Molecular and morphological diversity in species of Kronichthys (Teleostei, Loricariidae) from Atlantic coastal rivers of Brazil.

The Neotropical catfish genus Kronichthys contains three species distributed along coastal rivers of southern and southeastern Brazil. Although phylogenetic hypotheses are available, the molecular and morphological diversity and species boundaries within the genus remain unexplored. In this study, the authors generated mitochondrial data for 90 specimens combined with morphometric and meristic data to investigate species diversity, species boundaries and putative morphological signatures in Kronichthys. Phylogenetic and species delimitation results clearly show the presence of four genetic lineages, three within Kronichthys heylandi along the coast from Rio de Janeiro to southern São Paulo and a single lineage encompassing both the nominal species Kronichthys lacerta and Kronichthys subteres from the Ribeira de Iguape basin to Santa Catarina in southern Brazil. Nonetheless, morphological data show overlapped ranges in morphometrics and a definition of only two morphotypes, with clear phenotypic differences in the teeth number: K. heylandi differs from K. subteres + K. lacerta by the higher number of premaxillary teeth (30-52 vs. 19-28) and higher number of dentary teeth (28-54 vs. 17-28). Headwater captures and connections of paleodrainages because of sea-level fluctuations represent the two major biogeographic processes promoting species diversification and lineage dispersal of Kronichthys in the Atlantic coastal range of Brazil.

Genetic and Morphological Analyses Demonstrate That Schizolecis guntheri (Siluriformes: Loricariidae) Is Likely to Be a Species Complex.

Schizolecis is a monotypic genus of Siluriformes widely distributed throughout isolated coastal drainages of southeastern Brazil. Previous studies have shown that fish groups found in isolated river basins tend to differentiate over time in the absence of gene flow, resulting in allopatric speciation. In this study, we used partial sequences of the mitochondrial gene COI with the analysis of the General Mixed Yule Coalescent model (GMYC) and the Automatic Barcode Gap Discovery (ABGD) for single locus species delimitation, and a Principal Component Analysis (PCA) of external morphology to test the hypothesis that Schizolecis guntheri is a complex of species. We analyzed 94 samples of S. guntheri for GMYC and ABGD, and 82 samples for PCA from 22 coastal rivers draining to the Atlantic in southeastern Brazil from the Paraná State to the north of the Rio de Janeiro State. As a result, the GMYC model and the ABGD delimited five operational taxonomy units (OTUs - a nomenclature referred to in the present study of the possible new species delimited for the genetic analysis), a much higher number compared to the traditional alfa taxonomy that only recognizes S. guntheri across the isolated coastal rivers of Brazil. Furthermore, the PCA analysis suggests that S. guntheri is highly variable in aspects of external body proportions, including dorsal-fin spine length, pectoral-fin spine length, pelvic-fin spine length, lower caudal-fin spine length, caudal peduncle depth, anal width and mandibular ramus length. However, no exclusive character was found among the isolated populations that could be used to describe a new species of Schizolecis. Therefore, we can conclude, based on our results of PCA contrasting with the results of GMYC and ABGD, that S. guntheri represents a complex of species.

DNA barcoding techniques used to identify the shared ichthyofauna between the Pantanal floodplain and Upper Parana River.

The biological invasion process is widely debated topic, as the population depletion of some species and the extinction of others are related to this process. To accelerate the identification of species and to detect non-native forms, new tools are being developed, such as those based on genetic markers. This study aimed to use Barcode DNA methodology to identify fish species that had translocated between the Parana and Paraguay River Basins. Based on a database of two studies that were conducted in these regions, 289 sequences of Cytochrome Oxidase C subunit 1 (COI) were used for General Mixed Youle Coalecent (GMYC) analysis, including 29 morphospecies that were sampled in both river basins. As a result, we observed that while some morphospecies have low variation, demonstrating a recent occupation of the basins, other morphospecies probably represent species complexes. A third of the morphospecies had well-defined lineages but not enough to be treated as different Molecular Operational Taxonomic Units (MOTUs). These results demonstrate that human interventions possibly participated in the distribution of some lineages. However, biogeographical historical processes are also important for the morphospecies distribution. The data suggest that the number of species that are present in these two basins is underestimated and that human actions can irreversibly affect the natural history of the species in these regions.

ZZ/Z0: The New System of Sex Chromosomes in Eigenmannia aff. trilineata (Teleostei: Gymnotiformes: Sternopygidae) Characterized by Molecular Cytogenetics and DNA Barcoding.

The cytogenetic characteristics of Eigenmannia aff. trilineata were analyzed by basic and molecular cytogenetics, applying fluorescent in situ hybridization, with 18S and 5S rDNA and U2 snRNA probes. The species revealed a kind of polymorphism associated to ZZ/Z0 type sex chromosomes, with 2n = 32 (8m+2sm+22a, NF = 42) in all males under analysis, whereas females evidenced 2n = 31 (8m+1sm+22a, NF = 40). C-banding showed constitutive heterochromatin restricted to the pericentromeric region of all chromosomes and single-nucleolus organized regions on pair 11. A site for rDNA 5S was synthetic with a cluster of rDNA 18S near the centromere on the long arm of only one homologue of pair 11. Other clusters for 5S rDNA were sited on pairs 7, 10, 12, 13, and 16. Further, 5S rDNA was co-located with U2 cluster in the pericentromeric region of pair 12. Joint analysis of DNA barcoding from cytochrome c oxidase subunit I (COI) sequences, generated from the karyotyped samples of E. aff. trilineata, and sequences of other Gymnotiforms recognized E. aff. trilineata as an Operational Taxonomic Unit. Results foreground the hypothesis that cytotypes are independent evolution units as cryptic species with a low morphological differentiation level, although with high genetic/karyotype differentiation rates.

Using Different Methods to Access the Difficult Task of Delimiting Species in a Complex Neotropical Hyperdiverse Group.

The genus Rineloricaria is a Neotropical freshwater fish group with a long and problematic taxonomic history, attributed to the large number of species and the pronounced similarity among them. In the present work, taxonomic information and different molecular approaches were used to identify species boundaries and characterize independent evolutionary units. We analyzed 228 samples assembled in 53 distinct morphospecies. A general mixed yule-coalescent (GMYC) analysis indicated the existence of 70 entities, while BOLD system analyses showed the existence of 56 distinct BINs. When we used a new proposed integrative taxonomy approach, mixing the results obtained by each analysis, we identified 73 OTUs. We suggest that Rineloricaria probably has some complexity in the known species and several species not formally described yet. Our data suggested that other hyperdiverse fish groups with wide distributions can be further split into many new evolutionary taxonomic units.

Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

The fish species Synbranchus marmoratus has been reported to exist as a species complex due to high intraspecific karyotypic variability in spite of the difficulty or impossibility to distinguish them using morphological traits alone. The goal of this work was to use cytogenetic and molecular methods to determine the species delimitations and understand the karyoevolution of S. marmoratus using samples collected from distinct Brazilian localities. Among the analyzed specimens, a large degree of cytogenetic variation related to diploid numbers and karyotype structure was observed, with karyotypes showing 2n=42, 44 and 46 chromosomes. In addition, using sequences of three mitochondrial genes, the phylogenetic relationships between every sample with a known karyotype were determined, which revealed significant nucleotide divergence among the karyomorphs. Also, the analyses indicate that chromosomal rearrangements occurred independently within the distinct lineages of S. marmoratus complex, which resulted in the appearance of distinct karyotypic variants in a non-linear fashion related to diploid numbers and in the appearance of similar non-homologous chromosomes. Finally, the integration of both molecular cytogenetic and phylogenetic approaches allowed the determination of specific chromosomes possibly involved in rearrangements and a better understanding about the evolutionary processes involved in the differentiation of Synbranchus genus.

A espécie de peixe Synbranchus marmoratus tem sido reportada como um complexo de espécies devido à elevada variabilidade cariotípica intraespecífica a despeito da dificuldade ou impossibilidade de distingui-las usando apenas caracteres morfológicos. O objetivo deste trabalho foi utilizar métodos citogenéticos e moleculares para determinar a delimitação das espécies e compreender a carioevolução de S. marmoratus utilizando amostras coletadas em distintas localidades brasileiras. Dentre os espécimes analisados, um alto grau de variação citogenética relativo aos números diploides e estrutura cariotípica foi observado, com cariótipos mostrando 2n=42, 44 e 46 cromossomos. Adicionalmente, utilizando sequências de três genes mitocondriais, as relações filogenéticas entre cada amostra com cariótipo conhecido foram determinadas, revelando uma divergência nucleotídica significativa entre os cariomorfos. Além disso, as análises indicam que rearranjos cromossômicos ocorreram independentemente nas distintas linhagens do complexo S. marmoratus, o que resultou no aparecimento de distintas variantes cariotípicas de forma não linear em relação aos números diploides e no surgimento de cromossomos similares e não homólogos. Finalmente, a integração de uma abordagem citogenética molecular e filogenética permitiu a determinação de cromossomos específicos que, possivelmente, estão envolvidos em rearranjos e um melhor entendimento sobre os processos evolutivos envolvidos na diferenciação do gênero Synbranchus.

Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

The fish species Synbranchus marmoratus has been reported to exist as a species complex due to high intraspecific karyotypic variability in spite of the difficulty or impossibility to distinguish them using morphological traits alone. The goal of this work was to use cytogenetic and molecular methods to determine the species delimitations and understand the karyoevolution of S. marmoratus using samples collected from distinct Brazilian localities. Among the analyzed specimens, a large degree of cytogenetic variation related to diploid numbers and karyotype structure was observed, with karyotypes showing 2n=42, 44 and 46 chromosomes. In addition, using sequences of three mitochondrial genes, the phylogenetic relationships between every sample with a known karyotype were determined, which revealed significant nucleotide divergence among the karyomorphs. Also, the analyses indicate that chromosomal rearrangements occurred independently within the distinct lineages of S. marmoratus complex, which resulted in the appearance of distinct karyotypic variants in a non-linear fashion related to diploid numbers and in the appearance of similar non-homologous chromosomes. Finally, the integration of both molecular cytogenetic and phylogenetic approaches allowed the determination of specific chromosomes possibly involved in rearrangements and a better understanding about the evolutionary processes involved in the differentiation of Synbranchus genus.(AU)

A espécie de peixe Synbranchus marmoratus tem sido reportada como um complexo de espécies devido à elevada variabilidade cariotípica intraespecífica a despeito da dificuldade ou impossibilidade de distingui-las usando apenas caracteres morfológicos. O objetivo deste trabalho foi utilizar métodos citogenéticos e moleculares para determinar a delimitação das espécies e compreender a carioevolução de S. marmoratus utilizando amostras coletadas em distintas localidades brasileiras. Dentre os espécimes analisados, um alto grau de variação citogenética relativo aos números diploides e estrutura cariotípica foi observado, com cariótipos mostrando 2n=42, 44 e 46 cromossomos. Adicionalmente, utilizando sequências de três genes mitocondriais, as relações filogenéticas entre cada amostra com cariótipo conhecido foram determinadas, revelando uma divergência nucleotídica significativa entre os cariomorfos. Além disso, as análises indicam que rearranjos cromossômicos ocorreram independentemente nas distintas linhagens do complexo S. marmoratus, o que resultou no aparecimento de distintas variantes cariotípicas de forma não linear em relação aos números diploides e no surgimento de cromossomos similares e não homólogos. Finalmente, a integração de uma abordagem citogenética molecular e filogenética permitiu a determinação de cromossomos específicos que, possivelmente, estão envolvidos em rearranjos e um melhor entendimento sobre os processos evolutivos envolvidos na diferenciação do gênero Synbranchus.(AU)

Evolutionary and biogeographic history of the subfamily Neoplecostominae (Siluriformes: Loricariidae).

Freshwater fish evolution has been shaped by changes in the earth's surface involving changes in the courses of rivers and fluctuations in sea level. The main objective of this study is to improve our knowledge of the evolution of loricariids, a numerous and adaptive group of freshwater catfish species, and the role of geological changes in their evolution. We use a number of different phylogenetic methods to test the relationships among 52 representative taxa within the Neoplecostominae using 4676 bps of mitochondrial and nuclear DNA. Our analysis revealed that the subfamily Neoplecostominae is monophyletic, including Pseudotocinclus, with three lineages recognized. The first lineage is composed of part of Pareiorhina rudolphi, P. cf. rudolphi, and Pseudotocinclus; the second is composed of Isbrueckerichthys, Pareiorhaphis, Kronichthys, and the species Neoplecostomus ribeirensis; and the third is composed of Pareiorhina carrancas, P. cf. carrancas, Pareiorhina sp. 1, a new genus, and all the species of the genus Neoplecostomus, except N. ribeirensis. The relaxed molecular clock calibration provides a temporal framework for the evolution of the group, which we use for a likelihood-based historical biogeographic analysis to test relevant hypotheses on the formation of southeast Brazil. We hypothesize that headwater capture events and marine regressions have shaped the patterns of distribution within the subfamily Neoplecostominae throughout the distinct basins of southeast Brazil.