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Filogenia e Filogeografia gênero
Eurycheilichthys (Siluriformes: Loricariidae)
Ana Maria Rubini Liedke
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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO GRANDE DO SUL
FACULDADE DE BIOCIÊNCIAS
PROGRAMA DE PÓS-GRADUAÇÃO EM ZOOLOGIA
Filogenia e Filogeografia gênero Eurycheilichthys (Siluriformes:
Loricariidae)
Ana Maria Rubini Liedke
Orientador: Dr. Sandro Luis Bonatto
Co-Orientador: Dr Roberto E. Reis
DISSERTAÇÃO DE MESTRADO
PORTO ALEGRE - RS - BRASIL
2007
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3
Sumário
Agradecimentos...................................................................................................IV
Resumo.................................................................................................................V
Abstract................................................................................................................VI
Apresentação.........................................................................................................7
1. Histórico do Problema...........................................................................7
1.1 Introdução................................................................................7
1.2. Sistemática do grupo...............................................................8
1.4.1 O Gênero Eurycheilichthys...................................................9
1.3 As Bacias Hidrográficas do Rio Grande do Sul ....................11
1.4. Filogenia e Filogeografia.......................................................12
2. Referências Bibliográficas....................................................................14
Artigo científico....................................................................................................15
4
Agradecimentos
Chegar ao final desse trabalho me fez lembrar e agradecer todas as pessoas que
participaram direta e indiretamente e que sem a ajuda, dedicação, compreensão,
companheirismo, apoio e amor eu não teria finalizado.
Agradeço ao Sandro por ter me aceitado como aluna, por todos esses anos
trabalhando junto, por tudo que me ensinou, e também por ter compreendido meus maus
momentos. Ao Reis, professor que sempre esteve disposto a ensinar muitas coisas, com
alegria e disposição para conversar sobre tudo, inclusive o trabalho, dando muito apoio
e idéias novas.
Ao Tiago e Edson que ajudaram na coleta dos peixes, bem como Marco
Azevedo por colaborar com amostras. Agradeço ao Prof. Régis Lahm e ao Roger Santos
do Laboratório de Tratamento de Imagens e Geoprocessamento - PUCRS, pela ajuda e
confecção do mapa de distribuição dos peixes.
Aos colegas do Genoma, em especial a Cladinara R. Sarturi e Fernanda Britto,
por estarem sempre dispostas a ajudar, e também a Roberta Graboski pela amizade que
fizemos nesses anos!
Ao Felipe, que esteve junto o tempo todo, ajudando e sugerindo novas idéias,
sempre disposto, mesmo se tivesse milhões de coisas para fazer ao mesmo tempo,
mostrando o lado “bonito” das coisas, passando um “ar de tranqüilidade” e calma...
Agradeço aos meus pais, que me orientaram sempre, com muito amor, apoio e
compreensão, e nessa fase do mestrado me mostraram em vários momentos qual o
caminho a ser seguido.... Meus irmãos, Alice e Pedro, os quais também estão no
mestrado, por todo apoio, paciência e palavras que iluminam minha vida!
Não posso deixar de agradecer minhas amigas, Viviane e Roberta, que nos
encontros quase semanais discutíamos o futuro das nossas carreiras, e como boas
ouvintes, com elas dividi minhas angústias e alegrias!! E a Isabel, que mesmo estando
longe no último ano, esteve bastante presente no primeiro!
Agradeço ao Moysés, que com todo carinho e amor sempre me apoiou e
compreendeu a distância geográfica que nos separou nessa etapa; a paciência com
minhas angústias e crises, além de sempre ouvir - falta pouco!! Pelos momentos que
juntos estivemos, os quais foram essenciais para seguir, e claro, por acreditar que no fim
tudo da certo!!!
5
Resumo
A fauna de peixes de água doce da América do Sul é uma das mais ricas e, ao mesmo
tempo, uma das menos conhecidas do mundo. O gênero Eurycheilichthys é endêmico do
sul do Brasil, possuindo apenas duas espécies descritas na literatura. Eurycheilichthys
pantherinus ocorre na Bacia do Uruguai, enquanto E. limulus ocorre somente na
microbacia do alto Jacuí, na bacia do Guaíba. Atualmente, sete novas espécies estão
sendo descritas com base na morfologia, as quais ocorrem na microbacia do Taquari-
Antas, também na bacia do Guaíba. A área de ocorrência é restrita ao platô dos estados
do Rio Grande do Sul e Santa Catarina, acima de 600 m de altitude. O presente trabalho
estuda a filogenia e a filogeografia das espécies de Eurycheilichthys utilizando
evidências moleculares. Foram seqüenciados os genes mitocondriais COI e ND2 de 126
indivíduos sendo os dados concatenados para as análises, totalizando 1208pb. A
filogenia sugere estruturação entre as bacias hidrográficas, assim como para as
microbacias. Das espécies novas, notadamente ficaram monofiléticas apenas aquelas
três com distribuição restrita a pequenos rios, enquanto as espécies de ampla ocorrência
ficaram não-monofiléticas. Quando estimado o tempo de divergência entre as espécies,
utilizando duas taxas evolutivas (0,9% e 1,8% por milhão de anos), percebe-se que o
processo de diferenciação das novas espécies foi recente (entre 0.5-1.0 Mya), sugerindo
que a não-monofilia das espécies pode ser causada por compartilhamento de linhagens
ancestrais, uma vez que é possível notar que existe uma expansão quase simultânea de
todas espécies novas. A distribuição do gênero nessas bacias hidrográficas deve ter sido
modulada por vicariância, com a formação da bacia do Uruguai, datada do Plioceno.
6
PHYLOGENY AND PHYLOGEOGRAPHY OF
Eurycheilichthys (SILURIFORME: LORICARIIDAE)
Abstract
The Neotropical genus Eurycheilichthys is endemic to the southern Brazilian
plateau, with two described species: E. pantherinus, which occurs in the upper Uruguay
River basin, and E. limulus, that is found in the upper Jacuí River microbasin, which is
part of the Guaíba Lake drainage. Seven new species are being described based on
morphological characters and they are all endemic to the upper Taquari-Antas River
microbasin, which is also part of Guaíba Lake drainage. We conducted the first
molecular analysis concerning the phylogenetic relationships and phylogeography of the
whole genus Eurycheilichthys using two mtDNA genes (COI and ND2). The
phylogenetic trees and the network are structured by basin and microbasin and
presented both described species as monophyletic. However, only three of the new
species were monophyletic for these genes while some species showed several clades
with restrict geographical distribution which could perhaps indicate other highly
endemic cryptic species or the non-monophyly may be caused by incomplete lineage
sorting. The origin of the Taquari-Antas clade was estimated around 0.5-1.0 million
years ago while the divergence time among the species from this microbasin may have
occurred around half a million years ago. Population analyses showed signals of a
recent population growth for most of the species. The disjunct distribution among the
river basins likely happened between the late Pliocene and the early Pleistocene, with
the origin of the Uruguay basin. The high diversity and degree of endemicity of this
group in the Taquari-Antas microbasin may be explained by its high declivity
associated with the ecological restriction of the group.
KEYWORDS: molecular phylogeny, phylogeography, Loricariidae, Eurycheilichthys.
7
APRESENTAÇÃO
1. HISTÓRICO DO PROBLEMA
1.1 Introdução
A fauna de peixes de água doce da América do Sul é uma das mais ricas e, ao
mesmo tempo, uma das menos conhecidas do mundo (Vari & Malabarba, 1998). Em
2003 foi publicado o livro Check List of Freshwater Fishes of South and Central
America – CLOFFSCA, o qual listou as 4475 espécies e 1550 que estão por serem
descritas, totalizando 6025 espécies, reunidas em 71 famílias (Reis et al., 2003). Esse
levantamento permite perceber o quão desconhecida é a ictiofauna, podendo se inferir
que no estado do Rio Grande do Sul existe uma grande quantidade de espécies a serem
descritas.
O gênero Eurycheilichthys é endêmico do sul do Brasil, possuindo até o
momento apenas duas espécies descritas na literatura (Reis & Schaefer, 1992; Reis &
Schaefer, 1998). Porém, expedições realizadas no estado pela equipe do Laboratório de
Ictiologia do Museu de Ciências e Tecnologia da PUCRS com o objetivo de enriquecer
o conhecimento da ictiofauna do RS, apontam para a existência de ao menos sete novas
espécies. A área de distribuição do gênero é bastante restrita, ocorrendo apenas no
planalto do Rio Grande do Sul e parte de Santa Catarina, acima de 600 metros de
altitude. Até o momento nenhum estudo enfocando a variabilidade genética e a história
evolutiva do gênero foi realizada.
8
1.2. Sistemática do grupo
De acordo com Schaefer (1991) o gênero Eurycheilichthys encontra-se classificado
conforme abaixo:
Classe: Actinopterygii
Ordem: Siluriformes
Família: Loricariidae
Sub-família: Hypoptopomatinae
Tribo: Otothyrini
Genêro: Eurycheilichthys
A classe Actinopterygii é composta pelos peixes de nadadeiras raiadas, e está
dividida em 42 ordens. Os representantes da ordem Siluriformes ocorrem
principalmente em ambientes de água doce, porém, existem duas famílias, Ariidae e
Plotosidae, que possuem representantes em águas salinas. Os Siluriformes ocorrem em
todos os continentes quando incluído os registros fósseis do Eoceno ou Oligoceno
encontrados na Antártica. Algumas espécies são conhecidas por possuírem substâncias
venenosas, produzidas em células epidérmicas do tecido que revestem os espinhos das
nadadeiras. A maioria é passiva, utilizando o espinho para defesa contra predadores. A
ordem Siluriformes é dividida em 34 famílias (Nelson, 1994).
Dentre estas, a família Loricariidae é a que contém o maior número de espécies
de peixes na região Neotropical, e provavelmente no mundo. Atualmente é composta
por 683 espécies, e a cada ano novas espécies são descritas. Esses peixes estão
distribuídos desde o norte da Costa Rica até o sul da Argentina. A grande maioria das
espécies encontra-se no lado leste da Cordilheira dos Andes, mas existem espécies que
são restritas aos declives do lado oeste da mesma. Essa família apresenta-se dividida em
seis subfamílias, sendo enfatizada a subfamília Hypoptopomatinae (Reis et al, 2006).
A subfamília Hypoptopomatinae é composta por 16 gêneros e 79 espécies. Estão
distribuídos principalmente nas planícies cisandinas, desde a Venezuela até o norte da
Argentina e ocorrem em pequenos e médios rios e córregos. Quando adultos são
pequenos, variando entre 20mm e 75mm CP (comprimento padrão). Como outros
membros dessa família, esses peixes são revestidos por placas ósseas. A boca é
adaptada para sucção, sendo os lábios bastante carnudos. A alimentação é
9
predominantemente herbívora, e o hábito, diurno. A maioria é encontrada próxima à
superfície da água, associada à vegetação das margens do rio (Reis & Schaefer, 2003).
Entre as subfamílias de Loricariidae, a subfamília Hypoptopomatinae é a mais estudada
e compreendida. As relações entre os gêneros foram quase totalmente resolvidas por
Schaefer (1991), o qual posteriormente revisou alguns gêneros e dividiu essa subfamília
em duas tribos monofiléticas: Hypoptopomatini e Otothyrini (Vari & Malabarba, 1998).
Mais recentemente, no entanto, Lehmann (2006) não encontrou evidências para suporte
da tribo Otothyrini.
1.4.1 O Gênero Eurycheilichthys
O gênero Eurycheilichthys (Fig. 1) é endêmico do estado do Rio Grande do Sul,
possuindo somente duas espécies descritas na literatura: E. pantherinus (Reis &
Schaefer, 1992) e E. limulus Reis & Schaefer, 1998. A área de ocorrência se restringe
apenas ao planalto do estado, acima de 600 metros de altitude. A espécie E. pantherinus
é encontrada apenas na bacia do Uruguai, enquanto E. limulus está presente na bacia do
Guaíba (microbacia do alto rio Jacuí).
Expedições realizadas no estado pela equipe do Laboratório de Ictiologia do
Museu de Ciências e Tecnologia da PUCRS, com o objetivo de enriquecer o
conhecimento da ictiofauna do RS, possibilitaram verificar a existência de uma área
mais ampla da distribuição do gênero Eurycheilichthys, tendo sido encontrado também
na microbacia do Taquari-Antas, além das outras duas citadas acima (Fig. 2). A
variação morfológica desses espécimes coletados na bacia do Taquari é muito grande,
principalmente dos padrões da coloração. Esses indivíduos aparentemente são distintos
das espécies já descritas, e possivelmente existem no mínimo sete novas espécies. Para
todas as novas espécies, indivíduos foram coletados em diversos afluentes da bacia do
Taquari, e em alguns locais foi possível verificar a ocorrência de mais de um táxon.
Figura 1: Espécime de Eurycheilichthys
10
Figura 2: Área de distribuição de Eurycheilichthys pantherinus ,
Eurycheilichthys limulus e espécies novas
11
1.3 As Bacias Hidrográficas do Rio Grande do Sul
No Rio Grande do Sul é possível delimitar três bacias hidrográficas,
reconhecidas pelas direções de escoamento dos rios. São elas: a bacia hidrográfica do
Rio Uruguai, na parte norte e oeste, cujas águas desembocam no Rio da Prata; o sistema
da Lagoa dos Patos, na região central, sul e leste, e a bacia do Rio Tramandaí, no litoral
norte (Fontana et. al., 2003). É relevante detalhar as duas primeiras, a bacia do Rio
Uruguai e o sistema da Lagoa dos Patos, pois é nessas regiões que se encontra a área de
distribuição do gênero Eurycheilichthys.
A bacia do Rio Uruguai está subdividida em dez unidades, ou microbacias:
Apuaê / Inhandava; Passo Fundo; Várzea; Turvo / Santa Rosa / Santo Cristo; Ijuí; Butuí
/ Piratinim / Icamaquã; Ibicuí; Quaraí; a bacia do Rio Santa Maria e a bacia do Rio
Negro. A ocorrência do gênero Eurycheilichthys foi identificada em três microbacias:
dos Rios Apuaê / Inhandava, Várzea e do Rio Ijuí. Provavelmente existe a ocorrência
deste gênero nas demais microbacias, porém não há dados de coleta.
O sistema da Lagoa dos Patos é subdividido em bacias e o gênero
Eurycheilichthys ocorre somente na Bacia do Guaíba. Essa bacia também foi
subdividida, sendo formada por nove microbacias: Guaíba; Gravataí, Sinos, Caí e Baixo
Jacuí; Alto Jacuí, Taquari-Antas, Pardo, Vacacaí / Vacacaí-Mirim. Duas são relevantes
para o estudo, pois há registros de ocorrência de espécies do gênero Eurycheilichthys: a
microbacia do Alto Jacuí, que corresponde ao segmento inicial do Rio Jacuí, e a
microbacia do Taquari-Antas, que também desemboca no Rio Jacuí. Todas as bacias de
interesse estão localizadas no Planalto Meridional.
12
A Bacia do Uruguai está contida na Bacia do Paraná, a qual distribui-se por
quase toda porção meridional do Brasil, possuindo como leito as rochas formadas no
derrame basáltico que originou o Planalto Meridional do Brasil. Essa formação basáltica
provém de sucessivos eventos vulcânicos, entre 250 milhões de anos e 50 milhões de
anos, tendo ocorrido o pico de atividade vulcânica em torno de 135 milhões de anos, no
início do Cretáceo. Parte desse derrame basáltico está presente na costa da África, pois
nessa época os continentes estavam unidos na Gondwana, cuja fragmentação começou
cerca de 120 milhões de anos.
Após a separação dos continentes, houve a acomodação das placas tectônicas,
que derivou no soerguimento da Serra Geral do Brasil. Esse soerguimento resultou na
mudança na drenagem dos rios, escoando para o interior do continente até a confluência
do Rio Uruguai com o Rio Negro, que deságuam no Rio da Prata (Potter, 1997)
1.4. Filogenia e Filogeografia
Dados moleculares são de grande importância para a inferência filogenética,
devido à riqueza de informações. Esses dados são utilizados na sistemática como
ferramentas para o acesso a questões evolutivas tradicionais. Diversos tipos de dados e
de análises têm sido empregados, sendo que a abordagem mais direta é determinar a
seqüência de nucleotídeos de um ou mais genes homólogos nas espécies em que se
busca a filogenia.
Os dados de seqüências moleculares podem ser utilizados para estimar filogenias
por diversas técnicas. Embora existam divergências sobre qual a melhor técnica a ser
utilizada, alguns métodos como Máxima Verossimilhança, Máxima Parcimônia,
Neighbor-Joining e Análise Bayesiana são constantemente aplicados e os resultados
comparados (Holder & Lewis, 2003).
A filogeografia pode ser definida como estudo dos princípios e processos que
governam a distribuição geográfica de linhagens genealógicas (Avise, 2000). As bases
históricas desta ciência estão intimamente ligadas aos estudos empíricos com o DNA
mitocondrial de animais, iniciados nos anos 70 e que têm sido usadas intensivamente
para estudos filogenéticos, nos quais a distribuição dos grupos de haplótipos de mtDNA
13
através da área de uma espécie ou complexos de espécies é utilizado para inferir sobre a
história das populações (Avise, 2000)
A filogeografia supre uma ponte empírica e conceitual entre a genética de
populações e a biologia filogenética. E mesmo relações explicadas de forma
estritamente ecológica, podem, através de uma interpretação filogeográfica, explicitar as
bases filogenéticas para certas características da história natural de um grupo (ver
Avise, 2000).
A utilização da variação entre seqüências do DNA mitocondrial tem sido a
metodologia mais amplamente utilizada para inferir sobre os padrões filgeográficos das
espécies (Avise, 2000). A evolução de algumas regiões mitocondriais é extremamente
rápida comparada ao DNA nuclear (Nedbal & Flynn, 1998) o que habilita o mtDNA a
ser utilizado como um marcador molecular para microevolução (Avise, 2000).
Análises de padrões filogeográficos intraespecíficos conduzem a observação de
barreiras e estruturas geográficas (p.ex. Eizirik, 2001) e levam a um maior avanço em
nosso conhecimento dos processos históricos biogeográficos.
14
2. REFERÊNCIAS BIBLIOGRÁFICAS
Avise, L.C., 2000. Phylogeography: The history and formation of species. Harvard
University press, Cambridge.
Eizirik, E., Kim, J., Raymond, M.M., Crawshaw, P.G., O’Brien, S.J. and Johnson,
W.E., 2001. Phylogeography, population history and conservation genetics of
jaguars (Panthera onca, Mammalia, Felidae). Mol. Ecol. 10: 65-79.
Fontana, C. S. (Org.); Bencke, G. A. (Org.); Reis, R. E. (Org.), 2003. Livro vermelho
da fauna ameaçada de extinção no Rio Grande do Sul. 1. ed. Porto Alegre: Edipucrs.
v. 1. 632 p.
Lehmann, P.A., 2006. Anatomia e relações filogenéticas da família Loricariidae
(Ostariophysi: Siluriformes) com ênfase na subfamília Hypoptopomatinae. PhD
Thesis Pontifícia Universidade Católica do Rio Grande do Sul
http://www.pucrs.br/biblioteca/tede/381156.pdf
Holder, M., Lewis, P.O., 2003. Phylogeny estimation: traditional and Bayesian
approaches. Nature Reviews Genetics, 4, 275–284.
Nedbal, M.A., Flynn, J.J., 1998. Do combined effects of asymmetric process of
replication and DNA damage from oxygen radicals produce a mutation-rate
signature in the mitocondrial genome? Mol. Biol. Evol. 15:219-223.
Nelson, J.S., 1994. Fishes of the world. 3. ed. New York, NY : J. Wiley, 600 p. : il.
Potter, P.E., 1997. The Mesosoic and Cenozoic paleodreinage of South America: a
natural history. Journal of south American Sciences vol. 10 pp 331-344
Reis, R.E., Pereira, E.H.L., . Armbruster, J.W., 2006. Delturinae, a new loricariid
catfish subfamily (Teleostei, Siluriformes), with revisions of Delturus and
Hemipsilichthys] Zoological Journal of the Linnean Society , 2006, 147 , 277–299.
Reis, R.E., Schaefer, S.A., 1992. Eurycheilus pantherinus, a new genus and species of
Hypoptopomatinae from southern Brazil (Siluriformes, Loricariidae). Copeia, 1992:
215-223.
Reis, R.E., Schaefer, S.A., 1993. Eurycheilichthys Nom. Nov., a substitute name for
Eurycheilus Reis & Schaefer, 1992 (Siluroidei: Loricariidae). Copeia, 1993: 894.
Reis, R.E., Schaefer, S.A., 1998. New cascudinhos from southern Brazil: systematics,
endemism, and relationships (Siluriformes, Loricariidae, Hypoptopomatinae). Amer.
Mus. Novitates, 3254: 1-25.
Reis, R.E., Kullander, S.O., Ferraris Jr, C. J., 2003. Check list of freshwater fishes of
South and Central America. Porto Alegre : Edipucrs, c2003. 729 p
Schaefer, S.A., 1991. Phylogenetic analysis of the loricariid subfamily
Hypoptopomatinae (Pisces: Siluroidei: Loricariidae), with comments on generic
diagnoses and geographic distribution. Zoological Journal of the Linnean Society,
102:1-41.
Vari, R.P., Malabarba, L.R., 1998. Neotropical ichthyology: An overview. Pp. 1-11 in.
Malabarba, L.R., R.E. Reis, R.P. Vari, Z.M.S. Lucena & C.A.S. Lucena (eds)
Phylogeny and Classification of Neotropical Fishes, Edipucrs, Porto Alegre, 603p.
15
Artigo Científico
A ser enviado para a revista Molecular Phylogenetics and Evolution
16
Phylogeny and phylogeography of the endemic catfish genus Eurycheilichthys
(Siluriformes: Loricariidae) from the southern Brazilian plateau
Ana M. R. Liedke
1
, Felipe G. Grazziotin
1
, Roberto E. Reis
2
, Sandro L. Bonatto
1
1
Centro de Biologia Genômica e Molecular, Faculdade de Biociências, Pontifícia
Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil;
2
Laboratório de Ictiologia, Pontifícia Universidade Católica do Rio Grande do Sul,
Porto Alegre, RS, Brazil;
Corresponding author:
Dr. Sandro L. Bonatto
Centro de Biologia Genômica e Molecular
Faculdade de Biociências - PUCRS
Av. Ipiranga 6681, cep 90619-900 Porto Alegre, RS, Brazil
Telephone number: 55 (51) 3320.3500 Ext. 4717
Fax number: 55 (51) 3320.3612
Email: [email protected]
17
Abstract
The Neotropical genus Eurycheilichthys is endemic to the southern Brazilian
plateau, with two described species: E. pantherinus, which occurs in the upper Uruguay
River basin, and E. limulus, that is found in the upper Jacuí River microbasin, which is
part of the Guaíba Lake drainage. Seven new species are being described based on
morphological characters and they are all endemic to the upper Taquari-Antas River
microbasin, which is also part of Guaíba Lake drainage. We conducted the first
molecular analysis concerning the phylogenetic relationships and phylogeography of the
whole genus Eurycheilichthys using two mtDNA genes (COI and ND2). The
phylogenetic trees and the network are structured by basin and microbasin and
presented both already described species as monophyletic. However, only three out of
the seven new species were monophyletic for these genes while some species showed
several clades with restrict geographical distribution which could perhaps indicate other
highly endemic cryptic species or the non-monophyly may be caused by incomplete
lineage sorting. The origin of the Taquari-Antas clade was estimated around 0.5-1.0
million years ago while the divergence time among the species from this microbasin
may have occurred around the half a million years ago. Population analyses showed
signals of a recent population growth for most of the species of the genus. The disjunct
distribution among the river basins likely happened between the late Pliocene and the
early Pleistocene, with the origin of the Uruguay basin. The high diversity and degree of
endemicity of this group in the Taquari-Antas microbasin may be explained by its high
declivity associated with the ecological restriction of the group.
KEYWORDS: molecular phylogeny, phylogeography, Loricariidae, Eurycheilichthys.
18
1. Introduction
The Neotropical rivers of South and Central America host the most diverse
freshwater fish fauna of the world, with almost 6,000 of the world’s approximated
13,000 species (Reis et al., 2003). Within this huge diversity of species, the great
majority belongs to the superorder Ostariophysi, in which Siluriformes, or catfishes, is
the most diverse and widely distributed order (Nelson, 1994). The high diversity of
habits and shape of the body within siluriformes is noteworthy, especially in the
superfamily Loricarioidea, which contributes with more then two thirds of all catfish
species found in the Neotropics.
The Neotropical Eurycheilichthys (Reis and Schaefer, 1992) include small-body
species that belong to the family Loricariidae, subfamily Hypoptopomatinae and was
included in the tribe Otothyrini by Schaefer (1991). Eurycheilichthys is endemic to
southern Brazil, where it is restrictedly distributed throughout the plateaus of Rio
Grande do Sul (RS) and Santa Catarina (SC) states (above 600 meters of altitude, Fig.
1). Currently, the genus has only two described species: E. pantherinus (Reis and
Schaefer, 1992) and E. limulus Reis and Schaefer, 1998. Eurycheilichthys pantherinus
is present only in the Uruguay River basin, which drains into the La Plata River; on the
other hand E. limulus is found only in the upper Jacuí River microbasin, which is part of
the Guaíba Lake drainage (Fig. 1). However, recent extensive collection in these areas
have found at least seven additional new species for the genus (RER unpublished
results) based on morphological characters. The new species also belong to Guaíba
Lake drainage, but differently from E. limulus they are all endemic to the Taquari-Antas
microbasin (Fig. 1). This microbasin has a high average declivity with little riparian
forest left. It is geographically divided by altitude and declivity in three regions: The
first is in the highest portions of the microbasin (at almost 1,200m above sea level),
where the main river channel drains from east to west, and is deeply escavated with a
declivity of 4.8m/km. In the second region the river has a moderated declivity with only
1.6 m/km and a direction from northeast to southeast. In the last portion the river flows
out from the plateaus to a central depression in Rio Grande do Sul state, changing its
feature to a plain river (Fig. 1). Fishes of the genus Eurycheilichthys inhabit mostly
head streams with no more than 50 centimeters deep, in the first and second regions of
the microbasin. They stay under rocks, protecting themselves from the watercourse,
feeding the vegetation incrusted in the rock surfaces (Reis and Schaefer, 1992).
19
These new species are being described mainly based upon differences in
morphology and color patterns, and there are two principal distributional patterns
among them: some species are spread across several relatively distant streams (E. sp.
nov. 1, E. sp. nov. 2, E. sp. nov. 5; Fig. 1) while others are limited to only one or a few
near streams (E. sp. nov. 3, E. sp. nov. 4, E. sp. nov. 6, E. sp. nov. 7; Fig. 1). No genetic
diversity study has been done to date in the genus or in any other member of the
subfamily Hypoptopomatinae.
Fishes that present an endemic and speciously pattern have been helping us to
better understand questions about the evolutionary process of speciation, from the
classical works of D. E. Rosen with the fish fauna of the karst region in Guatemala
(reviewed in Rosen, 1979) and A. Meyer (Meyer et al., 1990) with cichlids from the
African lakes, to others works regarding similar patterns around the world (Robalo et
al., 2006; Cunha et al., 2004; Alves et al., 1997) and the new approaches to these same
classical problems (Won et al., 2006). These studies frequently presented complex
results, sometimes contrasting species delimitation based on morphology with
relationships based on gene trees (Funk et al., 2003; Pollard et al., 2006). This conflict
is know to be caused by many distinct speciation scenarios as described by different
phylogeographic patterns (e.g. Avise 2000).
In this present phylogeographic study of Eurycheilichthys we tried to better
understand the evolutionary processes that gave rise to a group with a so unusual
endemic diversity. We also explored the demography and biogeography of this group in
a restricted area of the southern Brazilian plateau.
2. Material and Methods
2.1 Sampling procedures
A total of 126 specimens of Eurycheilichthys were used in this study, of which
123 specimens were collected by us in 22 sampling points, and three samples were
supplied by FZB/RS (MCN in Table 1). We collected, when possible, around ten
specimens in each locality (Fig. 1). They were preserved in alcohol 90%, deposited in
MCP fish collection, and recorded under the catalog numbers as shown in Table 1. As
outgroups, we used samples of two related genera (same tribe according Schaefer,
1991): Epactionotus itaimbezinho and Hisonotus sp., supplied by MCP.
2.2 DNA amplification and sequencing
20
Muscular tissue was isolated and total DNA was prepared according to the protocol of
Ammonia Acetate (Sambrook et al., 1989). Fragments of NADH subunit 2 (ND2) and
cytochromo oxidase I (COI) mitochondrial DNA genes were amplified using the
polymerase chain reaction (PCR) and sequenced using the following primers pairs: L-
5216 and H-6313 (Sorenson et al, 1999) and L-1490 and H-2198 (Herbert et al., 2003),
respectively.
All PCR products were analyzed in 1% agarose gel, purified by standard
Polietilenoglicol (PEG) 8000 precipitation and sequenced using the DYEnamic ET Dye
Terminator Cycle Sequencing Kit (GE Healthcare) in a MegaBACE 1000 automated
sequencer (GE Healthcare) following the manufacturer’s protocols. The primers used
for sequencing were the same as those used in the PCR for COI, but an internal primer
was constructed to sequence the final portion of ND2 segment (FISH_ND2SEQ 5’-
GCA CCA ATA CAC TTC TGA AT-3´). Chromatograms were visually checked with
the CHROMAS 2.0 software (Technelysium) and sequences were aligned manually
using BIOEDIT 6.0.7 (Hall, 1999).
2.3 Phylogenetic Analyses
Phylogenetic analyses were performed using the criteria of maximum likelihood (ML),
maximum parsimony (MP), neighbor-joining (NJ), and Bayesian phylogenetic inference
(BI). When appropriate, the model of nucleotide substitution was used as estimated by
Modeltest 3.07 (Posada and Crandall, 1998) using the minimum theoretical information
criterion (AIC).
Maximum likelihood, MP and NJ trees were estimated using PAUP* 4.0b10
(Swofford, 2003). We inferred ML trees with heuristic searches with the tree-bisection-
reconnection (TBR) option and a NJ starting tree; statistical confidence was estimated
with 100 replications of bootstrap using the same search strategy. MP was performed
with heuristic searches (TBR) with starting tree produced by 100 replication of random
stepwise addition. To assess the MP statistical confidence, 1000 bootstrap replicates
were conducted using simple stepwise addition. The NJ analysis used the ML distance
under the evolutionary model selected by Modeltest and to access the statistical support
1000 bootstrap replicates were conducted.
21
The BI was performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001)
with 2,000.000 generations for the Markov chain Monte Carlo (MCMC) algorithm
using flat priors. Tracer 1.3 (http://evolve.zoo.ox.ac.uk/software/tracer/) was used to
determine the burn-in and a consensus tree was estimated from the remaining trees
using PAUP*. We consider bootstrap support (BS) higher than 70% and Bayesian
posterior probability (PP) higher than 95% as a significant support for a clade
(Felsenstein, 1985; Hillis and Bull, 1993; Huelsenbeck and Ronquist, 2001; Wilcox et
al., 2002).
The Kishino-Hasegawa test (Kishino and Hasegawa, 1993), calculated using
PAUP*, was used to test hypothesis of monophyly of the species as well as the
hypothesis of an alopatric pattern concerning the genetic structure in the basins (i.e., the
monophyly of the basins). A median-joining network (Bandelt et al., 1999) was
constructed using the program Network 4.2 (www.fluxusengineering.com) to better
investigate the relationship among the closely related haplotypes from the Guaíba
Basin.
To estimate a time frame for the results, we first tested the molecular clock
assumption comparing the likelihood of the best ML tree and the clock enforced tree
using Shimodaira-Hasegawa test (Shimodaira and Hasegawa, 2001) performed in
PAUP*. As there is neither a suitable calibration point for this group nor any
substitution rate for the mtDNA of any close related taxa, we used two extreme
substitution rates found in literature for fishes: 0.9%/My for COI described for
goodeines by Webb et al. (2004) and 1.8%/My for Centropomus proposed by Kocher
and Carleton (1997), based in the cichlid’s ND2 gene.
2.4 Phylogeographic Analyses
We used the Mantel test to try to distinguish between the influence of the
geographic distance and the species delimitation in the genetic diversity structure of the
new species from the Guaíba Lake basin. For this we used Arlequin 3.01 (Schneider et
al., 2000) to calculate the correlation between genetic distances matrix and two other
matrices: geographical distances along the rivers and a discrete matrix in which one or
zero were set to represent different or equal species, respectively. We also carried out an
analysis of molecular variance (AMOVA Excoffier et al., 1992) with these two
approaches using Arlequin 3.01. We set the populations using two approaches: first,
22
each species was assumed to be a population; and second, all specimens collected in the
same or in close streams were considered a population.
Genetic summary statistics as nucleotide and haplotype diversity, Tajima’s D
(Tajima, 1983) and Fu’s Fs (Fu, 1997) neutrality tests were estimated for each species.
We also computed a mismatch distribution analysis for all species and for each
microbasin to find out signals of population expansion. The summary statistics and
mismatch distribution were performed in Arlequin 3.01. To estimate the dynamics of
the population size along the time, we used the Bayesian Skyline Plot method
implemented in the program BEAST 1.4 (Drummond and Rambaut, 2003). This
Bayesian approach incorporates the uncertainty in the genealogy by using MCMC
integration under a coalescent model, where the timing of divergence dates provides
information about effective population sizes through time. It was used the evolutionary
model suggested by Modeltest, a length chain of 10,000,000 and the substitution rates
described above.
3. Results
3.1. Sequence analyses and alignments
Our alignments result in two matrices of 601bp and 607bp for the ND2 and COI
fragments, respectively. Both genes concatenated produced a matrix of 1208bp of which
204 sites were variable and 119 sites were parsimony informative. We could not
amplify the COI gene from the three specimens of Eurycheilichthys pantherinus, so we
coded these sequences as missing data for COI in the concatenated matrix. This matrix
resulted in 59 haplotypes with no shared haploype between species. Although it was not
possible to obtain homogeneous sample sizes across the species, as some species are
very rare, we compared summary statistics among those with more than 10 individuals.
The nucleotide diversity ranged for 0.01% to 1.14% and the haplotype diversity ranged
for 0.66 to 0.95 among the species (Table 2), with no correlation with the sample size.
It is remarkable the high diversity found in E. sp. nov. 1 and E. sp. nov. 5, the latter
presenting nucleotide diversity higher than the whole Taquari-Antas Microbasin. The
sequences obtained in this study were deposited in the GenBank (sequences not yet
submitted).
3.2. Phylogenetic analyses
23
For the whole data set the evolutionary model selected by Modeltest was
GTR+I+G, with a proportion of invariable sites of 0.6723 and an alpha parameter of
3.0062. All phylogenetic approaches produced a similar pattern (Fig. 2 A): the topology
presented two major monophyletic clades, one representing the Uruguay River basin
where only Eurycheilichthys pantherinus occurs and the other is formed by all species
of the Guaíba Lake basin, this latter clade presenting two subclades structured by
microbasin: one consisting of E. limulus from the upper Jacuí microbasin and the other
the seven new species from the Taquari-Antas microbasin. The Taquari-Antas
microbasin monophyly occurred in the MP, NJ and BI trees but with low support,
whereas in the ML trees two haplotypes from E. sp. nov. 5 (sampling point 18, Fig. 1)
grouped with haplotypes of E. limulus from the upper Jacuí microbasin (not showed).
However, the values of BS and PP support for this latter clade were very low and in the
Shimodaira-Hasegawa test comparing this best ML tree with a ML tree estimated with a
constrain where all haplotypes from the Taquari-Antas microbasin stayed monophyletic,
showed they are non-significantly different. Besides, the median-joining network also
supports the monophyly of the species from the Taquari-Antas microbasin (not shown).
The phylogenetic analyses highly support the monophyly of the two species
described by Reis and Schaefer (1992, 1998), Eurycheilichthys pantherinus and E.
limulus. On the other hand, only three of the new species of the Taquari-Antas
microbasin are monophyletic by the mitochondrial lineages (E. sp. nov. 3, E. sp. nov. 4,
and E. sp. nov. 7). To test the support for this result, we compared the best ML tree
against the best tree were all species were constrained to be monophyletic using the
Kishino-Hasegawa test. The monophyletic constrained trees found were significantly
worst than the ML tree. The relationship among the more closely related haplotypes
from the Guaíba Lake basin estimated using the median-joining network (Fig. 3) is
similar to the phylogenetic trees, supporting the non-monophyletic tree and showing
seven mutational steps between E. limulus and the other species of Taquari-Antas
microbasin, also in agreement with the phylogenetic tree (Fig.2).
Within the Taquari-Antas microbasin species we could distinguish two common
and two idiosyncratic phylogeographic patterns. First, there are the monophyletic
species with a very restrict area of distribution (E. sp. nov. 3, E. sp. nov. 4 and E. sp.
nov. 7); second there are non-monophyletic, geographically widespread species with
non-monophyletic sample points (E. sp. nov. 1 and E. sp. nov. 2). In this latter pattern
there is one haplotype of E. sp. nov. 1 shared by samples from two different and apart
24
locations: the sampling point 3 and 6 (Fig. 1). For all other species that have the same
haplotype in individuals from different locations they are, in fact, in the same
watercourse. There are also E. sp. nov. 6, that is also highly endemic but non-
monophyletic and E. sp. nov. 5, that is geographically widespread and non-
monophyletic but with the individuals from the same collecting point being
monophyletic.
As the Shimodaira-Hasegawa test did not reject the molecular clock for the data,
we used the slow (0.9%) and the fast (1.8%) rates of substitution to estimate divergence
times along the tree (Fig. 4). The clade comprising E. pantherinus from the Uruguay
River basin diverged from the clade found in the Guaíba Lake basin about 1.2 to 2.4
Mya (million years ago), while the divergence time between E. limulus from the upper
Jacuí microbasin and the clade of species from the Taquari-Antas microbasin was
estimated around 0.5 to 1.0 Mya. All internal clades of Taquari-Antas microbasin
showed a recent radiation around 167,000 – 80,000 years ago. This radiation is visible
in Fig.2 B, where all specimens are represented in a NJ tree.
3.3 Phylogeographic analyses
As the phylogenies showed that some species are separated in different clades
which represent haplotypes found in the same river while others represent the whole
species, we tried a simple test to see which variable (geographic distance along the
rivers or the species) present a better correlation with the genetic distance among the
haplotypes. The Mantel tests resulted in a correlation of 54% between genetic distance
and geographical distance matrices but of only 7% between the genetic distance and the
discrete matrix of species similarity (all statistics were significant at P = 0.01).
However, in the analysis of molecular variance (Table 3) both approaches for grouping
the haplotypes, by the morphospecies or by the river where they were found, resulted in
similar partitions of the variation.
The neutrality tests and the mismatch distributions were estimated only for the
species with sample size larger than 10 (Table 2). Fu’s F
s
, known to be more sensitive to
demographic changes, presented significantly negative values for all monophyletic
species calculated and for the whole clade from the Taquari-Antas Microbasin. As
expected, Tajima’s D being more conservative, was significantly negative for E. sp.
nov. 3 only. Furthermore, the two non-monophyletic species tested (E. sp. nov. 1 and E.
25
sp. nov. 5) showed non-significant values. The suggestions for recent population
expansion indicated by the F
s
test were corroborated by the single wave mismatch
distributions (not shown), while the other species presented a clear ragged distribution
(as expected in cases of the non-monophyletic species). Moreover, the distribution
estimated using the Taquari-Antas microbasin clade also resulted in a single wave
pattern that supports a bottleneck followed by a population expansion (Fig. 4) around
0.46 and 0.92 Mya (using the slow and fast rates on a tau of 10.1, respectively).
The demographic history of the whole Taquari-Antas microbasin species could
be estimated with more details using a Bayesian skyline plot (Fig. 5). Using both rates
(0.9% and 1.8%), the results suggest that this group was relatively stable since its origin
around one Mya but had suffered a population expansion about 70,000 – 130,000 years
ago preceded by a moderate bottleneck.
4. Discussion
Three major mitochondrial clades were found in Eurycheilichthys and these are
hierarchically structured in the drainages by basin and microbasin. Two of these clades
coincide with the two known species of the genus (E. pantherinus and E. limulus) while
the third clade is endemic to the Taquari-Antas microbasin but is now considered to
comprise seven undescribed species. While three of these seven new species are
monophyletic for the mitochondrial lineages, the other four are non-monophyletic. One
obvious question is the significance of these phylogenetic results to the reality of these
four species both as evolutionary units and as valid taxa.
First, although some of these species are quite similar, all of them could be
morphologically diagnosed by the standard taxonomic methods and parameters used for
the group (RER unpublished results). Moreover, it is now widely known that the
relationship between closely related species and mitochondrial phylogenies (or actually
any single gene tree) is complex. The non-monophyly in single gene trees of undisputed
valid species have been observed in a wide number of phylogeographical studies (e.g.,
Fry and Zink, 1998 Andolfatto et al., 2003; Gifford et al., 2004, Grazziotin et al., 2006;
Chang, 2007). Besides, there are rich theoretical demonstrations of speciation scenarios
that could result in true species that are non-monophyletic in single gene trees, such as
recent peripatric speciation that display paraphyletic gene tree patterns or sharing of
ancestral lineages caused by incomplete lineage sorting after recent allopatric
divergence in large ancestral populations (reviewed in Avise, 2000). This latter seems a
26
likely scenario to the Taquari-Antas microbasin endemic Eurycheilichthys species,
where most internal clades arise in a narrow and recent (<1 Mya) time frame (Fig. 4)
resembling an adaptive radiation scenario. This scenario could explain the
phylogeographic patterns we described for this group, such as the monophyletic
microendemic species (E. sp. nov. 3, E. sp. nov. 4, and E. sp. nov. 7) or the non-
monophyletic, geographically widespread species with clades with haplotypes from
different collecting points (E. sp. nov. 1 and E. sp. nov. 2). One additional hypothesis is
that some of the clades that are geographically restricted (clades A-G, Fig. 2) but are
found inside some widespread species, may perhaps represent cryptic species, what
would increase the already high microendemicity of this genus in this microbasin.
A radical alternative hypothesis is that the whole Taquari-Antas microbasin
clade is actually a single species with at least some of the undescribed morphospecies
and maternal lineages representing subspecific evolutionary units. However, as species
definitions are related to species concepts, and this is an unresolved and somewhat
subjective issue, we would not like to enter this debate. The phylogeographic results
presented here shed some light in the evolutionary history of the genus irrespective the
taxonomic status assigned to these evolutionary units.
Our results suggest that the divergence between the clades from the Uruguay
River and Guaíba Lake basins occurred between the late Pliocene and early Pleistocene
(1.2-2.4 Mya). However, as the geological and hydrological history of this particular
area is poorly known, it is difficult to know accurately the time and mode of the
evolution of this drainage system. Nevertheless, there are some evidences that the
Uruguay drainage basin was formed in the Pliocene (Maack, 1968; Bossi, 1969; Souza
et al., 2005). Therefore, using the upper and lower limits of the molecular and
geological estimates, respectively, it could not be rejected the hypothesis that a vicariant
event between basins was the process that shaped this first divergence within the genus.
However, we think it is more likely that these two drainages were established before our
estimated molecular divergence times suggesting an alternative scenario to simple basin
vicariance (see below). Interestingly, there are several other sister-taxa pair of fish
species between the Uruguay and the Jacuí basins, e.g. Gymnogeophagus (Reis and
Malabarba, 1988; Wimberger et al., 1998), Parapimelodus (Lucena et al., 1992),
Cnesterodon (Lucinda, 2005), and Hypostomus (Reis et al, 1990).
The divergence between the clades from the two Guaíba microbasins (upper
Jacuí and Taquari-Antas) was estimated to have occurred around the middle Pleistocene
27
(1.0-0.5 Mya) and the initial divergence of several species from the Taquari-Antas
microbasin started soon after that. Unfortunately, the absence of information about the
formation of the microbasins of the Guaíba basin precludes us, at this time, to draw
specific correlations of our evolutionary scenario with the geological history. However,
it is not likely that these microbasins, especially the Taquari-Antas, being constituted by
valleys deeply excavated in a huge volcanic plateau, were formed later than the Pliocene
(< 1.8 Mya).
Considering that there are no strong geographic barriers between these basins
and microbasins, that they are geographically very close at their headwaters, and that
several species are known to occur at these very close headstreams, another, perhaps
more likely scenario to explain the above results (at least the divergence between two
Guaiba microbasins clades) is past headwater capture between the basins and the
microbasins (Beurlen, 1970). Considering the monophyly of the three drainage systems
studied here under the above headwater capture scenario, only two headwater capture
events are necessary to parsimoniously explain the results. However, it was not possible
to establish the polarities of the unidirectional gene flow that these putative river
captures imply.
Interestingly, no haplotype was found to be shared between the seven species of
the Taquari-Antas microbasin, suggesting the absence of very recent gene flow across
the species barriers. On the other hand, clade E is very singular, as it comprises
haplotypes from four different species and it is widely distributed. This last pattern may
be explained by an ancestral widespread population or by some gene flow in a relatively
recent past. This last process may also explain the presence of haplotype 16 in two
relatively distant collecting sites (sites 3 and 6, Figs. 1 and 2).
We detected, using the Bayesian skyline plot, a signal of a moderate population
bottleneck followed by a significant populations size increase in the whole Taquari-
Antas microbasin clade around 70-130 thousand years ago, that were also detected
separately in some of the species and clades. This demographic fluctuation may be
related to the climatic changes that occurred in the late Pleistocene around this time
(Ledruet al. 1996; Behling and Lichte 1997), in special the more severe effect of the dry
period estimated to have occurred in the southern part of the Atlantic Forest.
Interestingly, a similar demographic fluctuation was estimated for the jararaca snake
(Bothrops jararaca) in this region (Grazziotin et al., 2006).
28
What could explain the unusually high degree of microendemicity in the
Taquari-Antas microbasin in contrast with the single species found in the other drainage
systems? One possible explanation is the natural features of the Taquari-Antas
microbasin which is distinct from those found in the other drainage systems included in
this study. In the Taquari-Antas microbasin the rivers present a high declivity and the
shallow headwaters where most species inhabit appear to be isolated from each other by
more slow-flowing, deeper sections of river. On the hand, the upper Uruguay basin and
the upper Jacuí microbasin are located mostly on the top of the plateaus, without clear
geographical barriers.
One final comment concerns a conservation issue related to the Taquari-Antas
microbasin, where three hydroelectric dams are being implanted
(http://www.ceran.com.br), and several others are planned. It is not clear how these
dams would affect those geographical barriers and consequentially the persistence of
these evolutionary units.
5. Acknowledgements
We thank T. Carvalho, E. H. L. Pereira (PUCRS), and M. A. Azevedo (FZB-RS) for
helping in collecting the fishes and R. A. Lahm and R. Santos (LTIG-PUCRS) for
design of the map. Financial support was provided by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq, 301748/2003-7) and Fundação de
Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).
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34
Table 1: Eurycheilichthys species, number of sampling point, haplotype, name of
drainage, coordinate points and voucher catalog number.
Species Sampling
point
Haplotype Drainage Latitude, Longitude Voucher
E. pantherinus 22 Haplo 1 Uruguay Basin 28º38’42”S, 50º17’01”W MCP 35042 A
22 Haplo 1 Uruguay Basin 28º38’42”S, 50º17’01”W MCP 35042 B
22 Haplo 2 Uruguay Basin 28º38’42”S, 50º17’01”W MCP 35042 C
22 Haplo 3 Uruguay Basin 28º38’42”S, 50º17’01”W MCP 35042 D
22 Haplo 1 Uruguay Basin 28º38’42”S, 50º17’01”W MCP 35042 E
E. limulus 12 Haplo 4 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 A
12 Haplo 5 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 C
12 Haplo 6 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 D
12 Haplo 8 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 E
12 Haplo 7 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 F
12 Haplo 8 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 G
12 Haplo 6 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 H
12 Haplo 9 Alto-Jacuí Microbasin 28º22’14”S, 52º30’37”W MCP 35120 J
11 Haplo 10 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 A
11 Haplo 10 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 B
11 Haplo 11 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 C
11 Haplo 12 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 D
11 Haplo 13 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 E
11 Haplo 8 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 F
11 Haplo 10 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 G
11 Haplo 14 Alto-Jacuí Microbasin 28°18’24”S, 52º28’13”W MCP 35118 H
E. sp. nov. 1 3 Haplo 15 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 A
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 B
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 C
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 D
3 Haplo 17 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 E
3 Haplo 15 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 F
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 G
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 I
3 Haplo 16 Taquari-Antas Microbasin 29º03’30”S, 52º30’58”W MCP 35058 J
4 Haplo 18 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 A
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 B
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 C
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 D
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 E
4 Haplo 20 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 F
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 G
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 H
4 Haplo 19 Taquari-Antas Microbasin 28º48’24”S, 52º18’14”W MCP 35057 J
5 Haplo 21 Taquari-Antas Microbasin 28º49’44”S, 52º14’35”W MCP 35122 A
5 Haplo 21 Taquari-Antas Microbasin 28º49’44”S, 52º14’35”W MCP 35122 B
5 Haplo 21 Taquari-Antas Microbasin 28º49’44”S, 52º14’35”W MCP 35122 C
5 Haplo 21 Taquari-Antas Microbasin 28º49’44”S, 52º14’35”W MCP 35122 D
6 Haplo 16 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 B
6 Haplo 22 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 C
6 Haplo 16 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 D
6 Haplo 23 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 E
6 Haplo 24 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 F
6 Haplo 25 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 G
6 Haplo 26 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 I
6 Haplo 27 Taquari-Antas Microbasin 28º34’06”S, 51º50’35”W MCP 35045 J
7 Haplo 28 Taquari-Antas Microbasin 29º01’06”S, 51º31’37”W MCP 36837 A
7 Haplo 28 Taquari-Antas Microbasin 29º01’06”S, 51º31’37”W MCP 36837 B
E. sp. nov. 2 17 Haplo 29 Taquari-Antas Microbasin 28º21’51”S, 51º17’53”W MCP 35119 A
35
17 Haplo 30 Taquari-Antas Microbasin 28º21’51”S, 51º17’53”W MCP 22800 A
17 Haplo 30 Taquari-Antas Microbasin 28º21’51”S, 51º17’53”W MCP 22800 B
21 Haplo 31 Taquari-Antas Microbasin 29º05’34”S, 50º37’30”W MCP 22374 A
E. sp. nov. 3 8 Haplo 32 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 B
8 Haplo 33 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 C
8 Haplo 34 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 D
8 Haplo 34 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 E
8 Haplo 34 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 F
8 Haplo 34 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 G
8 Haplo 34 Taquari-Antas Microbasin 28º31’36”S, 52º08’37”W MCP 35043 H
9 Haplo 34 Taquari-Antas Microbasin 28º21’29”S, 52º15’51”W MCP 35121 D
9 Haplo 35 Taquari-Antas Microbasin 28º21’29”S, 52º15’51”W MCP 35121 F
9 Haplo 34 Taquari-Antas Microbasin 28º21’29”S, 52º15’51”W MCP 35121 H
9 Haplo 34 Taquari-Antas Microbasin 28º21’29”S, 52º15’51”W MCP 35121 I
9 Haplo 34 Taquari-Antas Microbasin 28º21’29”S, 52º15’51”W MCP 35121 J
10 Haplo 36 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 A
10 Haplo 35 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 B
10 Haplo 34 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 C
10 Haplo 34 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 D
10 Haplo 34 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 E
10 Haplo 32 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 F
10 Haplo 35 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 G
10 Haplo 34 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 H
10 Haplo 32 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 I
10 Haplo 34 Taquari-Antas Microbasin 28º21’08”S, 52º15’56”W MCP 35049 J
E. sp. nov. 4 14 Haplo 39 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 A
14 Haplo 40 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 B
14 Haplo 37 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 D
14 Haplo 38 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 E
14 Haplo 39 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 F
14 Haplo 40 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 I
14 Haplo 39 Taquari-Antas Microbasin 28º38’04”S, 51º36’53”W MCP 35062 J
15 Haplo 40 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 A
15 Haplo 41 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 B
15 Haplo 42 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 D
15 Haplo 40 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 F
15 Haplo 39 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 G
15 Haplo 43 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 H
15 Haplo 40 Taquari-Antas Microbasin 28º39’35”S, 51º37’05”W MCP 35041 I
E. sp. nov. 5 13 Haplo 44 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 C
13 Haplo 45 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 D
13 Haplo 46 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 E
13 Haplo 46 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 F
13 Haplo 46 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 H
13 Haplo 46 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 I
13 Haplo 46 Taquari-Antas Microbasin 28º44’25”S, 51º41’15”W MCP 35037 J
16 Haplo 47 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 A
16 Haplo 48 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 B
16 Haplo 49 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 C
16 Haplo 50 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 D
16 Haplo 47 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 E
16 Haplo 49 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 G
16 Haplo 49 Taquari-Antas Microbasin 28º24’19”S, 51º29’25”W MCP 35125 I
18 Haplo 51 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 A
18 Haplo 52 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 B
18 Haplo 52 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 C
18 Haplo 52 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 D
18 Haplo 52 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 E
18 Haplo 52 Taquari-Antas Microbasin 28º23’26”S, 51º03’22”W MCP 35123 H
E. sp. nov. 6 19 Haplo 53 Taquari-Antas Microbasin 29º16’41”S, 50º14’42”W MCP 35044 A
36
19 Haplo 54 Taquari-Antas Microbasin 29º16’41”S, 50º14’42”W MCP 35044 C
19 Haplo 55 Taquari-Antas Microbasin 29º16’41”S, 50º14’42”W MCP 35044 E
20 Haplo 56 Taquari-Antas Microbasin 29º07’24”S, 50º21’29”W MCN 18563 A
20 Haplo 56 Taquari-Antas Microbasin 29º07’24”S, 50º21’29”W MCN 18563 B
20 Haplo 56 Taquari-Antas Microbasin 29º07’24”S, 50º21’29”W MCN 18563 C
E. sp. nov. 7 1 Haplo 57 Taquari-Antas Microbasin 29º02’53”S, 52º33’19”W MCP 35124 C
2 Haplo 58 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 A
2 Haplo 57 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 B
2 Haplo 59 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 D
2 Haplo 59 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 E
2 Haplo 58 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 F
2 Haplo 58 Taquari-Antas Microbasin 29º02’51”S, 52º34’06”W MCP 35071 H
37
Table 2. Summary statistics for the species of Eurycheilichthys.
n
h
H
d
π D F
s
E. pantherinus 5
3
0.700 (0.218) 0.09 (0.08) NA NA
E. limulus 16
11
0.958 (0.036) 0.20 (0.13) -1.436 -8.256*
Whole Taquari-Antas 105
45
0.979 (0.004) 1.03 (0.52) -0.869 -17.770*
E. sp. nov. 1 32
14
0.909 (0.030) 0.82 (0.43) 0.237 -0.066
E. sp. nov. 2 4
3
0.666 (0.314) 0.44 (0.36) NA NA
E. sp. nov. 3 22
5
0.878 (0.039) 0.02 (0.03) -1.729* -14.731*
E. sp. nov. 4 14
7
0.923 (0.050) 0.14 (0.10) -0.724 -5.329*
E. sp. nov. 5 20
9
0.873 (0.046) 1.14 (0.59) 2.077 3.403
E. sp. nov. 6 6
4
0.857 (0.137) 0.52 (0.32) NA NA
E. sp. nov. 7 7
3
0.809 (0.129) 0.01 (0.02) NA NA
n, number of sequences; h, number of haplotypes; Hd, Haplotype diversity; π,
Nucleotide diversity; D, Tajima’s D; Fs, Fu’s Fs. *P < 0.05; values in parentheses are
the confidence interval; NA, not applicable.
.
38
Table 3. AMOVA considering two different approaches to group the data
Species approach River approach
Source of variation d.f. % of variation d.f. % of variation
Among groups 8 49.27 9 46.2
Among populations
within groups
13 39.24 13 42.21
Within populations 103 11.49 103 11.58
39
Fig. 1. Geographic distribution of the Eurycheilichthys species with sampling
localities. Solid line separates the Uruguay (up and right) from the Guaíba basins, and
dashed line separate upper Jacuí (left) from Taquari-Antas microbasins. Symbols
represent each species as in the internal legend, and numbers identify the sample sites.
Fig. 2. A) Bayesian tree, with all haplotypes, with support values (BI, MP, NJ
and ML, respectively). * for <50 and – for nodes not found in the respective tree. B) NJ
tree with all specimens
Fig. 3. Median-joining network for the Guaíba Lake basin haplotypes. The
straight dashed line at the left demarcate the two microbasins. Each circle represents a
different haplotype with size proportional to its relative frequency. The different
shading patterns indicate the species as indicated in the internal legend. The
demarcation with the oval dashed lines indicate the number of the site were the
haplotypes were collected (see map in Fig. 1), showing only those that are not
monophyletic. The crossed marks are nucleotide substitutions inferred in that branch.
Fig. 4. ML tree with the molecular clock forced for estimating divergence times
using both substitution rate above 1.8%, bellow 0.9%.
Fig. 5. Mismatch distribution for the haplotypes of Taquari-Antas species. The
observed frequency is represented by the diamond and black line, and the expected
frequency under the expansion model is depicted by gray line connected by squares.
Fig. 6. Bayesian skyline plot showing the effective population size fluctuation
throughout time of the Taquari-Antas microbasin clade (heavy line, median estimation;
thin lines, confidence interval); above and below the x axis and left and right y axis are
the time estimated using the 1.8% and the 0.9% My rates, respectively.
40
Fig. 1
E. pantherinus
E. limulus
E. sp. nov.1
E. sp. nov.2
E. sp. nov.3
E. sp. nov.4
E. sp. nov.5
E. sp. nov.6
E. sp. nov.7
-52.14
-29.33 -51.64
-51.14
-50.64
-28.83
-28.33
41
Fig. 2
A)
Uruguay
Basin
Guaíba Basin
Upper Jacuí Microbasin Taquari-Antas Microbasin
Epactionotus sp.
Hisonotus sp.
Haplo 15
Haplo 1
Haplo 2
Haplo 3
Haplo 11
Haplo 10
Haplo 6
Haplo 12
Haplo 5
Haplo 9
Haplo 8
Haplo 7
Haplo 13
Haplo 4
Haplo 14
18
18
3
3
14,15
15
14
15
14
15
14,15
13
13
13
1, 2
2
2
19
19
20
3, 6
6
6
6
6
6
6
7
17
19
21
17
16
16
16
16
5
4
4
4
8, 9,10
8,10
10
9,10
8
22
22
22
11
11
11
12
12
12
11,12
12
12
11
11
100/98
98/98
96/84
87/87
100/86
84/84
100/92
97/90
100/81
70/69
100/77
67/65
100/90
93/97
100/89
81/85
100/82
77/82
100/98
94/99
100/99
100/100
100/73
70/ 73
78/*
67/65
100/98
98/99
100/92
71/84
100/93
87/90
100/98
97/95
100/100
100/100
100/80
93/80
*/-
-/*
Haplo 17
Haplo 16
Haplo 23
Haplo 24
Haplo 27
Haplo 22
Haplo 25
Haplo 26
Haplo 28
Haplo 21
Haplo 19
Haplo 18
Haplo 20
Haplo 29
Haplo 30
Haplo 31
Haplo 34
Haplo 32
Haplo 33
Haplo 36
Haplo 35
Haplo 40
Haplo 43
Haplo 38
Haplo 41
Haplo 37
Haplo 42
Haplo 39
Haplo 57
Haplo 58
Haplo 59
Haplo 55
Haplo 54
Haplo 56
Haplo 51
Haplo 52
Haplo 45
Haplo 44
Haplo 46
Haplo 53
Haplo 47
Haplo 50
Haplo 48
Haplo 49
E. pantherinus
E. limulus
Clade A
E. sp. nov. 4
Clade B
E. sp. nov. 7
Clade C
Clade D
Clade F
E. sp. nov. 3
Clade E
E. pantherinus
E. limulus
E. sp. nov.1
E. sp. nov.2
E. sp. nov.3
E. sp. nov.4
E. sp. nov.5
E. sp. nov.6
E. sp. nov.7
Clade G
100/93
-/-
*/*
-/-
Uruguay
Basin
Guaíba Basin
Upper Jacuí Microbasin Taquari-Antas Microbasin
Epactionotus sp.
Hisonotus sp.
Haplo 15
Haplo 1
Haplo 2
Haplo 3
Haplo 11
Haplo 10
Haplo 6
Haplo 12
Haplo 5
Haplo 9
Haplo 8
Haplo 7
Haplo 13
Haplo 4
Haplo 14
18
18
3
3
14,15
15
14
15
14
15
14,15
13
13
13
1, 2
2
2
19
19
20
3, 6
6
6
6
6
6
6
7
17
19
21
17
16
16
16
16
5
4
4
4
8, 9,10
8,10
10
9,10
8
22
22
22
11
11
11
12
12
12
11,12
12
12
11
11
100/98
98/98
96/84
87/87
100/86
84/84
100/92
97/90
100/81
70/69
100/77
67/65
100/90
93/97
100/89
81/85
100/82
77/82
100/98
94/99
100/99
100/100
100/73
70/ 73
78/*
67/65
100/98
98/99
100/92
71/84
100/93
87/90
100/98
97/95
100/100
100/100
100/80
93/80
*/-
-/*
Haplo 17
Haplo 16
Haplo 23
Haplo 24
Haplo 27
Haplo 22
Haplo 25
Haplo 26
Haplo 28
Haplo 21
Haplo 19
Haplo 18
Haplo 20
Haplo 29
Haplo 30
Haplo 31
Haplo 34
Haplo 32
Haplo 33
Haplo 36
Haplo 35
Haplo 40
Haplo 43
Haplo 38
Haplo 41
Haplo 37
Haplo 42
Haplo 39
Haplo 57
Haplo 58
Haplo 59
Haplo 55
Haplo 54
Haplo 56
Haplo 51
Haplo 52
Haplo 45
Haplo 44
Haplo 46
Haplo 53
Haplo 47
Haplo 50
Haplo 48
Haplo 49
E. pantherinus
E. limulus
Clade A
E. sp. nov. 4
Clade B
E. sp. nov. 7
Clade C
Clade D
Clade F
E. sp. nov. 3
Clade E
E. pantherinus
E. limulus
E. sp. nov.1
E. sp. nov.2
E. sp. nov.3
E. sp. nov.4
E. sp. nov.5
E. sp. nov.6
E. sp. nov.7
Clade G
100/93
-/-
*/*
-/-
Epactionotus sp.
Hisonotus sp.
Haplo 15
Haplo 1
Haplo 2
Haplo 3
Haplo 11
Haplo 10
Haplo 6
Haplo 12
Haplo 5
Haplo 9
Haplo 8
Haplo 7
Haplo 13
Haplo 4
Haplo 14
18
18
3
3
14,15
15
14
15
14
15
14,15
13
13
13
1, 2
2
2
19
19
20
3, 6
6
6
6
6
6
6
7
17
19
21
17
16
16
16
16
5
4
4
4
8, 9,10
8,10
10
9,10
8
22
22
22
11
11
11
12
12
12
11,12
12
12
11
11
100/98
98/98
96/84
87/87
100/86
84/84
100/92
97/90
100/81
70/69
100/77
67/65
100/90
93/97
100/89
81/85
100/82
77/82
100/98
94/99
100/99
100/100
100/73
70/ 73
78/*
67/65
100/98
98/99
100/92
71/84
100/93
87/90
100/98
97/95
100/100
100/100
100/80
93/80
*/-
-/*
Haplo 17
Haplo 16
Haplo 23
Haplo 24
Haplo 27
Haplo 22
Haplo 25
Haplo 26
Haplo 28
Haplo 21
Haplo 19
Haplo 18
Haplo 20
Haplo 29
Haplo 30
Haplo 31
Haplo 34
Haplo 32
Haplo 33
Haplo 36
Haplo 35
Haplo 40
Haplo 43
Haplo 38
Haplo 41
Haplo 37
Haplo 42
Haplo 39
Haplo 57
Haplo 58
Haplo 59
Haplo 55
Haplo 54
Haplo 56
Haplo 51
Haplo 52
Haplo 45
Haplo 44
Haplo 46
Haplo 53
Haplo 47
Haplo 50
Haplo 48
Haplo 49
E. pantherinus
E. limulus
Clade A
E. sp. nov. 4
Clade B
E. sp. nov. 7
Clade C
Clade D
Clade F
E. sp. nov. 3
Clade E
E. pantherinus
E. limulus
E. sp. nov.1
E. sp. nov.2
E. sp. nov.3
E. sp. nov.4
E. sp. nov.5
E. sp. nov.6
E. sp. nov.7
Clade G
100/93
-/-
*/*
-/-
42
B)
0.002
E. li mulus
Clade A
Clade B
Clade G
E. sp. nov. 4
E. sp. nov. 7
Clade C
Clade D
Clade E
E. sp. nov. 3
Clade F
0. 002
E. li mulus
Clade A
Clade B
Clade G
E. sp. nov. 4
E. sp. nov. 7
Clade C
Clade D
Clade E
E. sp. nov. 3
Clade F
43
Fig. 3
Taquari-Antas
Microbasin
Upper Jacuí
Microbasin
6
13
18
16
4
5
3
19
17
3
3
7
21
20
E. li mul us
E. sp. nov.1
E. sp.nov. 2
E. sp.nov. 3
E. sp. nov.4
E. sp.nov. 5
E. sp.nov. 6
E. sp.nov. 7
44
Fig. 4
E. li mul us
Clade A
Clade B
Clade G
E. sp. nov. 4
E. sp. nov. 7
Clade C
Clade D
Clade E
E. sp. nov. 3
Clade F
E. pantheri nus
0
1.0
2.0
0.25
0.5
01.0
2.0
0.5
3.0
45
Fig. 5
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
number of differences
frequency
observed
expected
46
Fig. 6
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
0
200,000
400,000
600,0
0
0
800,000
1
,0
00,0
0
0
1,200,000
0
10
3
10
4
10
5
10
6
10
7
10
8
0
10
4
10
5
10
6
10
7
10
8
10
3
0
.
2
0
.
2
0
.
3
0
.
4
0
.
5
0
.
6
0
.
4
0
.
6
0
.
8
1
.
0
1
.
2
0
.
1
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
0
200,000
400,000
600,0
0
0
800,000
1
,0
00,0
0
0
1,200,000
0
10
3
10
4
10
5
10
6
10
7
10
8
0
10
4
10
5
10
6
10
7
10
8
10
3
0
.
2
0
.
2
0
.
3
0
.
4
0
.
5
0
.
6
0
.
4
0
.
6
0
.
8
1
.
0
1
.
2
0
.
1
47
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