Integrative taxonomy reveals a new species of pacu (Characiformes: Serrasalmidae: Myloplus) from the Brazilian Amazon

Pacus of the genus Myloplus represent a formidable taxonomic challenge, and particularly so for the case of M. asterias and M. rubripinnis, two widespread and common species that harbor considerable morphological diversity. Here we apply DNA barcoding and multiple species discovery methods to find candidate species in this complex group. We report on one well-supported lineage that is also morphologically and ecologically distinct. This lineage represents a new species that can be distinguished from congeners by the presence of dark chromatophores on lateral-line scales, which gives the appearance of a black lateral line. It can be further diagnosed by having 25-29 branched dorsal-fin rays (vs. 18-24), 89-114 perforated scales from the supracleithrum to the end of hypural plate (vs. 56-89), and 98-120 total lateral line scales (vs. 59-97). The new species is widely distributed in the Amazon basin, but seems to have a preference for black- and clearwater habitats. This ecological preference and black lateral line color pattern bears a striking similarity to the recently described silver dollar Metynnis melanogrammus.(AU)

Pacus do gênero Myloplus representam um desafio taxonômico formidável, e particularmente o caso de M. asterias e M. rubripinnis, duas espécies amplamente distribuídas e comuns que abrigam uma considerável diversidade morfológica. Aplicamos aqui a tecnologia do DNA barcoding e múltiplos métodos de descoberta de espécies para encontrar possíveis espécies novas nesse grupo complexo. Registramos uma linhagem bem suportada que também é distinta morfológica e ecologicamente. Essa linhagem representa uma nova espécie que pode ser distinguida das demais congêneres por apresentar cromatóforos escuros nas escamas da linha lateral que conferem uma aparência de linha lateral preta. Ela pode ser adicionalmente diagnosticada por ter 25-29 raios ramificados na nadadeira dorsal (vs. 18-24), 89-114 escamas perfuradas do supracleitro até o final da placa hipural (vs. 56-89) e 98-120 escamas totais na linha lateral (vs. 59-97). A nova espécie é amplamente distribuída na bacia Amazônica, mas aparentemente possui preferência por habitats de água preta e clara. A preferência ecológica e o padrão de colorido escuro da linha lateral consistem em semelhanças impressionantes com o silver dólar recém descrito Metynnis melanogrammus.(AU)

Phylogenetic relationships of the neon tetras Paracheirodon spp. (Characiformes: Characidae: Stethaprioninae), including comments on Petitella georgiae and Hemigrammus bleheri

Neon tetras (Paracheirodon spp.) are three colorful characid species with a complicated taxonomic history, and relationships among the species are poorly known. Molecular data resolved the relationships among the three neon tetras, and strongly supported monophyly of the genus and its sister taxon relationship to Brittanichthys. Additionally, the sister-taxon relationship of the rummy-nose tetras Hemigrammus bleheri and Petitella georgiae was strongly supported by molecular and morphological data. Therefore, we propose to transfer the rummy-nose tetras H. bleheri and H. rhodostomus to the genus Petitella. Furthermore, Petitella georgiae is likely to be a species complex comprised of at least two species.(AU)

Os neon tetras (Paracheirodon spp.) são três espécies de caracídeos coloridos com uma complicada história taxonômica e as relações entre suas espécies são pouco conhecidas. Dados moleculares resolveram as relações entre os três neons tetras, suportando fortemente a monofilia do gênero e a relação de grupo-irmão com Brittanichthys. Adicionalmente, a relação de grupo-irmão entre os rodóstomos Hemigrammus bleheri e Petitella georgiae foi fortemente suportada por dados moleculares e morfológicos. Portanto, nós propomos transferir os rodóstomos H. bleheri e H. rhodostomus para o gênero Petitella. Além disso, é possível que Petitella georgiae seja um complexo de espécies composto por, pelo menos, duas espécies.(AU)

Evidence of polygamy in the socially monogamous Amazonian fish Arapaima gigas (Schinz, 1822) (Osteoglossiformes, Arapaimidae)

Arapaima gigas is one of the largest freshwater fishes of the world. It is socially monogamous, forming pairs, constructing a nest and providing parental care. We performed a paternity analysis under three scenarios in captive, semi-natural and natural areas using 10 microsatellite markers. As a positive control, we analyzed three pairs and their offspring isolated individually in artificial breeding ponds (a priori very high probability of monogamy). We then analyzed two samples of offspring from large artificial ponds with multiple adults but only one reproductive pair (a priori high probability of monogamy), two samples from semi-natural breeding station with multiple adults but only one reproductive pair (a priori high probability of monogamy), and a sample from a natural lake with multiple adults, some potentially breeding (a priori medium probability of monogamy). Analysis of patterns of Mendelian heredity suggested an extra-pair contribution for all broods except the positive controls. Similarly, results based on multilocus analysis estimated at least two sib-groups per nest. These results reject monogamy as a system of breeding in Arapaima gigas. From a management perspective, this behavior may be exploited to maintain genetic diversity in captive and as well in wild populations of Arapaima gigas.

O pirarucu Arapaima gigas é um dos maiores peixes de água doce do mundo. É socialmente monogâmico, forma casais, constrói ninhos e fornece cuidado parental. Com o objetivo de acessar o sistema de acasalamento do pirarucu, analisamos três cenários: em áreas de cativeiro, semi-naturais e naturais, utilizando 10 marcadores microssatélites. Como controle positivo, analisamos três casais e suas ninhadas isolados em açudes individuais (probabilidade a priori muito alta de monogamia). A seguir, analisamos duas amostras de ninhadas de um açude com vários adultos, mas somente um casal reprodutivo (probabilidade a priori alta de monogamia), duas amostras de estação de criação semi-natural com vários adultos mas somente um casal reprodutivo (probabilidade a priori alta de monogamia), e uma amostra de lago natural com vários adultos alguns potencialmente em fase de reprodução (probabilidade a priori média de monogamia). Análises de padrões mendelianos de hereditariedade sugerem contribuição extra-par para todas as ninhadas, exceto as do controle positivo. Similarmente, resultados baseados em análises multilocus realizadas no programa KINALYZER estimaram pelo menos dois grupos-irmãos por ninhada. Nossos resultados rejeitam a monogamia como sistema de acasalamento em Arapaima gigas. Da perspectiva de manejo, esse comportamento pode ser explorado para manter a diversidade genética em cativeiro assim como em populações naturais de Arapaima gigas.

Molecular diagnosis of the arowanas Osteoglossum ferreirai Kanazawa, 1966 and O. bicirrhossum (Cuvier, 1829) from the Orinoco and Amazon River basins

The arowanas, fishes of Gondwanan origin, are represented in South America by the genus Osteoglossum. All species were initially reported as being exclusive to the Amazon region, with O. ferreirai restricted to the Negro River basin and O. bicirrhosum to the Amazon and Essequibo Rivers basin. Starting in the mid 1970's it was reported that O. ferreirai also occurs in the Orinoco River basin. In all regions the arowanas assumed significant socio-economic importance due to their popularity in the international ornamental fish trade, leading to over-exploitation of both species in some areas. The Orinoco populations are particularly heavily exploited, and thus conservation and management measures are needed. Both depend on the clarification of taxonomic status, and phylogenetic distinctness of the Orinoco populations. With the goal of molecularly characterizing the two species of Osteoglossum, and comparing populations of Osteoglossum from the Orinoco and Amazon basins, we characterized individuals sampled from eight localities, one in the Orinoco River basin and seven in the Amazon River basin. We sampled 39 individuals, obtaining 1004 base pairs, of which 79 were synapomorphies. Genetic distance between the two species calculated using the HKY + G model of molecular evolution was 8.94%. Intraspecific distances ranged from 0.42% in O. bicirrhosum to 0.10% in O. ferreirai. The genetic characterization confirmed the taxonomic status of O. ferreirai in the Orinoco basin, and suggested that its distribution in the Orinoco basin is unlikely to be the result of vicariance or natural dispersal, but rather an anthropic introduction.

Os aruanãs, peixes de origem Gondwana, são representados na América do Sul pelo gênero Osteoglossum. Todas as espécies foram inicialmente reportadas como sendo exclusivamente da região Amazônica, com O. ferreirai restrito a bacia do Rio Negro e O. bicirrhosum para a bacia Amazônica e rio Essequibo. No meio dos anos 70 foi reportado que O. bicirrhosum também ocorre na bacia do rio Orinoco. Em todas as regiões os aruanãs são de significante importância socio-econômica devido a sua popularidade no comércio internacional de peixes ornamentais, levando a sobre-exploração de ambas as espécies em algumas áreas. As populações no rio Orinoco são particularmente muito exploradas, e assim medidas de conservação e manejo são necessárias. Ambas as medidas dependem de clarificações do estatus taxonômico, e distinções filogenéticas das populações do Orinoco. Com o objetivo de caracterizar molecularmente as duas espécies de Osteoglossum, e comparar as populações de Osteoglossum das bacias do Orinoco e Amazonas, nós caracterizamos indivíduos amostrados de oito localidades, uma da bacia do rio Orinoco e sete da bacia Amazônica. Nós amostramos 39 indivíduos, obtendo 1004 pares de bases, dos quais 97 foram sinapomorfias. A distância genética entre as duas espécies, calculadas usando-se o modelo HKY+G de evolução molecular foi de 8.94%. Distâncias intraespecíficas variaram de 0.42% em O. bicirrhosum a 0.10% em O. ferreirai. A caracterização genética confirmou o estatus taxonômico de O. ferreirai na bacia do rio Orinoco, e sugere que sua distribuição na bacia do rio Orinoco é improvavel de ser o resultado de um evento de vicariância ou dispersão natural, sendo melhor explicada como uma introdução antrópica.

Color pattern variation in Cichla temensis (Perciformes: Cichlidae): resolution based on morphological, molecular, and reproductive data

Morphological variants of Cichla temensis, readily differentiated by their striking color pattern differences, are found in several Amazon basin flood pulse river systems. The adult variants have at times been thought to represent different species or sexual dimorphism. A three part study was performed in two regions in Brazil (rio Igapó Açú and rio Caures) to elucidate the nature of the variants. In part one; selected diagnostic morphometric characters were compared intraspecifically among the variants and interspecifically with C. monoculus and C. orinocensis. All of the C. temensis variants were found to differ significantly from their sympatric congeners while not differing among each other. In part two, mitochondrial DNA samples were compared intraspecifically among the variants and interspecifically with their sympatric congeners. There were no diagnostic molecular synapomorphies that would unambiguously distinguish the variants and all C. temensis variants were clearly diagnosable and divergent from their sympatric congeners. In part three, color pattern variation in both sexes was compared to a gonadosomatic index (GSI). A significant correlation between color pattern variation and gonadosomatic index was found. The results of this study demonstrate that Cichla temensis variants are confirmed to be members of a single species and that the variation does not represent a sexual dimorphism. The color pattern variation is a cyclically occurring secondary sexual characteristic and is indicative of the specific degree of an individual's seasonal sexual maturation.

Variantes morfológicas de Cichla temensis, facilmente diferenciados por seus padrões de coloração marcantes, são encontrados em vários rios do sistema de inundação da várzea da bacia Amazônica. Variantes dos indivíduos adultos têm sido cogitados como pertencerem a uma diferente espécie ou apresentarem dimorfismo sexual. Um estudo em três etapas foi realizado em duas regiões no Brasil (rio Igapó Açú e rio Caures) para elucidar a natureza desta variação. Na primeira parte, caracteres morfométricos diagnósticos foram comparados intraespecificamente entre os variantes e interespecificamente com C. monoculus e C. orinocensis. Todos os variantes de C. temensis apresentaram diferenças significativas quando comparados com seus congêneres simpátricos enquanto não diferiram entre si. Na segunda parte, sequências de DNA mitocondrial foram comparadas intraespecificamente entre os variantes e interespeficamente com seus congêneres simpátricos. Não houve sinapomorfias moleculares diagnósticas para diferenciar inequivocamente os variantes e todos os variantes de C. temensis foram claramente divergentes de seus congêneres simpátricos. Na terceira parte, a variação no padrão de cor em ambos os sexos foi comparado ao índice gonadossomático (GSI). Foi encontrada uma correlação significativa entre a variação na coloração e o índice gonadossomático. Os resultados deste estudo demonstram que os variantes em Cichla temensis são membros de uma única espécie e que a variação não representa um dimorfismo sexual. A variação no padrão de cor é uma característica sexual secundária que ocorre em ciclos, sendo um indicativo do grau de maturação sexual sazonal dos indivíduos.

The complete mitochondrial genome of the pirarucu (Arapaima gigas, Arapaimidae, Osteoglossiformes)

We sequenced the complete mitochondrial genome of the pirarucu, Arapaima gigas, the largest fish of the Amazon basin, and economically one of the most important species of the region. The total length of the Arapaima gigas mitochondrial genome is 16,433 bp. The mitochondrial genome contains 13 protein-coding genes, two rRNA genes and 22 tRNA genes. Twelve of the thirteen protein-coding genes are coded on the heavy strand, while nad6 is coded on the light strand. The Arapaima gene order and content is identical to the common vertebrate form, as is codon usage and base composition. Its control region is atypical in being short at 767 bp. The control region also contains a conserved ATGTA motif recently identified in the Asian arowana, three conserved sequence blocks (CSB-1, CBS-2 and CBS-3) and its 3' end contains long series of di- and mono-nucleotide microsatellite repeats. Other osteoglossiform species for which control region sequences have been published show similar control region characteristics.

A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes)

DNA barcoding is a recently proposed global standard in taxonomy based on DNA sequences. The two main goals of DNA barcoding methodology are assignment of specimens to a species and discovery of new species. There are two main underlying assumptions: i) reciprocal monophyly of species, and ii) intraspecific divergence is always less than interspecific divergence. Here we present a phylogenetic analysis of the family Potamotrygonidae based on mitochondrial cytochrome c oxidase I gene, sampling 10 out of the 18 to 20 valid species including two non-described species. Potamotrygonidae systematics is still not fully resolved with several still-to-be-described species while some other species are difficult to delimit due to overlap in morphological characters and because of sharing a complex color patterns. Our results suggest that the family passed through a process of rapid speciation and that the species Potamotrygon motoro, P. scobina, and P. orbignyi share haplotypes extensively. Our results suggest that systems of identification of specimens based on DNA sequences, together with morphological and/or ecological characters, can aid taxonomic studies, but delimitation of new species based on threshold values of genetic distances are overly simplistic and misleading.

Genomic resources for the conservation and management of the harpy eagle (Harpia harpyja, Falconiformes, Accipitridae)

We report the characterization and optimization of 45 heterologous microsatellite loci, and the development of a new set of molecular sex markers for the conservation and management of the Neotropical harpy eagle (Harpia harpyja L. 1758). Of the 45 microsatellites tested, 24 were polymorphic, six monomorphic, 10 uncharacterizable due to multiple bands and five did not amplify. The observed gene diversity of the analyzed sample of H. harpyja was low and similar to that of other threatened Falconiformes. While a high proportion of the microsatellite markers were highly variable, individuals of H. harpyja could be differentiated by a joint analysis of just three (p = 2.79 x 10-4) or four markers (p = 2.89 x 10-5). Paternity could be rejected with 95.23 percent and 97.83 percent probabilities using the same three and four markers, respectively. The sex determination markers easily and consistently differentiated males from females even with highly degraded DNA extracted from naturally shed feathers. The markers reported in this study potentially provide an excellent set of molecular tools for the conservation and management of wild and captive H. harpyja and they may also prove useful for the enigmatic Neotropical crested eagle (Morphnus guianensis Daudin 1800).

Population genetic analysis of Caiman crocodilus (Linnaeus, 1758) from South America

The genetic structure of Caiman crocodilus was investigated using a 1085 bp mtDNA fragment of the cytochrome b gene. Inferences were based on 125 individuals from nine localities in Peru, Brazil and French Guiana. With the exception of Mamirauá Lake, Anavilhanas Archipelago and the Tapará Community which show a signal of demographic expansion, the sampled localities are in a mutation-drift genetic equilibrium. Divergence between the Amazon basin and extra-Amazon basin localities is significant; however, inference from Nested Clade Analysis cannot distinguish between continuous range expansion, long distance colonization or past fragmentation; however, past fragmentation is unlikely due to low number of mutational steps separating these two regions. The divergence is probably maintained by the reduced ability of C. crocodilus to cross salt water barriers. Within the Amazon basin, continuous range expansion without isolation-by-distance is the most likely process causing genetic structuring. The observed genetic patterns are compatible with the ecology of C. crocodilus, and history of human exploitation. As commercial hunting depleted more valuable species, C. crocodilus expanded its range and ecological niche, prompting hunters to harvest it. Following a period of intense hunting, C. crocodilus is now experiencing recovery and a second population expansion especially in protected areas.