(1) 6

Pt04 83.3(5) 16.7(1) 6

Pt05 100.0 6

Pt06 100.0 1

Pt07 * 4

Pt08 16.7(1) 66.7(4) 16.7(1) 6

Pt09 * 5

a. 93.9% of selected original YOY grouped cases correctly classified

b. 41.9% of unselected original adult grouped cases correctly classified

56

Adult peacock bass otolith core chemical analysis

Using 87Sr/86Sr ratio as response variable measured from the adults otoliths core, there

were differences between the otoliths chemistry of the adult peacock bass within sampling

sites (F8, 32 = 402.41, p < 0.0001). Also Sr/Ca presented values of F8, 32 = 12.97 with p <

0.0001, showing significant differences among groups of adult fish by sampling sites.

Contrary to Ba/Ca, ANOVA showed values of F8, 32 = 1.65 and p < 0.175, indicating that

there are no significant differences among fish groups. Tukey's HSD test for 87Sr/86Sr

presented no significant differences among sampling sites for Pt02 related to Pt06 (df = 32, p

= 0.936397) and to Pt08 (df = 32, p = 0.230759); Pt03 was correlated with Pt04 (df = 32, p =

0.994172); Pt05 was related to Pt06 (df = 32, p = 0.988656), Pt07 was related to Pt08 (df =

32, p = 0.121124) and to Pt09 (df = 32, p = 0.900394); Pt08 was related with Pt06 (df = 32, p

= 0.140686). Also for Sr/Ca no significant differences among sampling sites were found for

Pt02 and sampling sites Pt03 (df = 32, p = 1.000), Pt04 (df = 32, p = 0.999821), Pt06 (df = 32,

p = 1.000), Pt07 (df = 32, p = 0.999525), and Pt08 (df = 32, p = 0.998068); also between

sampling sites Pt05 and Pt09 (df = 32, p = 1.000), no significantly differences were found. As

well, Tukey's test for Ba/Ca were no significantly different among all sampling sites.

The evidence was apparent when looking at the values of 87Sr/86Sr isotopes from

sampling sites Pt03 and Pt04 (Aracá River) that presented the highest values of 87Sr/86Sr

isotopes. Intermediate values of 87Sr/86Sr were found from sampling sites Pt05 (Demeni

River), Pt07, Pt08 (Preto River) and Pt09 (Padauari River), also very similar to values of

87Sr/86Sr isotopes found in samplings Pt02 and Pt06 situated at the Negro River main channel.

On the other hand, the lowest radiogenic values of 87Sr/86Sr isotopes were found at the Cuiuni

River in sampling site Pt01 (Figure 3).

57

Figure 3. Adult Cichla temensis peacock bass’ otolith core 87Sr/86Sr isotope ratio distributed

by sampling sites. Identical letters mean homogeneous chemical values (ANOVA followed by

Tukey's test).

On the other hand, when comparing the values of Sr/Ca and Ba/Ca elemental

composition, only Sr/Ca presented notable differences in their chemical values with a

decreasing pattern occurring from sampling sites Pt01 (Cuiuni River) to Pt09 (Padauari

River). These variations occurred in three distinct situations: i) the samples collected at the

Cuiuni River (Pt01), situated at the southern margin of the Negro River, presented higher

elemental values than the others sampling sites, ii) all otolith cores sampled from black water

tributaries (Pt02 and Pt06), situated in the northern margin of the Negro River, presented

similar elemental composition values, iii) the otolith fingerprint from white water tributaries

(Pt05 and Pt09) exhibited homogeneous chemical values of Sr/Ca and Ba/Ca ratios, also

presenting lowest values of these elements/Ca when comparing its values with those of

nearby black water tributaries (Figure 4A and 4B).

58

Figure 4. Distribution of elemental compositions from adult Cichla temensis peacock bass’

otolith core. Identical letters mean homogeneous chemical values (ANOVA followed by

Tukey's test).

59

87Sr/86Sr isotope ratio analysis from otolith transects of adults peacock bass

A total of 43 otoliths from the adults fish were used to ablate a transect (line) from the

core to the edge in order to verify variations in 87Sr/86Sr isotopes. The results suggest that

55.8% (n=24) of adult C. temensis remained in their birth locations, especially fish from the

Cuiuni (Pt01) and Demeni (Pt05) Rivers, as well as fish from the confluence of the Preto and

Padauari Rivers (Pt07), which presented homogeneous values for 87Sr/86Sr isotopes. The

findings suggest that these fish did not move far away from their birth location (Figure 5). In

addition, other individual fish stayed in their birthplaces as observed in the Preto River (Pt08,

n=1), Padauari River (Pt09, n=3), Aracá River (Pt04, n=1), at the confluence of the Aracá and

Demeni Rivers (Pt03, n=1), and at the confluence of the Negro and Demeni Rivers (Pt02,

n=2).

Pt07

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,8

0

96

19

2

28

8

38

4

48

0

57

6

67

2

76

8

86

4

96

0

10

56

11

52

12

48

13

44

14

40

15

36

16

32

17

28

18

24

19

20

20

16

21

12

22

08

23

04

24

00

24

96

87Sr

/86Sr

Distance from core (µm)

A1 (31)

A2 (38.5)

A3 (26.5)

A4 (24.5)

A5 (25)

E1 (42.5)

E2 (26)

E3 (26)

E4 (24.5)

E5 (46.5)

E6 (32.5)

G1 (51)

G2 (43.5)

G3 (27.5)

G4 (57)

Fish code

Demeni River

Cuiuni River

Figure 5. Cichla temensis peacock bass’ movement history from their birth to the locations

where they were caught as adult individuals, based on variation in 87Sr/86Sr isotope ratio

values on the otolith transect. Pt07 refers to the confluence of the Preto and Padauari rivers.

The standard length values of fish are displayed between parentheses. The spots at the end of

lines represent the adult’s caught locations.

60

Differences in 87Sr/86Sr isotope ratio values from adult’s transects also suggest that

44.18% (n=19) of adult peacock bass moved away from their birth location to other places,

and also showed that they come back, at least once (n=16), to their natal sites (Figure 6A, 6B,

6C, 6D and 6E). A clear example of fish leaving and returning to their birth locations is

observed in the adult peacock bass caught at the Aracá River (fish code: P9L-76.5) (Fig.6C)

and other fish from the Padauari River (fish code: ID3-35.5) (Fig.6E). Among the movers,

three individuals (fish codes: CD1-59, Fig.6B; P9L-76.5, Fig.6C; and HD1-62.5, Fig.6D)

presented differences in their otolith cores, revealing that these fish belong to unknown birth

locations, and that they have moved into the YOY sampling sites.

The last part (dotted lines and spot) at the end of each transect line correspond to the

adult catch locations (YOY chemical fingerprint reference). The exception were the fish from

the Padauari River (Fig.6E), where the last part of the transect line "reference location" comes

from average values of 87Sr/86Sr of the fish (ID1-21), due to its consistent values of 87Sr/86Sr

presented in its transect line. This procedure was made due to the nonexistence of YOY

reference material for that sampling site.

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,8

0

96

19

2

28

8

38

4

48

0

57

6

67

2

76

8

86

4

96

0

10

56

11

52

12

48

13

44

14

40

15

36

16

32

17

28

18

24

19

20

20

16

21

12

22

08

23

04

24

00

24

96

87

Sr/86

Sr


BD1 (46)

BD2 (28.5)

BD3 (29)

P3 (28)

Fish code

A

61

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,8

0

14

4

28

8

43

2

57

6

72

0

86

4

10

08

11

52

12

96

14

40

15

84

17

28

18

72

20

16

21

60

23

04

24

48

25

92

27

36

28

80

30

24

87Sr

/86Sr


CD1 (59)

CD2 (28)

CD4 (27.5)

P4 (27.5)

P5 (26)

P6 (24)

Fish code

B

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,80

0

96

19

2

28

8

38

4

48

0

57

6

67

2

76

8

86

4

96

0

10

56

11

52

12

48

13

44

14

40

15

36

16

32

17

28

18

24

19

20

20

16

21

12

22

08

23

04

24

00

24

96

25

92

87Sr

/86Sr


DD1 (25.5)

DD2 (26)

DD3 (24.5)

P7 (24)

P8 (22.5)

P9L (76.5)

C

Fish code

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,8

0

96

19

2

28

8

38

4

48

0

57

6

67

2

76

8

86

4

96

0

10

56

11

52

12

48

13

44

14

40

15

36

16

32

17

28

18

24

19

20

20

16

21

12

22

08

23

04

24

00

24

96

87Sr

/86Sr


HD1 (62.5)

HD2 (56)

HD3 (51)

P14 (49)

P15 (46.5)

P16 (52.5)

Fish code

D

62

Figure 6. Transect lines displaying Cichla temensis peacock bass’ movement histories from

their birth to catch locations. Where: A = Pt02; B = Pt03; C = Pt04-Aracá River; D = Pt08-

Preto River; E = Pt09-Padauari River. In parentheses are the standard lengths (cm) of fish.

Water chemical analysis

We used the discriminate function analysis to quantify the limnological variables from

sample pairs for all studied tributaries. We also applied ANOVA to verify differences in water

parameters among sample locations and a post hoc Tukey analysis when differences we

found. The limnological values in means and standard deviation are contents in Table 2.

0,71

0,72

0,73

0,74

0,75

0,76

0,77

0,78

0,79

0,8

0

14

4

28

8

43

2

57

6

72

0

86

4

10

08

11

52

12

96

14

40

15

84

17

28

18

72

20

16

21

60

23

04

24

48

25

92

27

36

28

80

30

24

31

68

87Sr

/86Sr


ID1 (21)

ID2 (46.5)

ID3 (35.5)

P17 (34.5)

P18 (50)

Fish code

E

63

Table 2. Limnological variables from samples locations distributed at the Negro River

basin (Means ± standard deviation)

Sampling

sites pH TEMP DO COND

Pt01 4.68 ± 0.37 28.28 ± 1.62 1.86 ± 0.70 11.50 ± 2.12

Pt02 4.92 ± 0.22 27.86 ± 0.87 5.22 ± 2.05 11.50 ± 3.53

Pt03 4.69 ± 0.00 28.67 ± 1.03 1.97 ± 2.50 17.50 ± 0.70

Pt04 4.52 ± 0.00 28.79 ± 1.20 1.13 ± 0.89 17.00 ± 0.00

Pt05 5.38 ± 0.00 29.01 ± 2.26 1.02 ± 0.85 15.00 ± 5.65

Pt06 4.52 ± 0.11 26.42 ± 0,76 3.86 ± 0.55 18.50 ± 2.12

Pt07 4.34 ± 0.00 26.53 ± 1.56 3.23 ± 1.07 16.50 ± 4.94

Pt08 4.20 ± 0.14 26.22 ± 1.14 3.18 ± 0.45 26.00 ± 4.24

Pt09 4.70 ± 0.24 27.02 ± 2.19 3.35 ± 1.76 10.00 ± 2.82

ANOVA p = 0.02 p = 0.45 p = 0.16 p = 0.02

Tukey HSD test

pH: Pt05 ≠ Pt07 with p = 0.01 and Pt05 ≠ Pt08 with p = 0.03

CON: Pt08 ≠ Pt01, Pt02 with p = 0.03 and Pt08 ≠ Pt09, with p = 0.01

Where: DO, Dissolved oxygen (mg·l-¹); TEMP, temperature (oC); pH; COND,

conductivity (µS·cm-¹).

Discussion

The results confirm that otolith microchemistry presents spatial geochemistry

differentiation that is associated with stream beds’ geology, and can be used as a natural tag to

track individual fish movement among populations situated in habitats with different water

types (e.g. Humston et al., 2010; Walter and Limburg, 2012).

This method was effective and reveals the usual departure of Cichla temensis from

their nursery locations. It also shows that peacock bass may return to its birth location at

mature ages. These results are observed by analyzing 87Sr/86Sr isotope values from a transect

64

line ablated in the otoliths (from the core to the edge) of adult fish. Many studies using the

same technique have successfully tracked movements of small (Humston, 2004) and large fish

(Thorrold et al., 2001), proving that otoliths’ microchemistry really functions as an

environmental fingerprint (Walter et al., 2008) in terms of correctly identifying fish’s natal

places (Thorrold et al., 2001; Hamann and Kennedy, 2012), and inter-stream movements

(Gillanders, 2002; Muhlfeld et al., 2012; Wolff et al., 2012) thereby successfully surpassing

the conventional tagging techniques.

The peacock bass C. temensis and its congeners are included in the group of K-

strategist behaviour species (Pianka, 1970). Due to their particular parental care

characteristics, however, Winemiller (1991) classified them as equilibrium strategist species.

Thus far, Cichla spp. is recognized as a sedentary fish due to its short distance movements

between rivers and lakes (Hoeinghaus et al., 2003; Granado-Lorencio et al., 2005; Kehrig et

al., 2008; Olivares et al., 2013).

K-strategist fish commonly has a long life, reproduces on a seasonal basis, and

provides parental care (Pianka, 1970) meanwhile equilibrium strategists’ concept emphasizes

the elevated offspring survivorship as a result of the fathers’ rigorous parental care

(Winemiller and Rose, 1992). Our results also bring new information that could help to

improve C. temensis classification. We find that this fish species leaves their nursery grounds

and returns with a high level of geographic fidelity, once 55.8% of the fish stayed and 44.18%

returned at least once to their birth locations. Mayr (1963) theorizes this pattern as philopatric

behavior. This theoretical evolution of the movement of peacock bass C. temensis is

demonstrated in Figure 7, where i) fish did not move, ii) fish moved slightly and, iii) birth

place fidelity behavior where the fish left and returned to their birth places.

65

Figure 7. Organizational chart with C. temensis movements. Where: N.A = Nursery area.

References: a = Pianka (1970), b = Winemiller (1989), c = Holley et al. (2008), d =

Hoeinghaus et al. (2003), e = the present study. The narrow full line represents the fish

moving out of their nursery area; the dotted lines are the fish returning.

Recent studies also report that peacock bass moved out to their natal areas.

Hoeinghaus et al. (2003) studied C. temensis at the Cinaruco River (Venezuela), and collected

four tagged individuals approximately 21 kilometers from their initial marking places. Holley

et al. (2008), using the same mark-and-recapture (M-R) technique, caught one peacock bass

40 kilometers from the marking place in the Middle Negro River basin (Brazil). In addition,

other studies reported little information that had been collected on the movement of wild C.

temensis populations (Taphorn & Barbarino Duque, 1993; Thorstad et al., 2001).

However, these studies inconclusively indicate a movement pattern for C. temensis;

they only show dispersal movements from the fish’s tagged and catch locations. This situation

may be attributed to problems with M-R technique because traditional tagging methodology is

not appropriate for tracking small fish and neither for reconstructing adult fish’s movement

history (Thorrold et al., 2002; Hendry et al., 2004).

66

Cichla temensis movements, reproduction and phylopatric behaviour synchronism.

Our results showed a strong correlation between adult and YOY otoliths’ isotope and

elemental compositions from C. temensis, confirming that both fish groups belong to the same

nursery area. Further, by examining just otoliths’ 87Sr/86Sr isotope transect, variation in its

chemistry values were observed, showing that these fish left their nursery areas at some point

in their lives and returned later, at least once, to the same birth location. Previous studies with

introduced (Zaret, 1980) and native (Hoeinghaus et al. 2003) Cichla populations hypothesized

that most of fish remain close to their natal or capture locations (Macrander, 2010).

In contrast, our results showed that C. temensis move extensively to regions distant

from their nursery areas, but that they return with high levels of fidelity. This movement

pattern may be indicative of philopatry, which is a strong affection for the birth location that

sustains the individual fish permanently in its nursery ground or causes it to return repeatedly

(Mayr, 1963; Hueter et al., 2004).

This supposition is in agreement with Weatherhead et al. (1994), who reported that

philopatry is the return of a species to their birthplace in order to breed. Also, the philopatric

concept rests on the assumption that an individual moves out of its nursery area (known

location) to explore other environments and later navigates back to its original location with

high frequency. This behaviour was supposed to improve individuals’ familiarization with the

food and others resources in the local area, which would allow it to obtain greater advantages

than individual dispersers (Weatherhead and Forbes, 1994; Robertson and Cooke, 1999).

The degree of homecoming found in this study for C. temensis from otolith

fingerprints suggests a philopatric behaviour for this species in the Negro River basin. A

reasonable explanation to support this assumption for C. temensis is the possibility of these

individuals having to return to their nursery grounds in order to breed. This supposition is

supported by the premise that most of the C. temensis caught in this study occurred during the

dry season that corresponds with their reproduction period (Taphorn and Duque, 1996;

Winermiller et al., 1997; Jepsen et al., 1999; Hoeinghaus et al., 2003; Morales-Nin & Panfili,

2005; Montana et al., 2006; Gomiero et al., 2009). Another reason to sustain this assertion is

that all adult peacock bass were caught close to the YOY nests; and this could presumably be

associated with parental care, such as effectively protecting their nests (Taphorn and Duque,

1996), which is suggestive of recent breeding time.

67

Hydrological barrier and C. temensis metapopulation structure

The Negro River basin connects tributaries with significantly different water types,

including black-water and white-water. Many physical and chemical changes in aquatic

environments can interfer directly on fish movements between different locations, mainly

when such chemical variation are large enough to work as hydrochemical barriers

(Winemiller et al., 2008; Duncan and Fernandes, 2010; Willis et al., 2010).

Recent studies in the Amazon basin have hypothesized the existence of a biological

filter for gene flow between the Negro and Orinoco Rivers for C. temensis, and between the

Negro and Amazon rivers for C. monoculus (Macrander, 2010). Winemiller et al. (2008) and

Willis et al. (2010) also suggest that the gradient of environmental conditions along a river’s

course might create an ecological barrier for the dispersal of many fish species (see also

Torrente-Vilara et al., 2011).

The tributaries of the Negro River also differ greatly in 87Sr/86Sr isotopes ratio and

elemental compositions (Allègre et al., 1996), and these chemical differences in aquatic

habitats could work combined with others limnological parameters as a hydrochemical barrier

for fish groups in terms of spatial distribution (Winemiller et al., 2008; Macrander, 2010).

Corroborating with this statement, our results show that based on 87Sr/86Sr isotopes from

peacock bass otoliths’ transect, the occupancy of fish in tributaries with homogeneous values

of 87Sr/86Sr isotopes or with a soft increasing of Sr isotopes in gradients waters, permits C.

temensis to do movement dispersion among location. Contrary, when differences in values of

87Sr/86Sr isotopes occurs abruptly in the aquatic environment the fish did not leave their

nursery ground.

The chemical similarity between otoliths and ambient water is strongly correlated

when comparing the fish otolith environmental fingerprint from the Cuiuni River with its

87Sr/86Sr isotope values from sediment load, sampled by Allègre et al. (1996). The Cuiuni

River (Pt01) headwaters are located at the Solimões Formation where it receives sediments

from the Solimões River during the flood period, containing low 87Sr/86Sr isotope values,

around 0.71319 ± 0.00002 (Allègre et al., 1996), which closely resemble to the 87Sr/86Sr

isotopes found in the otolith core of both peacock bass YOY (0.71314 ± 0.00066) and adults

(0.71365 ± 0.00233) sampled in the same area. Also, analogous microchemistry from

68

surrounding habitats was found in samples from tributaries with catchment areas situated at

the Guyana Shield that presented high radiogenic 87Sr/86Sr isotopes with equivalent values

between YOY and adult peacock bass as observed in the Aracá River (Pt04) with 87Sr/86Sr

isotope values for YOY equal to 0.78292 ± 0.00101 and for adults equal to 0.78089 ±

0.00158. Samples from the Demini River (Pt05) presented values of 87Sr/86Sr isotopes from

YOY equal to 0.73494 ± 0.00016; these were very similar to those of adult fish with 0.73557

± 0.00055. Also, samples from the Preto River (Pt08) presented 87Sr/86Sr isotope values for

YOY equal to 0.74211 ± 0.00100, and these were very similar to values from adult fish with

0.74535 ± 0.00390.

According to otolith microchemistry, we can deduce that C. temensis from the Negro

River retained themselves away from aquatic environments with high geochemical

differentiation represented by the 87Sr/86Sr isotopes values, and it’s are not related directly

with water color, but straight with water geochemical and limnological composition. This

chemical discrepancy could be a indicative of local hydro-chemical barriers for C. temensis as

reported in several studies in the Amazon basin for Cichla spp. and for others fish species as

well (e.g. Winemiller et al., 2008; Macrander, 2010; Willis et al., 2010).

The main argument to support this assumption is that C. temensis move between

tributaries with similar geochemical compositions, but they do not leave their habitats if large

geochemical differences between tributaries exist. Accordingly, to Jepsen et al. (1997) and

Macrander (2010), Cichla species exhibit distinct habitat preferences and appear to be able to

disperse across habitats with relatively similar chemical compositions (Figure 8).

≈ ≈

≠

≈WW

BW

BW BW

Movements

Isolation Isolation

69

Figure 8. The organograme illustrates C. temensis movement behaviour among different water

types in relation to differences in otoliths’ geochemical composition (87Sr/86Sr isotopes). With

≈ (similar) or ≠ (dissimilar) 87Sr/86Sr isotopes values; BW = Black water and WW = white

water habitats.

According to Sr isotopes in this study, the peacock bass groups became isolated by the

local geochemical composition as presented from individuals caught at the Cuiuni (Pt01) and

Demeni (Pt05) rivers, which presented high microchemical differentiation from the

surrounding aquatic habitats, with the main Negro River channel and Aracá River,

respectively, that probably work as a hydro-chemical barrier for C. temensis. Contrastingly,

peacock bass movements occurred in other Negro River tributaries with similar watershed

chemical compositions but not with the same water types, such as the Preto River (black-

water) and Padauari River (white-water). This chemical homogeneity among the

environments, despite the differences in water type, allows C. temensis from the Preto River

to enter and leave the Padauari River. On the other hand, the fish group from the Padauari

River did not show a dispersal movement in the direction of the Preto River, but only moving

up Padauary River. (Figure 9).

70

22

31 11

1

21 1 11

1 4 1

3

1

1

?

?

Cuiuni River

b

c

a

d

e

f

g

h

Legend

i

Figure 9. Theoretical spatial movement distribution of Cichla temensis in the middle Negro

River basin. Where: ? = unknown locations; a = birth and catch place; b = catch place; c =

birth place; d = fish move out direction; e = homecoming direction; f = white water; g = black

water; h = water flow direction; and i = isolated fish group. Obs: the areas under the

confluences betweem Aracá and Demeni, and Padauari and Preto rivers are Mixed water.

The results also shows that the main factor that could acts as a hydrochemical barrier

for C. temensis, are both the local geological and limnological parameters, and not the water

types. One example that could corroborates with this assumption is the existence of an

isolated C. temensis population from location Pt07 that shows no movement dispersion, its

happened maybe due to the limnological parameter differences from surround locations as

Preto and Padauari Rivers, differing from the others isolated groups as Pt01 and Pt05 that

appears to be separated by the geochemical compositions as 87Sr/86Sr isotopes values. This

supposition is based on the premise that fish assemblages reacts when variations in water

physical and chemical parameters occurs (Garcez and Freitas, 2008), and can works as a

hydro-chemical barrier for many fish species (Winemiller et al., 2008; Duncan & Fernandes,

2010).

71

The small geochemical variation from the Preto River with low 87Sr/86Sr isotopes and

high values of Ba/Ca and Sr/Ca ratios in relation to the Padauari River (white water)

permitted the fish group from this area to engage in dispersal movements between both rivers.

According to variations in 87Sr/86Sr isotopes, the fish from the Padauari River moved up and

down in the main channel to unknown locations. Similar situations have been reported by

Winemiller et al. (2008) who inferred that only a few species are able to move from black

water in the Negro River to white water in the upper Orinoco River, this fish movements

limitations are linked directly with limnological differences in aquatic environments (Willis et

al. (2010).

Our results show that the presence of peacock bass populations established inside and

between tributaries with different water types and geochemical composition in the Negro

River micro-basins, predicts a possible homecoming behavior and spatial population

distribution with the existence of connectivity among groups that maybe works as a

metapopulation organization. To better understand the peacock bass as a metapopulation

structure, however, the aquatic environment needs to be considered as patches that are

seasonally linked by the Negro River through the flood pulse (Junk, 1997; Parolin et al.,

2004) and by the movements of fish between patches (aquatic habitats).

The fish’s ability and adaptability to navigate among habitats with different water

chemistry will determine which population will persist locally. An isolated population will be

more vulnerable to extinction than connected populations, especially when considering the

environmental conditions and fisheries activities in those areas (Ricklefs, 1996). In order to

support this affirmation, it is necessary to gather robust sampling data with sufficient

information about fish movement among sampling sites over a large time scale.

However, we have yet to address water microchemical analysis encompassing the

hydrological gradient in the sampling sites. The otoliths’ microchemical data set suggests that

water chemistry may be a factor that is influencing the dispersal movement of C. temensis and

the formation of isolated and connected populations, with changes in the water chemistry

directly affecting fish’s physiological conditions (Val & Almeida-Val, 1995) and its otolith

compounds as well (Walter and Thorrold, 2006). This affirmation is in agreement with many

studies realized in the Amazon basin, which suggest that some rivers with environmental

heterogeneity operate as a hydro-chemical barrier for fish (e.g. Willis et al., 2007; Toffoli et

al., 2008; Winemiller et al., 2008; Duncan & Fernandes, 2010).

72

Our results show much more spatial and temporal dynamics in the C. temensis

movements than reported in previous studies (e.g. Holley et al., 2008; Hoeinghaus et al.,

2003; Thorstad et al., 2001), and that this information may be useful when considering the

peacock bass population’s movement dynamics in the Negro River basin.

Peacock bass fisheries management proposition

The Negro River basin is composed of many tributaries, and its headwaters are located

on a different geologic formation where the water’s chemical composition differs from other

parts of the basin (Queiroz et al. 2009). As a result, if an individual fish passes across these

different geochemical environments its otolith will record this information as an

environmental fingerprint (Elsdon and Gillander, 2005). Thus, knowing the fish movements

within and between tributaries, we presume that it is possible to use this movement

information for fisheries management planning.

The fish’s movement dynamics are fundamental to understanding spatial distribution,

which is the first step in considering how to adequately manage fishery sectors. Our results

show that based on variation in the 87Sr/86Sr isotope, C. temensis exhibit dispersion

movements among Negro River tributaries, and that part of the individuals may return to their

birthplaces with a high level of fidelity. Also were observed in this study that some groups

and individuals of C. temensis did not leave their nursery grounds, and this may be a

consequence of a hydrochemical barrier between habitats for this fish species (e.g. Duncan

and Fernandes, 2010; Willis et al., 2010).

This scenario also, maybe interpreted as a metapopulation structure that rests on fish’s

ability to enter or leave an aquatic environment in a colonization process (Kritzer and Sale,

2004). Considering the existence of connected and isolated peacock bass populations, we can

view the possibility of managing the fisheries in those areas differently on a spatial and

temporal scale.

Spatially, fisheries management needs to consider the peacock bass’ dispersal

movements among network tributaries as fundamental information to the protection of the

host of habitats used by the fish, as a metapopulation space that is formed by a gathering of

73

fish populations (Levins, 1969). On the other hand, when a school of fish faces a

hydrochemical barrier between tributaries, this group of fish needs to be supervised

differently as an isolated population because these populations struggle to recover in a short

period, mainly when the fisheries effort occurs in those areas with high intensity and

frequency (Kritzer and Sale, 2004). On a temporal scale, the hydrological conditions also

need to be measured, mainly during the drought, period when peacock bass’ spawn

(Winermiller et al., 1997; Gomiero et al., 2009) that corresponds with the high season of sport

fishing in the Negro River basin (Freitas and Rivas, 2006).

For both, connected and isolated populations of peacock bass, the hydrological cycle

and fish physiological and biological conditions should be taken into consideration for

fisheries management procedures. This thinking is important to decide the areas where fishing

should be authorized, and during which times of the year, in order to avoid conflicts between

sport, commercial and subsistence fisheries (e.g. Sobreiro, 2007) and preserve the fish stock

as well.

We believe that mainly 87Sr/86Sr isotopes found in otoliths of peacock bass represent

an excellent, and yet underused record of the geochemical properties of aquatic environments

in the Negro River basin. The C. temensis otolith geochemical fingerprints also highlight the

potential to demarcate fisheries sectors and guide effective fisheries management plans in

freshwater environments.

Acknowledgments

We thank Mr. Julio. A. D. Siqueira for his assistance in the field collecting the

samples. Financial support of this study was provided by Coordination for the Improvement

of Higher Education Personnel (CAPES-PDSE 0909/12-2), National Council for Scientific

and Technological Development (CNPq), Amazonas Research Foundation (FAPEAM), and

INCT-Adapta. Laboratory and logistic support also was provided by Federal University of

Amazonas (UFAM), National Institute of Amazonian Research (INPA), Intelligence Social

Environment Strategic Petroleum Industry in the Amazon region (I-PIATAM), and

Washington and Lee University (WLU). Fish were collected under research license 25606-

1/2010 from Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).

74

Conclusões

O uso da microquímica dos otólitos de tucunaré como marcadores ambientais, mostrou

ser eficiente para esclarecer os padrões de distribuição espacial da espécie C. temensis na

bacia do Médio Rio Negro. Com base nas informações dos isótopos de 87Sr/86Sr e da

concentração dos elementos traços Ba/Ca e Sr/Ca encontrados nos otólitos dos indivíduos

jovens e adultos, concluiu-se que:

i) É possível mapear a distribuição espacial de indivíduos jovens, menores que um

ano, através das diferenças microquímicas existentes em seus otólitos, que, por sua vez,

refletem a geoquímica da área de nascimento de cada grupo estudado;

ii) A microquímica da parte central dos otólitos dos tucunarés jovens foi similar à

encontrada nos otólitos de peixes adultos coletados nos mesmos locais, de forma que a

microquímica dos otólitos pode ser utilizada como impressão digital do local de nascimento

para prever a origem de indivíduos adultos capturados em diferentes locais da bacia do Rio

Negro;

iii) Os isótopos e elementos traços da parte central dos otólitos de tucunarés se

diferenciam nitidamente quando comparados à microquímica dos tributários de água preta

com os de água branca na bacia do Médio Rio Negro;

iv) A variação dos isótopos de 87Sr/86Sr em otólitos de tucunarés adultos, indicou a

existência de movimentos de dispersão de indivíduos entre os tributários do Rio Negro,

possivelmente formando metapopulações nessa região. Ainda, a microquímica dos otólitos

somadas as variações limnológicas ambientais, mostraram que as diferenças hidroquímicas

entre ambientes distintos podem atuar como barreiras hidroquímicas isolando algumas

populações;

v) Com o uso dos isótopos de 87Sr/86Sr, foi possível avaliar parte da história dos

movimentos de dispersão de cada tucunaré individualmente, onde foi verificado que parte dos

indivíduos estudados realizaram movimentos de dispersão entre os ambientes estudados, e

que outros permanecem em seus locais de origem. dos tucunarés adultos retornaram para seus

locais de nascimento com alto grau de filopatria.

75

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