Rediscovery of Leptopanchax splendens (Cyprinodontiformes: Aplocheilidae): a seasonal killifish from the Atlantic Forest of south-eastern Brazil that was recently considered extinct.

A recent collection of the seasonal killifish found Leptopanchax splendens c. 5 km from the type locality, 74 years after its last record. The species was historically common in its type locality, the Estrela River basin in south-eastern Brazil, until 1950, after which it was not encountered and thought to have become extinct due to widespread deforestation and urbanization in the region. Despite the rediscovery, this study finds that other recently published reports of L. splendens are misidentifications.

Molecular phylogeny and timing of diversification in South American Cynolebiini seasonal killifishes.

The rich biological diversity of South America has motivated a series of studies associating evolution of endemic taxa with the dramatic geologic and climatic changes that occurred during the Cainozoic. The organism here studied is the killifish tribe Cynolebiini, a group of seasonal fishes uniquely inhabiting temporary pools formed during the rainy seasons. The Cynolebiini are found in open vegetation areas inserted in the main tropical and subtropical South American phytogeographical regions east of the Andes. Here, we present the first molecular phylogeny sampling all the eight genera of the Cynolebiini, using fragments of two mitochondrial and four nuclear genes for 35 species of Cynolebiini plus 19 species as outgroups. The dataset, 4448bp, was analysed under Bayesian and maximum likelihood approaches, providing a relatively well solved tree, which retrieves high support values for the Cynolebiini and most included clades. The resulting tree was used to estimate the time of divergence in included lineages using two cyprinodontiform fossils to calibrate the tree. We further investigated historical biogeography through the likelihood-based DEC model. Our estimates indicate that divergence between the clades comprising New World and Old World aplocheiloids occurred during the Eocene, about 50Mya, much more recent than the Gondwanan fragmentation scenario assumed in previous studies. This estimation is nearly synchronous to estimated splits involving other South American and African vertebrate clades, which have been explained by transoceanic dispersal through an ancient Atlantic island chain during the Palaeogene. We estimate that Cynolebiini split from its sister group Cynopoecilini in the Oligocene, about 25Mya and that Cynolebiini started to diversify giving origin to the present genera during the Miocene, about 20-14Mya. The Cynolebiini had an ancestral origin in the Atlantic Forest and probably were not present in the open vegetation formations of central and northeastern South America until the Middle Miocene, when expansion of dry open vegetation was favoured by cool temperatures and strike seasonality. Initial splitting between the genera Cynolebias and Simpsonichthys during the Miocene (about 14Mya) is attributed to the uplift of the Central Brazilian Plateau.

Molecular phylogeny and evolution of internal fertilization in South American seasonal cynopoeciline killifishes.

Internal fertilization is a widespread mode of reproduction in chondrichthyans and tetrapods, but uncommon in actinopterygian fishes. In killifishes of the suborder Aplocheiloidei, internal fertilization is restricted to two genera, Campellolebias and Cynopoecilus, both containing species adapted to life in seasonal pools of subtropical South America and exhibiting elaborated inseminating structures. Phylogenetic studies involving these genera are scarce and limited to morphological characters and fragments of mitochondrial DNA sequences available for a few taxa, providing incongruent results and thus impeding hypotheses on the evolution of insemination and related morphological traits. We analyzed three nuclear loci (GLYT1, ENC1, Rho) for 13 aplocheiloid taxa obtaining the first well-supported phylogeny for cynopoecilines, thus providing a significant background to interpret evolutionary changes within the group. Like in killifishes of the suborder Cyprinodontoidei, the evolution of internal fertilization in aplocheiloids is associated with deep changes in the structure of male anal fin. The phylogenetic analyses indicate that internal fertilization corresponds to a single evolutionary event during the evolution of aplocheiloid killifishes. The analyses also indicate that male specialized muscle characters, comprising a muscular ejaculatory pump in the urogenital region and hypertrophied inclinatores and depressores anales, arose in the ancestor of the clade comprising Campellolebias and Cynopoecilus. On the other hand, anal fin specialized structures including the male inseminating tube of Campellolebias and the male inseminating fan of Cynopoecilus evolved independently in each genus.

Genetic Structure of the Mangrove Killifish Kryptolebias hermaphroditus Costa, 2011 (Cyprinodontiformes: Aplocheiloidei) Supports A Wide Connection among its Populations.

The Kryptolebias marmoratus species group is composed of the only three vertebrate species that lack females. These species present only males and simultaneously hermaphroditic individuals; that are able to reproduce by allogamy, with males, or by autogamy, performing self-fertilization and generating clones of themselves. The proportion of males is variable among those species and even among their populations. Kryptolebias hermaphroditus has the smallest proportion of males. Indeed, no males have been recorded in most known populations. This is a mainly autogamous species, with small populations having a disjunct distribution along the eastern and northern coast of Brazil. Species presenting such adaptations would be expected to have an elevated rate of genetic population structure, reflecting any barriers that obstruct gene flow between populations. Partial sequences of the mitochondrial cytochrome c oxidase I (COI) gene from 335 individuals were sampled to perform a population analysis. Only a single haplotype of COI, widely distributed throughout all the sampled populations, was recovered for K. hermaphroditus. Here we hypothesize that the high degree of communication within populations is probably the main biological feature leading to this pattern.

Integrative Taxonomy, Distribution and Ontogenetic Colouration Change in Neotropical Mountain Catfishes of the Trichomycterus nigroauratus Group (Siluriformes: Trichomycteridae).

Catfishes of the genus Trichomycterus comprise the most diverse fish group in mountain river basins crossing the Atlantic Forest of south-eastern Brazil, with a great concentration of species in the Rio Paraíba do Sul basin. The present study is directed to the T. nigroauratus group, endemic to the Rio Paraíba do Sul basin, comprising species commonly found associated with bottom leaf litter. Field studies revealed two sympatric, distinct colour morphs, one comprising small specimens with a black longitudinal stripe over a pale brown ground colour on the flank and another comprising a single larger specimen with small dark browns spots scattered over a pale-yellow ground colour. These specimens were found in the upper Rio Paraitinga drainage, an area disjunct from the area inhabited by the other species of the group. We performed coalescent single-locus analyses for species delimitation using a cytochrome b fragment (1088 bp) for specimens from eight localities, including sequences taken from specimens exhibiting different colouration morphs and topotypes of all the three nominal species of the T. nigroauratus group. The analyses supported the two colouration morphs as belonging to a single species that is herein described. It is distinguished from other congeners by its unique colour pattern, dorsal and anal fins fin more posteriorly placed, and by the morphology of the opercle, metapterygoid and pelvic bone. The Maximum Likelihood analysis indicated the new species as sister to a clade containing all other congeners of the group, which is corroborated by osteological data. The occurrence of different lineages in neighbouring areas of the upper Rio Paraíba do Sul basin is interpreted as a possible vestige of a past complex paleogeographical scenario during the Cenozoic. The present record of striking ontogenetic colouration change, revealed only after checking DNA sequences of individuals exhibiting different colouration phenotypes, again highlights the importance of combining morphological and molecular data in taxonomical studies.

Multigene phylogeny supports diversification of four-eyed fishes and one-sided livebearers (Cyprinodontiformes: Anablepidae) related to major South American geological events.

The high diversity of Neotropical fishes has been attributed to major South American palaeogeographic events, such as Andean uplift, rise of the Isthmus of Panama and marine transgressions. However, the unavailability of temporal information about evolution and diversification of some fish groups prevents the establishment of robust hypotheses about correlations between species diversification and proposed palaeogeographical events. One example is the Anablepidae, a family of teleost fishes found mostly in coastal habitats of Central and South America, but also in some inner river basins of South America. Historical aspects of the distribution patterns of the Anablepidae were never analysed and no accurate estimation of time of its origin and diversification is presently available. A multi-gene analysis was performed to estimate Anablepidae phylogenetic position, age and biogeography, comprising seven nuclear genes. The suborder Cyprinodontoidei was recovered in three major clades, one comprising all the Old World Cyprinodontoidei and two comprising New World lineages. Anablepidae was recovered as the sister group of the New World Poeciliidae, with the Amazonian genus Fluviphylax as their sister group. The ages found for the origin and diversification of Cyprinodontiformes were congruent with the pattern recorded for other vertebrate groups, with an origin anterior to the Cretaceous-Paleogene (K-Pg) transition and diversification during the Paleogene. The age estimated for the split between the Atlantic and Pacific lineages of Anableps was congruent with the rise of Panamanian Isthmus. The results suggest Miocene marine transgressions as determinant to the current distribution of Jenynsia.

Cryptic species diversity in the Hypsolebiasmagnificus complex, a clade of endangered seasonal killifishes from the São Francisco River basin, Brazilian Caatinga (Cyprinodontiformes, Aplocheilidae).

A great diversity of animal species adapted to life in the semi-arid Caatinga of northeastern Brazil, including seasonal killifishes, has been reported in the last three decades. More recently, field and molecular data have shown a high occurrence of cryptic species. The killifish group herein analysed, the Hypsolebiasmagnificus species complex, is endemic to the middle and southern portion of the Caatinga, occupying about 120 km along the floodplains of the middle São Francisco River and some adjacent tributaries. Species of this complex are rare and presently considered threatened with extinction, being uniquely found in pools protected by trees and bushes. Single-locus delimitation methods were used to test species limits of populations displaying different colour patterns along the whole distribution of the complex. All analyses consistently supported the three nominal species and two new, herein described: H.gardneri Costa, sp. n., from the floodplains of the middle São Francisco River and H.hamadryades Costa, sp. n., from the Gorotuba River floodplains. The phylogenetic analysis highly supports H.hamadryades as sister to a clade comprising H.gardneri and H.harmonicus. Our field observations suggest that H.hamadryades is a miniature species. This study indicates that the H.magnificus complex comprises cryptic species apparently endemic to small areas and extremely vulnerable to environmental changes, deserving high concern.

Synchronic historical patterns of species diversification in seasonal aplocheiloid killifishes of the semi-arid Brazilian Caatinga.

The Caatinga is the largest nucleus of seasonally dry tropical forests in South America, but little is known about the evolutionary history and biogeography of endemic organisms. Evolutionary diversification and distribution of terrestrial vertebrates endemic to the Caatinga have been explained by palaeogeographical Neogene episodes, mostly related to changes in the course of the São Francisco River, the largest river in the region. Our objective is to estimate the timing of divergence of two endemic groups of short-lived seasonal killifishes inhabiting all ecoregions of the Caatinga, testing the occurrence of synchronic events of spatial diversification in light of available data on regional palaeogeography. We performed independent time-calibrated phylogenetic molecular analyses for two clades of sympatric and geographically widespread seasonal killifishes endemic to the Caatinga, the Hypsolebias antenori group and the Cynolebias alpha-clade. Our results consistently indicate that species diversification took place synchronically in both groups, as well as it is contemporary to diversification of other organisms adapted to life in the semi-arid Caatinga, including lizards and small mammals. Both groups originated during the Miocene, but species diversification started between the Late Miocene and Early Pliocene, when global cooling probably favoured the expansion of semi-arid areas. Synchronic diversification patterns found are chronologically related to Tertiary palaeogeographical reorganizations associated to continental drift and to Quaternary climatic changes, corroborating the recent proposal that South American biodiversity has been continuously shaped between the Late Paleogene and Pleistocene.

A new species of inseminating seasonal killifish of the Cynopoecilus melanotaenia complex from southern Brazil (Cyprinodontiformes: Rivulidae).

BACKGROUND: The Cynopoecilus melanotaenia complex is a morphologically homogeneous killifish group, endemic from an area encompassing southern Brazil and northeastern Uruguay. It presently comprises four valid species: C. melanotaenia, the type species of the genus, and C. fulgens, C. intimus, and C. nigrovittatus. NEW INFORMATION: Cynopoecilus feltrini, n. sp., from the lower Tubarão river basin, southern Brazil, is distinguished from all congeners of the C. melanotaenia complex by having frontal E-scales medially overlapped, branchiostegal region orangish red in males and dorsum with few dark brown spots above opercular region. A phylogenetic tree derived from the analysis of a fragment of the mitochondrial gene cytochrome c oxidase subunit I (681 bp) indicates that C. feltrini is a member of the clade that includes all species of the C. melanotaenia complex except C. melanotaenia, as well as that C. feltrini is the sister group of a clade comprising C. fulgens and C. nigrovittatus.