Repetitive DNA Mapping in Five Genera of Tree Frogs (Amphibia: Anura) from the Atlantic Forest: New Highlights on Genomic Organization in Hylidae.

INTRODUCTION: The tribes Cophomantini, Scinaxini, and Dendropsophini are anurans that belong to Hylidae, with wide distribution in tropical and subtropical regions around the world. The taxonomy and systematics of this family remain in a state of ongoing revision. Previous cytogenetic analyses of genera Boana, Bokermannohyla, Ololygon, Scinax, and Dendropsophus described some karyotypic characters such as conventional staining, C-banding and NORs, and FISH with specific probes. METHODS: This study describes for the first time the karyotypes of four species: Bokermannohyla ibitipoca, Ololygon luizotavioi, Dendropsophus bipunctatus, and Dendropsophus ruschii. Furthermore, we map CA(15) and CAT(10) microsatellite sites for the aforementioned species and six more species from the same genera for insight into the chromosomal evolution within the subfamily Hyalinae. RESULTS: B. ibitipoca and O. luizotavioi had 2n = 24 and karyotypic formulas 18m + 4sm + 2st and 8m + 12sm + 4st, while D. bipunctatus and D. ruschii showed 2n = 30 and karyotypic formulas 12m + 12sm + 4st + 2t and 10m + 10sm + 6st + 4t, respectively. The diploid numbers and karyotypic formulas revealed here follow the previously reported trend for Hylidae, except B. ibitipoca has a particularity of eight metacentric chromosomes, more than what is commonly found in species of this genus. The microsatellites probes CA(15) and CAT(10) had markings accumulated in blocks in the centromeric, pericentromeric, and terminal regions that were more specific for some species, as well as markings scattered along the chromosomes. We present a comprehensive review table of current data on cytogenetics of these genera. CONCLUSION: Our findings showed that the karyotypes of the hylids studied here majority fit the postulated conserved diploid number (2n = 24) and morphological chromosome patterns, while the mapping of the microsatellites enabled us to detect differences between species that share similar chromosomal morphologies.

Cytogenetic analysis of Hypomasticus copelandii and H. steindachneri: relevance of cytotaxonomic markers in the Anostomidae family (Characiformes).

Recent phylogenetic hypotheses within Anostomidae, based on morphological and molecular data, resulted in the description of new genera (Megaleporinus Ramirez, Birindelli et Galetti, 2017) and the synonymization of others, such as the reallocation of Leporinus copelandii Steindachner, 1875 and Leporinus steindachneri Eigenmann, 1907 to Hypomasticus Borodin, 1929. Despite high levels of conservatism of the chromosomal macrostructure in this family, species groups have been corroborated using banding patterns and the presence of different sex chromosome systems. Due to the absence of cytogenetic studies in H. copelandii (Steindachner, 1875) and H. steindachneri (Eigenmann, 1907), the goal of this study was to characterize their karyotypes and investigate the presence/absence of sex chromosome systems using different repetitive DNA probes. Cytogenetic techniques included: Giemsa staining, Ag-NOR banding and FISH using 18S and 5S rDNA probes, as well as microsatellite probes (CA)15 and (GA)15. Both species had 2n = 54, absence of heteromorphic sex chromosomes, one chromosome pair bearing Ag-NOR, 18S and 5S rDNA regions. The (CA)15 and (GA)15 probes marked mainly the subtelomeric regions of all chromosomes and were useful as species-specific chromosomal markers. Our results underline that chromosomal macrostructure is congruent with higher systematic arrangements in Anostomidae, while microsatellite probes are informative about autapomorphic differences between species.

Impacts of the Samarco Tailing Dam Collapse on Metals and Arsenic Concentration in Freshwater Fish Muscle from Doce River, Southeastern Brazil.

On November 2015, Samarco tailings dam in Mariana, Minas Gerais, Brazil, collapsed, releasing 62 million tons of tailings that advanced through 668 km of the Doce River and adjacent floodplain. Although the collapse was the worst environmental disaster in Brazil, little is known about its consequences to aquatic biota. Here we evaluate the effects of the tailings mudflow on metal and As concentration in fish and how concentration correlates with water and fish characteristics. We quantified semitotal amounts of Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn in fish muscle tissue using inductively coupled plasma mass spectrometry (ICP-MS) in 255 individuals (34 species) sampled in unaffected and affected areas along the Doce River basin. Arsenic and Hg were higher in fish from affected sites, likely due to turbulent mixing of previously sedimented material by the giant tailings wave. Silver and Zn concentrations were higher in unaffected sites. Arsenic concentration in Geophagus brasiliensis decreased with increasing fish weight. Copper and Zn decreased with increasing fish weight considering the whole assembly of fish. The tailings mudflow increased water conductivity, and conductivity increased Al concentration in fish, so we expected a larger Al concentration in fishes from affected sites. However, the observed Al concentration in fishes from affected sites was lower than expected by water conductivity. Thus, the tailings mudflow reduced Al uptake or accumulation in fishes. Mercury decreased with increasing water conductivity in both unaffected and affected sites considering all species and in G. brasiliensis alone. Despite the relatively low concentration range of metals and As found in fish, fishes from sites affected by the Fe ore tailings mudflow showed higher As and Hg concentration, compared to fishes from unaffected sites. The higher As and Hg in affected sites require further detailed monitoring to ensure safeguards of human health by fishing activity along the Doce River. Integr Environ Assess Manag 2020;16:622-630. © 2020 SETAC.

Em novembro de 2015, a barragem de rejeitos da Samarco em Mariana, MG, Brasil, rompeu, liberando 62 milhões de toneladas de rejeitos de minério de ferro que avançaram por 668 km do rio Doce e planície adjacente. Embora este tenha sido considerado o pior desastre ambiental do país, pouco se sabe sobre as consequências da liberação do fluxo de lama para a biota aquática. Aqui avaliamos os efeitos do fluxo de lama de rejeitos sobre a bioacumulação de metais e arsênio em várias espécies de peixes coletadas na bacia do rio Doce e como a bioacumulação se correlaciona com as características da água e dos peixes. Quantificamos as quantidades semitotais de Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb e Zn no tecido muscular de peixes usando ICP-MS em um total de 255 indivíduos (representando 34 espécies), amostrados em locais não afetados e afetados ao longo da bacia do rio Doce. As concentrações de As e Hg foram maiores nos peixes dos locais afetados, provavelmente devido à mistura turbulenta de material previamente sedimentado pela onda gigante de rejeitos. As concentrações de Ag e Zn foram maiores nos peixes de locais não afetados. A concentração de As na espécie mais abundante nos locais não afetados e afetados (Geophagus brasiliensis) diminuiu com o aumento do peso dos peixes. A concentração de Cu e Zn diminuiu com o aumento do peso dos peixes, considerando toda a assembleia de espécies. O fluxo de lama de rejeitos aumentou a condutividade da água e a condutividade aumentou a concentração de Al nos peixes, portanto esperávamos uma maior concentração de Al nos peixes de locais afetados. No entanto, a concentração de Al observada nos peixes de locais afetados foi menor do que o esperado pela condutividade da água. Assim, o fluxo de lama de rejeitos reduziu a assimilação ou o acúmulo de Al nos peixes. O mercúrio diminuiu com o aumento da condutividade da água nos locais não afetados e afetados, considerando todas as espécies e também dentro de G. brasiliensis. Apesar da faixa de concentração relativamente baixa de metais nos peixes, os peixes de locais afetados pelo fluxo de lama de rejeitos de minério de ferro apresentaram maior concentração de As e Hg, em comparação com peixes de locais não afetados. A maior concentração de As e Hg nos locais afetados requer um monitoramento mais detalhado para garantir segurança alimentar em relação à atividade pesqueira ao longo do rio Doce. Integr Environ Assess Manag 2020;16:622-630.

Karyotypic Divergence of Two Co-Occurring Species of Andean Climbing Catfishes (Siluriformes: Loricarioidei: Astroblepidae).

The monotypic Astroblepidae fish family includes 81 species distributed along Central and Northern Andes in South America and Panamá in Central America; most aspects regarding its biology, taxonomy, and chromosomal features remain largely unknown. This study reports the karyotype of two sympatric Astroblepus species from the Colombian Andes, aiming to provide novel information on karyotype organization and reveal possible chromosomal rearrangements occurred on these species, through mapping of different repetitive DNA classes, including microsatellites and ribosomal DNA multigene families. The results showed differences in the chromosome number and karyotypic formula: Astroblepus grixalvii had 2n = 52 (28m+8sm +12st +4a) with the metacentric pair number 1 as the largest chromosome pair, whereas Astroblepus homodon had 2n = 54 (30m+8sm +8st +8a) and less evident chromosome size differences. Microsatellite probes marked the tips of all chromosomes in both species except the short arms of acrocentric pair numbers 24 and 25 in A. homodon. Each ribosomal probe marked different chromosome pairs in both species. Microsatellite patterns suggest that the 2n increase probably involved a centric fission event that occurred during the evolutionary history of these species. This is the first karyotype description of an Astroblepus species and it contributes to the theoretical framework about the karyoevolutionary trends within Loricarioidei.

An Unusual Accumulation of Ribosomal Multigene Families and Microsatellite DNAs in the XX/XY Sex Chromosome System in the Trans-Andean Catfish Pimelodella cf. chagresi (Siluriformes:Heptapteridae).

This work constitutes the first cytogenetic characterization of a trans-Andean species of Heptapteridae. The catfish Pimelodella cf. chagresi from the Upper Rio Magdalena was studied, applying standard cytogenetic techniques (Giemsa, C-banding, and argyrophilic nucleolar organizer region [Ag-NOR]) and fluorescence in situ hybridization techniques using repetitive DNA probes: microsatellites (CA15 and GA15) and ribosomal RNA (rRNA) multigene families (18S and 5S recombinant DNA [rDNA] probes). The species showed a unique diploid chromosome number 2n = 50 (32m [metacentrics] +14sm [submetacentrics] +4st [subtelocentrics]) and a XX/XY sex chromosomal system, where the heteromorphic Y-chromosome revealed a conspicuous accumulation of all the assayed domains of repetitive DNA. P. cf. chagresi karyotype shares common features with other Heptapteridae, such as the predominance of metacentric and submetacentric chromosomes, and one pair of subtelomeric nucleolar organizer regions (NORs). These results reflect an independent karyological identity of a trans-Andean species and the relevance of repetitive DNA sequences in the process of sex chromosome differentiation in fish; it is the first case of syntenic accumulation of rRNA multigene families (18S and 5S rDNA) and microsatellite sequences (CA15 and GA15) in a differentiated sex chromosome in Neotropical fish.

First chromosome data on Steindachneridion doceanum (Siluriformes: Pimelodidae): a critically endangered catfish endemic of the Doce River basin, Brazil

The present report represents the first cytogenetic description of Steindachneridion doceanum, great catfish which is currently at high extinction risk and it is listed as threatened on the red list of the Brazilian Ministry of the Environment, also are suggested karyotype relationships with other species of the same genus endemic from other river basins. The results revealed a diploid number of 2n = 56 and the karyotype composed of 18 metacentric, 20 submetacentric, 10 subtelocentric and 8 acrocentric chromosomes (NF = 104). The AgNORs and CMA3 signals were coincident in location occupying the short arm of an acrocentric chromosome pair (25th), in a secondary constriction. The 5S rDNA genes were localized on the short arms of one subtelocentric pair. C-banding revealed terminal blocks on the short arms on many chromosomes as well as terminal positive bands at the both ends of a submetacentric pair. C banding also revealed a large heterochromatic block in the secondary constriction (25th) region that was coincident with the AgNORs sites and CMA3+ bright bands. In spite S. doceanum represent an endemic taxon, in spite their geographic isolation their cytogenetic characteristics show similarities with other species of the genus.(AU)

O presente trabalho apresenta a primeira descrição citogenética de Steindachneridion doceanum, grande bagre que se encontra atualmente em alto risco de extinção e listado como ameaçado na lista vermelha do Ministério do Meio Ambiente, também sugere relações cariotípicas com outras espécies do mesmo gênero, endêmicas de outras bacias hidrográficas. Os resultados revelaram um número diplóide de 56 cromossomos e o cariótipo composto por 18 elementos metacêntricos, 20 submetacêntricos, 10 subtelocêntricos e 8 acrocêntricos (NF = 104). As marcações AgNORs e CMA3 foram coincidentes ocupando o braço curto de um par de cromossomos acrocêntricos (par 25), em uma constrição secundária. Os genes 5S rDNA foram detectados nos braços curtos de um par subtelocêntrico. A banda C revelou blocos terminais nos braços curtos em vários cromossomos, bem como blocos terminais nas duas extremidades de um par submetacêntrico. A banda C também evidenciou um grande bloco heterocromático na constrição secundária (par 25) coincidente com os sítios AgNORs e as bandas CMA3 positivas. Apesar de S. doceanum representar um táxon endêmico, suas características citogenéticas mostram semelhanças com outras espécies do gênero das quais se encontra geograficamente isolado.(AU)

Sequencing and comparative analysis of the Amblyomma sculptum mitogenome.

The mitogenome of Amblyomma sculptum was sequenced, providing important information for understanding the evolutionary relationships among species of the A. cajennense complex. The mitochondrial genome has a circular structure with 37 genes, including 13 coding DNA sequences, two ribosomal RNA genes (12S rRNA and 16S rRNA) and 22 tRNA genes. Comparative analysis with the mitogenomes of six reference species of the genus Amblyomma revealed that the ND5 gene, which is related to energy metabolism, and control regions 1 and 2 of the mitogenomes have polymorphisms that can be exploited as molecular markers to differentiate A. sculptum from other tick species in the Amblyomma cajennense complex as well as other Amblyomma species.

Cytogenetic characterization of Hoplias malabaricus (Bloch, 1794) from the Ctalamochita River (Córdoba, Argentina): first evidence for southernmost populations of this species complex and comments on its biogeography.

Hoplias malabaricus (Bloch, 1794), a predatory freshwater fish with a wide distribution throughout South America, represents a species complex with seven well characterized karyomorphs at the cytogenetic level. Although this species has been extensively studied in several Brazilian basins, data are still scarce for hydrographic systems from other South American countries. This study aims to characterize cytogenetically the Hoplias malabaricus populations from the Argentinean Central Region, close to the southernmost distribution of this species complex. A total of 32 specimens from the Ctalamochita River, a tributary of Lower Paraná Basin located in the province of Córdoba, were analyzed using cytogenetic techniques (Giemsa staining, C- and Ag-NOR banding and fluorescent in situ hybridization with 18S rDNA). All the specimens showed diploid number 2n=42, chromosomic formula 22m + 20sm and absence of sexual chromosomes. Thus, the analyzed populations belong to the karyomorph named A. These populations showed a remarkable degree of divergence in their cytogenetic traits such as karyotypic formula, C-banding, NORs and 18S rDNA patterns for Hoplias malabaricus from other populations bearing the same karyomorph in the Middle and Upper Paraná Basin. These findings are consistent with molecular data from a recent study (where specimens collected in the present work were included), which indicate a closer phylogenetic relationship of Hoplias malabaricus populations from the Ctalamochita River with those from the Uruguay basin and the coastal regions of South Brazil than with populations from the Middle and Upper Paraná Basin. Overall, these pieces of evidence highlight the distinctive features of Hoplias malabaricus from the Ctalamochita River, and also reveal a complex history of dispersion of these populations. The present work is the first to provide cytogenetic information and include some phylogeographic aspects of Hoplias malabaricus populations living in close proximity to the southernmost extreme of its distribution area. Therefore, this study expands significantly upon the previously known geographical coverage for karyomorph A and contributes to a better understanding of the karyotypic diversification within this species complex.

B chromosome dynamics in Prochilodus costatus (Teleostei, Characiformes) and comparisons with supernumerary chromosome system in other Prochilodus species.

Within the genus Prochilodus Agassiz, 1829, five species are known to carry B chromosomes, i.e. chromosomes beyond the usual diploid number that have been traditionally considered as accessory for the genome. Chromosome microdissection and mapping of repetitive DNA sequences are effective tools to assess the DNA content and allow a better understanding about the origin and composition of these elements in an array of species. In this study, a novel characterization of B chromosomes in Prochilodus costatus Valenciennes, 1850 (2n=54) was reported for the first time and their sequence complementarity with the supernumerary chromosomes observed in Prochilodus lineatus (Valenciennes, 1836) and Prochilodus argenteus Agassiz, 1829 was investigated. The hybridization patterns obtained with chromosome painting using the micro B probe of P. costatus and the satDNA SATH1 mapping made it possible to assume homology of sequences between the B chromosomes of these congeneric species. Our results suggest that the origin of B chromosomes in the genus Prochilodus is a phylogenetically old event.

Complete mitochondrial genome sequence of neotropical fish Astyanax giton Eigenmann 1908 (Ostariophysi; Characidae).

Here we report, for the first time, the complete mitochondrial genome of Astyanax giton which, together with other species, are popularly known as tetras. The mitogenome's length is 16,643 bp, containing 13 protein-encoding genes (CDS), two ribosomal RNAs (rRNA), 22 RNA transfer (tRNA) and one control region (D-loop). As for other vertebrates, all genes are encoded on the heavy strand except for ND6 and eight tRNA genes. In the phylogenetic analyses, this species and other Astyanax were paraphyletic.

Closely Related Syntopic Cytotypes of Astyanax taeniatus (Jenyns, 1842) from the Upper Piranga River, Upper Doce Basin in Southeastern Brazil.

Astyanax taeniatus occurs in coastal areas of southeastern Brazil, and it is very abundant in the Upper Doce River Basin. Our objective was to study C-, argyrophilic nucleolar organizer region (Ag-NOR) and fluorescent in situ hybridization (FISH) banding patterns using 5S, 18S, CA(15), and GA(15) repetitive DNA probes on a population of A. taeniatus present in the Piranga River, in the Doce Basin. Two syntopic cytotypes were found, both with 2n = 50: cytotype A (14m + 12sm + 16st + 8t) and cytotype B (10m + 14sm + 18st + 8t). In both cytotypes, heterochromatic blocks occurred in all the chromosomes; Ag-NOR sites were multiple, ranging from four to eight. The 5S rDNA probe marked eight chromosomes in both cytotypes, a unique condition within Astyanax, suggesting a recent divergence between these cytotypes. The 18S rDNA probe differed between the cytotypes, marking 10 and 8 chromosomes in cytotypes A and B, respectively. CA(15) and GA(15) FISH patterns were mainly subtelomeric, but CA(15) showed centromeric markings that were diagnostic for each cytotype. Although overall cytogenetic evidence suggests that these cytotypes are closely related, morphological and molecular data in progress will provide further hypothesis test on their phylogenetic relationship.

The karyotypes of five species of the Scinax perpusillus group (Amphibia, Anura, Hylidae) of southeastern Brazil show high levels of chromosomal stabilization in this taxon.

Based on morphological, bioacoustics, and morphological traits, the genus Scinax has been subdivided into two major clades: S. catharinae and S. ruber. The first clade includes S. catharinae and S. perpusillus groups, whereas the second clade includes S. rostratus and S. uruguayus groups. Chromosome morphology, NOR and C-banding patterns of variation support these clades. This study aims the cytogenetic characterization of five species currently included in the S. perpusillus group: Scinax sp. (gr. perpusillus), S. arduous, S. belloni, S. cosenzai, and S. v-signatus, including standard cytogenetic techniques and repetitive DNA FISH probes. All species had 2n = 24 chromosomes. Nucleolar organizing regions occurred in chromosome pair 6 in all species, but differed in their locations among some species, suggesting a putative synaponomastic character for the clade. In S. belloni, the first chromosome pair was a metacentric, contrasting with the submetacentric first pair reported in all other species of the genus. Scinax sp. (gr. perpusillus) and S. v-signatus had similar karyotypic formulae, suggesting they are related species. Scinax cosenzai had a divergent C-banding pattern. Repetitive DNA probes hybridized more frequently in chromosomal subtelomeric regions in all species indicating recent cladogenesis in these species. Karyotypic evidence indicates unreported high levels of stabilization within S. perpusillus and in S. catharinae clade, resulting in a wealth of characters potentially informative for higher phylogenetic analyses.

Cytogenetic and Molecular Data Demonstrate that the Bryconinae (Ostariophysi, Bryconidae) Species from Southeastern Brazil Form a Phylogenetic and Phylogeographic Unit.

Brycon spp. occur in Neotropical watersheds to the west and east of the Andes, and as they are sensitive to anthropogenic changes, many these species are endangered in southeastern Brazil. Coastal rivers in southeastern Brazil are characterized by the presence of relatively few freshwater fish species and high endemism of this fauna. The objective of this study was to examine whether Brycon spp. occurring in the coastal basins of southeastern Brazil are monophyletic, using cytogenetic data, mitochondrial, and nuclear molecular markers. All the species showed a diploid number of 50 chromosomes, a conserved number within the subfamily Bryconinae. However, the karyotypic formulas were unique to most species, including Brycon devillei (26m+22sm+2st), Brycon ferox (26m+12sm+12st), Brycon insignis (22m+20sm+8st), Brycon opalinus, and Brycon vermelha (24m+20sm+6st), indicating the prevalence of pericentric and paracentric inversions in the chromosomal evolution of these species. All of them had nucleolar organizer regions in the first pair of subtelocentric chromosomes and no equilocal distribution of heterochromatin in the first pair of chromosomes of the karyotype. These two features, not seen in any other Brycon spp. examined to date, indicate that Bryconinae species from the Brazilian southeastern coastal basins, including the monotypic genus Henochilus, are monophyletic. Also, this is the first study that reports NOR location and C-banding patterns as synapomorphies for a Neotropical fish species group. The monophyly was also supported by a phylogenetic analysis of 16S rDNA (16S), cytochrome oxidase subunit I (COI), alpha-myosin (MYH6) genes and S72 intron molecular data. Our results partially corroborate the "Brycon acuminatus" group proposed by Howes in 1982: our proposed clade keeps B. devillei, B. ferox, and B. insignis; but it also includes B. opalinus, B. vermelha, and H. weatlandii whereas it excludes B. nattereri. The phylogeographic unit formed by Bryconinae species in southeastern Brazil reflects the long and isolated paleohydrological history of these coastal basins relative to the continental watersheds.

Cytogenetic and Molecular Data Suggest Deuterodon pedri Eigenmann, 1907 (Teleostei: Characidae) Is a Member of an Ancient Coastal Group.

The genus Deuterodon has been taxonomically recently redefined. Seven of its species have been included in this genus, whereas three others are regarded as Incertae Sedis in Characidae. One of latter is Deuterodon pedri, an endemic species from Santo Antônio River in Minas Gerais. Cytogenetic and molecular data on 21 specimens of D. pedri allowed to reevaluate the phylogenetic position of this taxon. The specimens showed karyotypic formula 12m+12sm+20st+6a, low heterochromatin content, nucleolar organizer regions present in two chromosome pairs, and 10 and 2 labeled regions by fluorescence in situ hybridization with 18S and 5S probes respectively, a karyotype similar to Deuterodon stigmaturus. The phylogenetic tree generated by a cytochrome oxidase subunit I fragment revealed that D. pedri and Deuterodon singularis are closely related and are the sister-group of Deuterodon parahybae, Deuterodon iguape, Astyanax giton, Astyanax intermedius, Astyanax ribeirae, Astyanax scabripinnis, and Probolodus heterostomus. The same arrangement was partially recovered with the nuclear gene RAG2. Our data indicate the genus Deuterodon is not monophyletic and that it is closely related to other species that occur in Brazil coastal basins.

Evolutionary divergence among Oligosarcus spp. (Ostariophysi, Characidae) from the São Francisco and Doce River basins: Oligosarcus solitarius Menezes, 1987 shows the highest rates of chromosomal evolution in the Neotropical Region.

The Doce River, in southeastern Brazil, is a coastal drainage, configured since the Late Cretaceous, when South America separated from Africa. Of the 77 native fish species of the Doce River Basin, 37 are potentially endangered-Oligosarcus solitarius, Menezes 1987, is the only endemic species of the quaternary lakes in the middle portion of this drainage and Oligosarcus argenteus, Günther 1864, is distributed in the Doce River channel and headwaters. This study characterizes the morphological, cytogenetic, and mitochondrial DNA variation in the Oligosarcus spp. populations from the Doce and São Francisco River Basins. The principal component analysis indicates three morphological groups. Cytogenetic data corroborate existence of the O. solitarius and O. argenteus fish species in the Doce River Basin, with high levels of population cytogenetic polymorphism. Taking into consideration the Pleistocene-Holocene formation of the lacustrine system in the middle Doce River, with low molecular differentiation and high levels of chromosomal variation among the O. solitarius populations, we concluded that O. solitarius has the highest rate of chromosomal evolution observed in Neotropical freshwater fishes. The morphological and cytogenetic patterns of the Oligosarcus sp. population collected at the Das Velhas River headwaters suggest that it may represent an undescribed species.

Cryptic speciation within the Neotropical cichlid Geophagus brasiliensis (Quoy & Gaimard, 1824) (Teleostei Cichlidae): a new paradigm in karyotypical and molecular evolution.

The family Cichlidae is one of the most species-rich taxa in the Neotropics. However, the factors that determine these high levels of biodiversity remain unexplored. We have analyzed the morphological, cytogenetic, and molecular data from 62 specimens of a widespread cichlid, Geophagus brasiliensis, from three adjacent basins in southeastern Brazil. Morphological analyses did not show differences among specimens. The cytogenetic data indicate the occurrence of multiple nucleolar organizer regions and four sympatric karyotypes that differ in the first pair of chromosome morphology, in the Doce River Basin; whereas the karyotype from the Paraíba do Sul Basin is widely divergent. The molecular data--616 bp fragment of cytochrome oxidase subunit I--revealed two haplogroups with the deepest genetic divergence (6.4%) ever reported within a nominal species in the Neotropical Region: One of the haplogroups is restricted to the quaternary lakes in the middle portion of the Doce Basin and the Mucuri River, whereas the other haplogroup is composed of haplotypes from elsewhere in the Doce Basin and the Paraíba do Sul Basin. These patterns suggest that G. brasiliensis undergoes a cryptic speciation process involving three major lineages that differ from the African explosive cichlid radiation.

Pleistocene karyotypic divergence in Hoplias malabaricus (Bloch, 1794) (Teleostei: Erythrinidae) populations in southeastern Brazil

The lacustrine system of the middle rio Doce basin is considered a paradigm of Pleistocene geomorphology. In these lakes, two Hoplias malabaricus karyomorphs (2n = 42A and 2n = 42B) live in sintopy in Carioca Lake. Cytogenetic analyses were performed on 65 specimens from 8 lakes (including Carioca Lake) to determine the distribution and relative frequency of these karyomorphs and the degree of cytogenetic divergence caused putatively by recent geographic isolation. All fish were 2n = 42B karyomorphs, except for 1 specimen from the Marola Lake, which was 2n = 42A. Among-population variation was especially high for C-banding patterns. Other characters such as X chromosome size and CMA3/DAPI also varied among populations. Our results suggested that the karyotype of H. malabaricus is able to respond rapidly to geographic isolation, and revealed that heterochromatic variation may represent the lowest hierarchical level of chromosomal evolution.

O sistema lacustre da bacia do médio rio Doce é considerado um paradigma da geomorfologia do Pleistoceno. Nestes lagos, dois cariomorfos de Hoplias malabaricus (2n = 42A e 2n = 42B) vivem em sintopia na lagoa Carioca. Análises citogenéticas foram realizadas em 65 amostras de 8 lagos (incluindo lagoa Carioca) para determinar a distribuição e frequência relativa destes cariomorfos e o grau de divergência citogenética aparentemente causada pelo isolamento geográfico recente. Todos os peixes apresentaram o cariomorfo 2n = 42B, com exceção de 1 espécime da lagoa Marola, que foi 2n = 42A. Entre as populações, a variação foi especialmente elevada nos padrões de bandamento C. Outros caracteres como o tamanho do cromossomo X e os padrões de CMA3/DAPI também variaram entre as populações. Nossos resultados sugerem que o genoma de H. malabaricus é capaz de responder rapidamente ao isolamento geográfico, revelando que a variação de heterocromatina pode representar o nível hierárquico mais baixo de evolução cromossômica.

Biogeography and comparative cytogenetics between two populations of Hoplias malabaricus (Bloch, 1794) (Ostariophysi: Erythrinidae) from coastal basins in the State of Bahia, Brazil

The species Hoplias malabaricus is a predator fish found in nearly all cis-Andean basins. From a cytogenetic point of view, this species comprises, at least, seven differentiated karyomorphs. Several localities have been formerly analyzed in Brazil, however, some regions, such as Bahia State, remain underrepresented. Recently, the Brazilian Environment Ministry classified both Itapicuru and Contas river basins (entirely located within Bahia territory) as priority conservation areas, whose biodiversity status lacks enough information. Therefore, the goal of the present work was to characterize, cytogenetically, populations of H. malabaricus from both basins, by using conventional staining, Ag-NOR and C-banding techniques. All specimens presented a diploid number of 2n = 40 with metacentric/submetacentric chromosomes, without differences between sexes, thereby representing the so-called "karyomorph F". The first metacentric pair presented a remarkably larger size in relation to the other pairs. The NORs were multiple, comprising the terminal region on long arms of two chromosomal pairs in both populations. However, the C-banding pattern was somewhat distinguishable between samples. Although sharing heterochromatic blocks at centromeric region of all chromosomes, the population from Itapicuru River basin appeared to have some more conspicuous blocks than those observed in the population from Contas River basin. The similar karyotype observed in both populations suggests a common geological history between them. The present results represent an advance in the knowledge about the cytogenetic pattern of H. malabaricus populations from poorly studied basins.(AU)

A espécie Hoplias malabaricus é um predador que ocorre em praticamente todas as bacias cis-andinas. Sob o ponto de vista citogenético, ela compreende, pelo menos, sete cariomorfos diferenciáveis. Várias localidades já foram previamente analisadas no Brasil, porém, algumas regiões, como o Estado da Bahia, permanecem pouco amostradas. Recentemente, o Ministério de Meio Ambiente classificou as bacias do rio Itapicuru e Contas (inteiramente localizadas na Bahia), como áreas prioritárias de conservação, cuja biodiversidade carece de informações suficientes. Neste sentido, o objetivo do presente trabalho foi caracterizar citogeneticamente populações de H. malabaricus nessas bacias, por meio de técnicas de coloração convencional, Ag-RON e bandamento C. Todos os espécimes e populações analisadas apresentaram número diploide 2n = 40 com cromossomos metacêntricos/submetacêntricos, sem diferenças entre os sexos, representando assim o denominado "cariomorfo F". O primeiro par metacêntrico apresentou tamanho notavelmente maior que os demais pares. As RONs foram múltiplas, ocupando a região terminal do braço longo de dois pares cromossômicos em ambas populações. Entretanto, os padrões de heterocromatina foram relativamente diferenciáveis entre as bacias hidrográficas estudadas. Apesar de compartilharem blocos heterocromáticos na região centromérica de todos os cromossomos, a população da bacia do Itapicuru apresentou alguns blocos mais conspícuos em relação aos da bacia do rio de Contas. O cariótipo similar encontrado em ambas as populações parece indicar uma história geológica em comum. Os dados obtidos representam um avanço no conhecimento dos padrões citogenéticos de populações de H. malabaricus provenientes de bacias pouco estudadas(AU)

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini).

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.