Phylogenetic relationships and the origin of New World soles (Teleostei: Pleuronectiformes: Achiridae): The role of estuarine habitats.

Even though the monophyletic status of Achiridae has been supported by morphological and molecular data, the interrelationships within the representatives of this family are poorly resolved. In the present study, we carried out the most complete molecular phylogenetic analysis of this group, encompassing all genera and employing both nuclear (Rhodopsin, Recombination activator [Rag 1], Mixed - lineage Leukemia [MLL] and Early Growth Response Protein 3 [EGR3]) and mitochondrial (Cytochrome C Oxidase Subunit I [COI], Cytochrome B [CytB], ATPase 6.8, 16S and 12S RNAr) genes. All topologies based on Maximum Likelihood, Bayesian inferences and Bayesian Inference of the Multispecies Coalescent confirmed the monophyletism of Achiridae, in spite of some incongruences in relation to Achirus mucuri, A. lineatus, Apionichthys finis and Trinectes microphthalmus. In fact, Achirus and Trinectes proved to be non-monophyletic genera while Hypoclinemus mentalis was closely related to A. achirus, suggesting this species should be reevaluated. We provided evidence that Achiridae has first arisen in estuaries (about 23.5 million years ago) and some lineages have evolved independently to either marine or freshwater habitats. Furthermore, we propose a diversification scenario of New World soles involving at least two events of marine incursions during Miocene and Pliocene - Pleistocene associated with natural geographic barriers (Victoria-Trindade chain), the width and exposure of continental shelf and headwater capture along the Amazon basin. Finally, the evolutionary dependence of Achirid soles on estuaries, characterized as highly dynamic environments, has probably driven the recent divergence of many species of Achiridae.

Out of the shadows: Multilocus systematics and biogeography of night monkeys suggest a Central Amazonian origin and a very recent widespread southeastward expansion in South America.

Night monkeys (Aotus, Cebidae) are a widely distributed genus of Neotropical primates with a poorly understood taxonomy and biogeography. The number of species in the genus varies from one to nine, depending on the author, and there are at least 18 known karyotypes, varying from 2n = 46 to 2n = 58. Historically, night monkeys are divided into two species groups: red- and grey-necked groups from south and north of the Amazon-Solimões River, respectively. Here, we used 10 nuclear and 10 mitochondrial molecular markers from a wide taxonomic and geographic sample to infer phylogeny, divergence times, and biogeography of the genus. For phylogenetic reconstruction we used Maximum Likelihood (ML) and Bayesian Inferences (BI). Biogeographic models were generated using the 'BioGeoBEARS' software. We found support for nine taxa of Aotus and rejected the existence of monophyletic "red necked" and "grey necked" species groups. We suggest a taxonomic reclassification of the genus, which is better represented by two clades named northern group, which contains Aotus miconax, A. nancymae, A. trivirgatus, A. vociferans, A. lemurinus, A. griseimembra, A. zonalis, and A. brumbacki, and southern group, which contains A. nigriceps, A. boliviensis, A. infulatus, and A. azarae. The results suggest that the most recent common ancestor of all species of Aotus arose in the central Amazon basin in the Early Pliocene. The evolutionary history of night monkeys was guided by dispersal, vicariance and founder events. The end of the Andean uplift and the subsequent changes in the Amazon landscape, as well as the Amazon-Solimões and Tapajós rivers may have played an important role in the origin and diversification of Aotus, respectively. However, most of the Amazonian rivers seem not to have been geographical barriers to dispersal of night monkeys. The herein named southern group is fruit of a very recent diversification guided by dispersal, crossing the Tapajós, Xingú, Tocantins, and Guapore rivers and reaching the Cerrado in the last 1.6 My.

Phylogenetics and an updated taxonomic status of the Tamarins (Callitrichinae, Cebidae).

Traditionally, Saguinus has been organized into six taxonomic groups: bicolor, inustus, midas, mystax, nigricollis, and oedipus. After recent revisions, taxonomic reclassifications were proposed, including (1) the recognition of Leontocebus as a new genus, and (2) the subdivision of Saguinus into three subgenera. Nonetheless, the contradictory nature of these results reinforces the inconsistency concerning the monophyletic status of tamarins and its interspecific phylogeny. Therefore, in this study, we carried out phylogenetic inferences of Saguinus based on 44 molecular markers, of which 37 were from nuclear DNA and seven from mitochondrial DNA. A final dataset of 24,202 base pairs (bp) was obtained from 60 specimens of all recognized species of Saguinus and, also representatives of two main lineages of Leontocebus. Phylogenetic hypothesis was obtained from Maximum Likelihood (ML) and Bayesian inference (BI) methods. We also construct a Species Tree and a fossil-calibrated multi-locus phylogeny to estimate the time of divergence of Tamarins. Our phylogenetic results validated Leontocebus, or nigricollis group, as monophyletic, and recovered additionally three main clades within Saguinus. Same topology was obtained by the Species Tree. These clades correspond to (1) inustus + mystax groups, (2) oedipus group and (3) bicolor + midas group. Our results show support for a 10.5-million-year-old split between Leontocebus and the remaining Saguinus, followed by two other cladogenetic events, around 9.3 and 7.2 mya, which lead to the rise of the main clades of Saguinus. These phylogenetic data, in concert with the consistent morphological, ecological behavior and biogeographic evidence suggest a new classification for the Amazonian and trans-Andean tamarins. Therefore, we support the validation of Leontocebus as genus and recommend the split of Saguinus into three genera: (1) Tamarinus (inustus and mystax groups), (2) Oedipomidas (oedipus group), and (3) Saguinus (bicolor and midas groups).

New insights about species delimitation in red snappers (Lutjanus purpureus and L. campechanus) using multilocus data.

Lutjanus campechanus and Lutjanus purpureus are two commercially important lutjanid fishes (snappers) with non-sympatric distribution throughout Western Atlantic. Even though both taxa have traditionally been regarded as valid species, their taxonomic status remains under debate. In the present study, we used phylogeographic approaches and molecular methods of species delimitation to elucidate the taxonomic issues between both species, based on 1478 base pairs from four genomic regions. We found haplotypes shared between the two species, particularly in relation to nuclear DNA (nuDNA) sequences. The molecular delimitation of species supported the discrimination of L. purpureus and L. campechanus as distinct evolutionary units. Nonetheless, a unidirectional gene flow was found from L. campechanus towards L. purpureus. Therefore, it seems plausible to infer that L. campechanus and L. purpureus are two evolutionary units in which the apparent sharing of haplotypes should be driven by introgression.

Phylogenetic relationships in the genus Cheracebus (Callicebinae, Pitheciidae).

Cheracebus is a new genus of New World primate of the family Pitheciidae, subfamily Callicebinae. Until recently, Cheracebus was classified as the torquatus species group of the genus Callicebus. The genus Cheracebus has six species: C. lucifer, C. lugens, C. regulus, C. medemi, C. torquatus, and C. purinus, which are all endemic to the Amazon biome. Before the present study, there had been no conclusive interpretation of the phylogenetic relationships among most of the Cheracebus species. The present study tests the monophyly of the genus and investigates the relationships among the different Cheracebus species, based on DNA sequencing of 16 mitochondrial and nuclear markers. The phylogenetic analyses were based on Maximum Likelihood, Bayesian Inference, and multispecies coalescent approaches. The divergence times and genetic distances between the Cheracebus taxa were also estimated. The analyses confirmed the monophyly of the genus and a well-supported topology, with the following arrangement: ((C. torquatus, C. lugens), (C. lucifer (C. purinus, C. regulus))). A well-differentiated clade was also identified within part of the geographic range of C. lugens, which warrants further investigation to confirm its taxonomic status.

Genetic diversity and structuring in the arapaima (Osteoglossiformes, Osteoglossidae) population reveal differences between the Amazon and the Tocantins-Araguaia basins.

Arapaima is a widely-distributed fish of enormous economic importance in the Amazon region. In the present study, a total of 232 specimens were sampled, 121 from five sites in the Amazon basin and 111 from five sites in the Tocantins-Araguaia basin. The analyses investigated fragments of the Cytochrome b, Control Region, Cytochrome Oxidase I, NADH dehydrogenase subunit 2 and seven loci microsatellites. The analyses revealed the existence of two mitochondrial lineages within the general area, with no haplotypes shared between basins, and genetic variability significantly higher in the Amazon than in the Tocantins-Araguaia basin. Two divergent, but sympatric mitochondrial lineages were found in the Amazon basin, but only a single lineage in the Tocantins-Araguaia basin. The existence of these two mitochondrial lineages indicates that past events, probably occurring during the Pleistocene, resulted in the separation of the populations of this species and molded its evolutionary history, which is reflected directly in its mitochondrial DNA. The analysis of the arapaima population structure identified distinct levels of diversity within the distribution of the species, indicating specific geographic regions that will require special attention for the development of conservation and management strategies.

Phylogeny of the genus Hypophthalmus Cuvier, 1829 (Pimelodidae - Siluriformes), based on a multilocus analysis, indicates diversification and introgression in the Amazon basin.

The genus Hypophthalmus encompasses four valid South American freshwater catfish species: H. marginatus, H. edentatus, H. fimbriatus, and H. oremaculatus. More recently two new species were proposed Hypophthalmus n. sp. 1 and Hypophthalmus n. sp. 2. While Hypophthalmus species are a fundamentally important resource for the commercial fisheries that operate in the continental waters of the Amazon basin, their phylogenetic relationships and the true diversity of the genus have yet to be defined conclusively. Given this, the present study analyzed sequences of the mitochondrial COI gene and four nuclear markers (RAG2, Myh6, Plagl2 and Glyt) to evaluate the phylogenetic relationships and the diversity of the species of this genus. All the analyses showed that Hypophthalmus is monophyletic, and the species delimitation tests recovered all the Hypophthalmus taxa as distinct species. The putative new species Hypophthalmus n. sp. 1 and Hypophthalmus n. sp. 2 presented mean genetic divergence similar to or greater than that observed between valid Hypophthalmus taxa. All the analyses showed that H. oremaculatus is the sister group of H. n. sp. 1, which together group with H. fimbriatus. This clade is the sister group of the clade containing H. edentatus and H. n. sp. 2. One specimen, morphologically identified as H. oremaculatus, presented the nuclear genome of this species and the mitochondrial genome of H. n. sp. 1; while another specimen, morphologically identified as H. n. sp. 2, presented the nuclear Myh6 of H. n. sp. 2 and the mitochondrial and RAG2 genome of H. edentatus. These results indicate that hybridization and introgression has occurred between species in Hypophthalmus. The findings of this study indicate that the diversity of the Hypophthalmus is underestimated, emphasize the need for a taxonomic review of the genus, and a more systematic evaluation of the hybridization patterns found, to understanding the role of hybridization and introgression in the evolution of the genus.

On a new species of titi monkey (Primates: Plecturocebus Byrne et al., 2016), from Alta Floresta, southern Amazon, Brazil.

The taxonomy of the titi monkeys (Callicebinae) has recently received considerable attention. It is now recognised that this subfamily is composed of three genera with 33 species, seven of them described since 2002. Here, we describe a new species of titi, Plecturocebus, from the municipality of Alta Floresta, Mato Grosso, Brazil. We adopt an integrative taxonomic approach that includes phylogenomic analyses, pelage characters, and locality records. A reduced representation genome-wide approach was employed to assess phylogenetic relationships among species of the eastern Amazonian clade of the Plecturocebus moloch group. Using existing records, we calculated the Extent of Occurrence (EOO) of the new species and estimated future habitat loss for the region based on predictive models. We then evaluated the species' conservation status using the IUCN Red list categories and criteria. The new species presents a unique combination of morphological characters: (1) grey agouti colouration on the crown and dorsal parts; (2) entirely bright red-brown venter; (3) an almost entirely black tail with a pale tip; and (4) light yellow colouration of the hair on the cheeks contrasting with bright red-brown hair on the sides of the face. Our phylogenetic reconstructions based on maximum-likelihood and Bayesian methods revealed well-supported species relationships, with the Alta Floresta taxon as sister to P. moloch + P. vieirai. The species EOO is 10,166,653 ha and we predict a total habitat loss of 86% of its original forest habitat under a "business as usual" scenario in the next 24 years, making the newly discovered titi monkey a Critically Endangered species under the IUCN A3c criterion. We give the new titi monkey a specific epithet based on: (1) clear monophyly of this lineage revealed by robust genomic and mitochondrial data; (2) distinct and diagnosable pelage morphology; and (3) a well-defined geographical distribution with clear separation from other closely related taxa. Urgent conservation measures are needed to safeguard the future of this newly discovered and already critically endangered primate.

Contrasting Rates of LINE-1 Amplification among New World Primates of the Atelidae Family.

LINE-1 (L1) retrotransposons constitute the dominant category of transposons in mammalian genomes. L1 elements are active in the vast majority of mammals, and only a few cases of L1 extinction have been documented. The only possible case of extinction in primates was suggested for South American spider monkeys. However, these previous studies were based on a single species. We revisited this question with a larger phylogenetic sample, covering all 4 genera of Atelidae and 3 species of spider monkeys. We used an enrichment method to clone recently inserted L1 elements and performed an evolutionary analysis of the sequences. We were able to identify young L1 elements in all taxa, suggesting that L1 is probably still active in all Atelidae examined. However, we also detected considerable variations in the proportion of recent elements indicating that the rate of L1 amplification varies among Atelidae by a 3-fold factor. The extent of L1 amplification in Atelidae remains overall lower than in other New World monkeys. Multiple factors can affect the amplification of L1, such as the demography of the host and the control of transposition. These factors are discussed in the context of host life history.

Mitochondrial introgression obscures phylogenetic relationships among manakins of the genus Lepidothrix (Aves: Pipridae).

Lepidothrix is the most diverse genus of the family Pipridae, with eight recognized species. Although the genus' monophyly has been supported by both molecular and morphological characters, phylogenetic relationships and species limits within Lepidothrix remain uncertain. In the present study, we combined molecular sequences of mitochondrial (ND2 and COI) and nuclear (MYO, G3PDh and I5BF) markers in a multilocus analysis, to evaluate relationships and inter-specific limits among L. iris, L. nattereri, and L. vilasboasi, which are known to hybridize in eastern Amazonia. The results revealed a complex pattern, whereby events of secondary contact and gene flow after isolation and genetic and phenotypic differentiation prevented the recuperation of reciprocal monophyly among the studied taxa. The mitochondrial data indicate that L. nattereri is divided into two non-sister groups, one monophyletic, and the other, paraphyletic, with L. iris iris being more closely related to one of the two L. nattereri groups, while L. iris eucephala forms an undifferentiated clade with L. vilasboasi, probably resulting from an extensive process of mitochondrial introgression. In agreement with a previous study based on Single Nucleotide Polymorphism (SNP) data, mitochondrial haplotype networks also support that L. vilasboasi does not represent a recent "hybrid swarm" between L. iris and L. nattereri, but instead a genetically divergent lineage with a separate species status. Finally, the sister relationship recovered herein between L. iris iris and some western populations of L. nattereri currently in allopatry is also apparently explained by mitochondrial introgression, as also supported for nuclear genes by SNP data, indicating a complex scenario of past contact and gene flow between currently geographically distant Lepidothrix lineages.

Phylogeny, molecular dating and zoogeographic history of the titi monkeys (Callicebus, Pitheciidae) of eastern Brazil.

The titi monkeys belong to a genus of New World primates endemic to South America, which were recently reclassified in three genera (Cheracebus, Plecturocebus and Callicebus). The genus Callicebus, which currently includes five species, is endemic to eastern Brazil, occurring in the Caatinga, Savanna, and Atlantic Forest biomes. In the present study, we investigated the validity of these species and inferred their phylogenetic relationships, divergence times, and biogeographic patterns based on the molecular analysis of a concatenated sequence of 11 mitochondrial and nuclear DNA markers, derived from 13 specimens. We ran Maximum Likelihood (ML) and Bayesian Inference (BI) analyses, and estimated genetic distances, divergence times. Ancestral areas were estimated on BioGeoBears. Our results suggest that at about twelve million years ago, the ancestor of all titi monkeys inhabited a wide area that extended from the Amazon forest to the South of the Atlantic forest. A first vicariant event originated Cheracebus in the West of the Amazon and the ancestor of Callicebus and Plectorocebus which, later were separated by a second one. The diversification of Callicebus occurred during the Plio-Pleistocene (beginning at 5 Ma) probably influenced by climatic fluctuations and geological events. Therefore, the results of the present work confirmed the existence of five species that currently inhabit forested areas under increasing threat from human activities. Thus, a reliable diagnosis of the taxonomic status of species living in endangered environments is extremely important for the development of conservation measures.

Phylogeny of the subfamily Stelliferinae suggests speciation in Ophioscion Gill, 1863 (Sciaenidae: Perciformes) in the western South Atlantic.

Phylogenies based on morphological and molecular data confirm the monophyly of the subfamily Stelliferinae; however, there is no consensus on the intergeneric and interspecific relationships in the group. Previous studies suggested the non-monophyly of Ophioscion and Stellifer, and possible cryptic species in Ophioscion punctatissimus. Therefore, we used mitochondrial (16S rDNA and COI) and nuclear (Rhodopsin, EGR1, and RAG1) regions to examine phylogenetic relationships among species of this subfamily. Our results confirmed the monophyly of Stelliferinae and supports the close relationship among Bardiella, Corvula and Odontoscion, which form a sister group to Stellifer and Ophioscion. Notwithstanding, all the results support the non-monophyly of Stellifer and Ophioscion and we suggest that a taxonomic revision should consider Ophioscion as a junior synonym of Stellifer. Moreover, O. punctatissimus was grouped into two clades, with the O. punctatissimus lineage I (LI) being closer to O. scierus from the eastern Pacific than to the O. punctatissimus lineage II (LII). The most recent common ancestor (TMRCA) for the O. scierus and O. punctatissimus LI and O. punctatissimus LII clade dates from 7.2 (HPD: 4.3-10.5) Ma, whereas TMRCA for the O. scierus and O. punctatissimus LI clade dates from 5.3 (HPD: 2.4-8.6) Ma, indicating that speciation processes may be related to the rise of the Isthmus of Panama. Phylogeographic analyses corroborate the hypothesis of speciation in O. punctatissimus. These results suggest that lineages of O. punctatissimus originated from distinct ancestors and, by morphological similarity, were considered the same taxon. A taxonomic revision should be performed to validate the species status of such lineages.

How many pygmy marmoset (Cebuella Gray, 1870) species are there? A taxonomic re-appraisal based on new molecular evidence.

The pygmy marmoset, Cebuella pygmaea, the smallest of the New World monkeys, has one of the largest geographical distributions of the Amazonian primates. Two forms have been recognized: Cebuella pygmaea pygmaea (Spix, 1823), and C. p. niveiventris Lönnberg, 1940. In this study, we investigated if the separation of pygmy marmosets into these two clades can be corroborated by molecular data. We also examine and compare coloration of the pelage in light of the new molecular results. We analyzed the mtDNA cytochrome b gene and, for the first time for any Neotropical primate, we used a reduced representation genome sequencing approach (ddRADseq) to obtain data for recently collected, geographically representative samples from the Rio Japurá, a northern tributary of the Rio Solimões and from the Javarí, Jutaí, Juruá, Madeira and Purus river basins, all tributaries south of the Solimões. We estimated phylogenies and diversification times under both maximum likelihood and Bayesian inference criteria. Our analysis showed two highly supported clades, with intraclade divergences much smaller than interclade divergences, indicating two species of Cebuella: one from the Rio Japurá and one to the south of Solimões. The interpretation of our results in light of the current taxonomy is not trivial however. Lönnberg stated that the type of Spix's pygmy marmoset (type locality 'near Tabatinga') was obtained from the south of the Solimões, and his description of the distinct niveiventris from Lago Ipixuna, south of the Solimões and several hundred kilometres east of Tabatinga, was based on a comparison with specimens that he determined as typical pygmaea that were from the upper Rio Juruá (south of the Solimões). As such it remains uncertain whether the name pygmaea should be applicable to the pygmy marmosets north of the Rio Solimões (Tabatinga type locality) or south (near Tabatinga but across the Solimões). Finally, our analysis of pelage coloration revealed three phenotypic forms: (1) south of the Rio Solimoes, (2) Eirunepé-Acre, upper Juruá basin; and (3) Japurá. More samples from both sides of Solimões in the region of Tabatinga will be necessary to ascertain the exact type locality for Spix's pygmaea and to resolve the current uncertainties surrounding pygmy marmoset taxonomy.

Mutations in the melanocortin-1 receptor (MC1R) gene have no influence on the distinct patterns of melanic plumage found in the manakins of the genus Antilophia (Aves: Pipridae).

The melanocortin-1 receptor gene is the most widely-used marker for the investigation of the genetic determination of melanic plumage patterns. Studies of a number of wild bird species have shown an association between non-synonymous mutations of the MC1R gene and the presence of melanic variants. The genus Antilophia (Pipridae) includes only two manakin species (A. galeata and A. bokermanni), which are distinguished primarily by the differences in the pattern of melanic coloration of the plumage of the mantle in the adult males. In A. galeata, this plumage is black, while in A. bokermanni, it is predominantly white. This study investigates the possible association between mutations of the MC1R marker and the variation in plumage coloration observed in the two species. The MC1R sequences of the two species was analyzed, and the observed nucleotide variation was compared. Six polymorphic sites were identified, representing seven distinct genotypes. Five of these polymorphic mutations were non-synonymous, but were not related to the different phenotypes. Neutral evolution and the absence of any systematic association between the variants of the MC1R and plumage coloration in the Antilophia species indicate that alternative mechanisms regulate the expression of the coloration of the plumage in the adult males.

Identification and characterization of the expression profile of the microRNAs in the Amazon species Colossoma macropomum by next generation sequencing.

Colossoma macropomum is a resistant species native of Amazonas and Orinoco river basins. It is regarded as the second largest finfish of Solimões and Amazon rivers, representing a major fishery resource in Amazonas and an important species in tropical aquaculture. MicroRNAs are non-coding endogenous riboregulators of nearly 22 nucleotides that play a key role in post-transcriptional gene regulation of several organisms. We analyzed samples of liver and skin from specimens of C. macropomum using next generation sequencing. The dataset was evaluated using computational programs to check the quality of sequences, identification of miRNAs, as well as to evaluate the expression levels of these microRNAs and interaction of target genes. We identified 279 conserved miRNAs, being 257 from liver and 272 from skin, with several miRNAs shared between tissues, with divergence in the number of reads. The strands miR-5p and miR-3p were observed in 72 miRNAs, some of them presenting a higher number of 3p reads. The functional annotation of the most expressed miRNAs resulted in 27 pathways for the liver and skin mainly related to the "biological processes" domain. Based on the identified pathways, we visualized a large gene network, suggesting the regulation of selected miRNA over this interactive dataset. We were able to identify and characterize the expression levels of miRNAs in two tissues of great activity in C. macropomum, which stands out as the beginning of several studies that can be carried out to elucidate the influence of miRNAs in this species and their applicability as biotechnological tools.

Phylogenetic relationships among Capuchin (Cebidae, Platyrrhini) lineages: An old event of sympatry explains the current distribution of Cebus and Sapajus.

Capuchin monkeys are currently represented by four species of Cebus and eight of Sapajus. This group is taxonomically complex and several questions still need to be clarified. In the current study, using mtDNA markers and a larger sample representation than in previous studies, we seek to understand the phylogenetic relationships among the capuchin lineages and their historical biogeography. All 12 species of capuchins were analyzed for the mitochondrial Control Region and Cytochrome b to test two biogeographical hypotheses: "Reinvasion of the Amazon (ROA)" and "Sympatric Evolution (SEV)". The phylogenetic relationships among distinct lineages within genera is consistent with an evolutionary diversification pattern probably resulting from an explosive process of diversification and dispersal between 2.0 Ma and 3.0 Ma. Also, the analyses show that the ancestral capuchins were distributed in a wide area encompassing the Amazon and Atlantic Forest. Our results support the SEV hypothesis, showing that the current syntopic distribution of Cebus and Sapajus can be explained by a sympatric speciation event in the Amazon. We also indicate that the recently proposed species taxonomy of Cebus is not supported, and that S. cay and S. macrocephalus are a junior synonym of S. apella.

Divergence of cryptic species of Doryteuthis plei Blainville, 1823 (Loliginidae, Cephalopoda) in the Western Atlantic Ocean is associated with the formation of the Caribbean Sea.

Although recent years have seen an increase in genetic analyses that identify new species of cephalopods and phylogeographic patterns, the loliginid squid of South America remain one of the least studied groups. The suggestion that Doryteuthis plei may represent distinct lineages within its extensive distribution along the western Atlantic coasts from Cape Hatteras, USA (36°N) to northern Argentina (35°S) is consistent with significant variation in a number of environmental variables along this range including in both temperature and salinity. In the present study D. plei samples were obtained from a large number of localities along the western Atlantic coasts to investigate the distribution of these possible species in a phylogeographic context. Phylogeographic analyses were performed using the mitochondrial Cytochrome Oxidase I gene and nuclear Rhodopsin gene. Divergence times were estimated using Bayesian strict clock dating with calibrations based on fossil records for divergence from the lineage containing Vampyroteuthis infernalis (162mya), the probable origins of the North American loliginids (45mya), and the European loliginids (20mya) and fossil statolith from Doryteuthis opalescens (3mya). Our results suggest a deep genetic divergence within Doryteuthis plei. The currently described specie consists of two genetically distinct clades (pair-wise genetic divergence of between 7.7 and 9.1%). One clade composed of individuals collected in northwestern Atlantic and Central Caribbean Atlantic waters and the other from southwestern Atlantic waters. The divergence time and sampling locations suggest the speciation process at approximately 16Mya, which is in full agreement with the middle Miocene orogeny of the Caribbean plate, ending up with the formation of the Lesser Antilles and the adjacent subduction zone, coinciding with a particularly low global sea level, resulting in the practical absence of continental shelves at the area, and therefore an effective geographic barrier for D. plei. Furthermore, this study also provides evidence of previously undocumented sub-population structuring in the Gulf of Mexico.

Development of EPIC-PCR Markers for Lutjanus purpureus (Lutjanidae-Perciformes) and their Potential Applicability in Population Analyses.

In the present study, a novel set of eight EPIC primers were developed for Lutjanus purpureus and assayed in five other marine teleosts including three lutjanids, one scianid and one anablepid. Most of the genomic regions used in this study presented genetic diversity indexes equal or greater than the intragenic regions commonly used in population genetics studies. Moreover, six out of eight markers showed cross-amplification with other taxa. Thus, the primers described here may be used to elucidate questions at the intraspecific level for a large number of taxa.

Molecular analyses reveal the occurrence of three new sympatric lineages of velvet worms (Onychophora: Peripatidae) in the eastern Amazon basin.

In the present study, we investigated the possible existence of new lineages of peripatids through comparisons between known Neotropical species and specimens obtained from two locations in Pará, a state in eastern Brazilian Amazonia using a molecular approach based on sequences of the mtDNA genes COI, 16Sr RNA, and 18S RNA. The analyses included also sequences of Asian and African taxa for a more systematic understanding of the phylogenetic relationships within the group. The analysis of the COI, 16S rRNA and 18S RNA sequences permitted the identification of three distinct lineages (A, B and C) based on two different phylogenetic approaches (Bayesian methods and ML). The three lineages presented here are completely distinct from all other peripatid taxa so far defined by molecular data. The presence of specimens of three independent onychophoran lineages occurring in sympatry in the Amazon basin was confirmed in all the analyses, providing consistent support for the phylogenies presented in this study. These findings reinforce the importance of the Amazon region in the diversification of Neotropical peripatids, and indicate that onychophoran diversity is much greater than previously thought, given that the number of taxa found at a single site was equivalent to the total number of allopatric species described for the entire region.

Molecular evidence of two new species of Eleotris (Gobiiformes: Eleotridae) in the western Atlantic.

Fishes of the genus Eleotris present highly conserved morphology, which may make their recognition difficult. Here, two cryptic Eleotris lineages from five locations along the coast of Brazil were identified using the COI gene and two nuclear fragments. High bootstrap and posteriori values supported those lineages, and the genetic distance of COI varied from 6% between the two lineages to 14.1% from other western Atlantic Eleotris species, such as E. pisonis, E. amblyopsis and E. perniger. The reciprocal monophyly for both types of markers, the divergences between those lineages and the other Eleotris species from the Brazilian coast may, in fact, represent two new cryptic species. The cryptic lineages and currently recognized species were collected in distinct environments, reinforcing the need for further sampling to understand the real distribution of each taxon.