Detecting population structure of Paleosuchus trigonatus (Alligatoridae: Caimaninae) through microsatellites markers developed by next generation sequencing.

We isolated and characterized 10 new microsatellites loci for Paleosuchus trigonatus using ION TORRENT Sequencing Technology. We tested the transferability of these loci to three related species of the subfamily Caimaninae, and used these bi-parental markers to test population structure and genetic diversity of two populations of P. trigonatus impacted by hydroelectric dam construction on the Madeira (N = 16) and Xingu (N = 16) rivers. We also investigated the transferability of these markers to three related species: Paleosuchus palpebrosus (N = 5), Caiman crocodilus (N = 6) and Melanosuchus niger (N = 6). The genetic diversity of P. trigonatus was low in both the Madeira (He: 0.535 ± 0.148) and Xingu (He: 0.381 ± 0.222) populations, but the loci were sufficiently polymorphic to be used in system of mating and kinship studies in P. trigonatus. DAPC analysis with our set of microsatellites loci was able to separate the four species of Caimaninae studied and to detect a shallow genetic structure between Madeira and Xingu populations of P. trigonatus. AMOVA and STRUCTURE analyses using locprior model corroborate this shallow genetic structure. These novel molecular markers will be also useful in conservation genetics and phylogeographic studies of P. trigonatus, since they improve our ability to monitor the putative effects of dams on the loss of genetic diversity and allow us to investigate population dynamics and microevolutionary processes that occurred in the species.

Polyandrous behavior in an overexploited giant South American turtle (Podocnemis expansa) population in Central Amazon, Brazil.

The aim of the present study was to investigate the reproductive behavior of the giant Amazon River turtle (Podocnemis expansa) in the Amazon. This was carried out by estimating the degree of polymorphism in five DNA microsatellites in a sample of 359 hatchlings from 12 nests in the Mamirauá Sustainable Development Reserve in the municipality of Tefé, state of Amazonas, Brazil. An analysis of allele frequency and variation in the five microsatellite loci allowed for determination of multiple paternity in all nests sampled. Contributions of at least four male turtles to one nest, three male turtles to six nests, and two male turtles to five nests were identified. Knowledge on the reproductive behavior of P. expansa can assist in the establishment of management and conservation strategies for the population in the Mamirauá Reserve.

Microsatellite markers for Amazon pellona Pellona castelnaeana (Clupeiformes: Pristigasteridae).

The Amazon pellona is one of the few species of Pristigasteridae with recognized commercial value in the Amazon. We isolated 24 and characterized 8 microsatellite loci for this species. The number of alleles ranges from 2-8 per locus. Observed heterozygosities ranged from 0.052-0.823, while expected heterozygosities from 0.052-0.836. These 8 microsatellites are potentially valuable tools for characterizing the levels and distribution of genetic diversity, population structure, and gene flow. They may also be important parameters for the genetic conservation of this species, as well as for its sister taxon Pellona flavipinnis.

Evolutionary dynamics of repetitive DNA in semaprochilodus (characiformes, prochilodontidae): a fish model for sex chromosome differentiation.

Distribution of 6 microsatellites and 5 transposable elements on the chromosomes of Semaprochilodus taeniurus and S. insignis, commonly referred to as Jaraqui, was performed using their physical mapping with fluorescence in situ hybridization. In this study, we aim to understand the evolutionary dynamics in genomes of S. taeniurus and S. insignis by comparing the position, abundance and contribution of the repetitive sequences in the origins and differentiation of a ZZ/ZW sex chromosome system in S. taeniurus. Results revealed that distribution patterns of repetitive DNAs along the chromosomes varied considerably. Hybridization signals were observed on several autosomes in both species; however, in S. taeniurus genome, the repetitive sequences were more abundant. In addition, large clusters of known repetitive sequences were detected in sex chromosomes of S. taeniurus. This observation is notable because the accumulation of repetitive DNAs could reflect the degradation of genetic activities and the differentiation of protosex chromosomes, evolving into the heteromorphic ZW pair observed in S. taeniurus.

Population genetics analysis of Podocnemis sextuberculata (Testudines, Podocnemidae): lack of population structure in the central Amazon Basin.

The chelonians are, in general, important for the economy of the traditional populations of the Amazon region, especially as a source of animal protein. Furthermore, sub-products, such as eggs and fat, are utilized in the manufacture of cosmetics, and the plastron and carapace are used in the manufacture of adornments. The freshwater turtle species Podocnemis sextuberculata, locally known as "iaçá" or "pitiú", is widely distributed in the Amazon Basin in Brazil and also in Colombia and Peru. This species is on the International Union for Conservation of Nature Red List in the category of vulnerable species. We examined the genetic variability and population structure of three populations represented by 64 individuals sampled from Reserva Federal de Abufari, Tapauá, Amazonas State; Reserva de Desenvolvimento Sustentável Mamirauá, Tefé, Amazonas State, and Terra Santa, Pará State. All of these are over 1000 km from each other. A partial 415-bp sequence of the mitochondrial gene ND1 was utilized as a molecular marker. Seven haplotypes were observed; the most common haplotype was shared by all the areas sampled, while the rarest haplotypes were represented by a single individual and were thus restricted to a single locality. The sharing of the most common haplotype, the high number of migrants (Nm) and the AMOVA results indicate a lack of genetic structure among the sampling localities. The levels of genetic variability observed were homogeneous among the sampling localities. These results (Ó¨(ST) and Nm) are compatible with what is known about the ecology of this species, which has a great migratory capacity.

Prevalence of human papillomavirus (HPV) type 16 variants and rare HPV types in the central Amazon region.

Infection by human papillomavirus (HPV) is one of the primary causes of mortality by cancer in northern Brazil. Sexually active women from Manaus, Amazonas, without cytological alterations and women with pre-malignant and malignant cytological alterations were examined for HPV virus, identified via PCR and sequencing. The target region for this study was part of the L1 capsid gene of HPV. Twenty-three samples that were PCR-positive were sequenced. Analysis of 336 bp demonstrated a high incidence of high-risk HPV types in the population of Manaus, identified as HPVs 16, 33, 58, 66, 68. HPV type 16 was the most prevalent, presenting two variants similar to the Asian-American (AA) and East-Asian type (As) variants. A rare HPV type 13 related to "Heck's disease" was also detected. This preliminary provides important information about the HPV circulating in Amazonas State.

Polyandry in the red-headed river turtle Podocnemis erythrocephala (Testudines, Podocnemididae) in the Brazilian Amazon.

The genus Podocnemis comprises six living species, including P. erythrocephala (irapuca-red-headed river turtle). Data are available concerning the reproductive biology of the species of the genus Podocnemis, but little is known about their reproductive strategies. Considering the total lack of such data for P. erythrocephala, and with the goal of contributing information on their mode of reproduction, we examined the relationships among individuals of nests of this turtle, using microsatellite markers. Using four microsatellite loci, we analyzed the progeny in six nests from two localities in the Brazilian Amazon (Santa Isabel do rio Negro and Parintins). All juveniles from each nest were analyzed. The genotypes of each juvenile from each nest were identified, and because a sample of female DNA was not available, the maternal genotype was inferred from homozygous individuals in each nest. We found that this species is promiscuous; there was multiple paternity in five of the six nests analyzed. In addition to being important for the understanding of evolutionary and genetic processes, this type of information will be useful for chelonian management projects. Our data suggest one possible difference between reproductive patterns of the different populations. This multi-paternal condition may be a natural reproductive strategy for the preservation of the genetic diversity of this species.

The Casiquiare river acts as a corridor between the Amazonas and Orinoco river basins: biogeographic analysis of the genus Cichla.

The Casiquiare River is a unique biogeographic corridor between the Orinoco and Amazonas basins. We investigated the importance of this connection for Neotropical fishes using peacock cichlids (Cichla spp.) as a model system. We tested whether the Casiquiare provides a conduit for gene flow between contemporary populations, and investigated the origin of biogeographic distributions that span the Casiquiare. Using sequences from the mitochondrial control region of three focal species (C. temensis, C. monoculus, and C. orinocensis) whose distributions include the Amazonas, Orinoco, and Casiquiare, we constructed maximum likelihood phylograms of haplotypes and analyzed the populations under an isolation-with-migration coalescent model. Our analyses suggest that populations of all three species have experienced some degree of gene flow via the Casiquiare. We also generated a mitochondrial genealogy of all Cichla species using >2000 bp and performed a dispersal-vicariance analysis (DIVA) to reconstruct the historical biogeography of the genus. This analysis, when combined with the intraspecific results, supports two instances of dispersal from the Amazonas to the Orinoco. Thus, our results support the idea that the Casiquiare connection is important across temporal scales, facilitating both gene flow and the dispersal and range expansion of species.

Conservation strategies for Arapaima gigas (Schinz, 1822) and the Amazonian várzea ecosystem

In the present study we report a spatial autocorrelation analysis of molecular data obtained for Arapaima gigas, and the implication of this study for conservation and management. Arapaima is an important, but critically over-exploited giant food fish of the Amazonian várzea. Analysis of 14 variable microsatellite loci and 2,347 bp of mtDNA from 126 individuals sampled in seven localities within the Amazon basin suggests that Arapaima forms a continuous population with extensive genetic exchange among localities. Weak effect of isolation-by-distance is observed in microsatellite data, but not in mtDNA data. Spatial autocorrelation analysis of genetic and geographic data suggests that genetic exchange is significantly restricted at distances greater than 2,500 km. We recommend implementing a source-sink metapopulation management and conservation model by proposing replicate high quality várzea reserves in the upper, central, and lower Amazon basin. This conservation strategy would: 1) preserve all of the current genetic diversity of Arapaima; 2) create a set of reserves to supply immigrants for locally depleted populations; 3) preserve core várzea areas in the Amazon basin on which many other species depend. We stress that conservation strategies should not only preserve current genetic diversity, but also the evolutionary processes which have generated the observed patterns.

No presente estudo, é descrita uma análise de autocorrelação espacial de dados moleculares obtidos para Arapaima gigas e a implicação deste estudo para sua conservação e manejo. Arapaima é uma espécie de peixe gigante da várzea Amazônica de grande importância econômica, e criticamente sobre-explorada. A análise de 14 locos microssatélites e de 2.347 pb do DNAmt de 126 indivíduos amostrados em sete localidades na bacia Amazônica sugere que Arapaima forma uma população contínua com grande fluxo genético entre localidades. Um pequeno efeito de isolamento por distância é observado através dos dados de microssatélites, mas não através dos dados de DNAmt. A análise de autocorrelação espacial de dados genéticos e geográficos sugere que o fluxo gênico é significativamente restrito em distâncias maiores que 2.500 km. É recomendada a implementação de um modelo de manejo e conservação de metapopulação no padrão doador-receptor, replicando as reservas de várzea na bacia Amazônica superior, central e inferior. Esta estratégia de conservação iria: 1) preservar toda a atual diversidade genética da Arapaima; 2) criar um conjunto de reservas para fornecer imigrantes para populações locais reduzidas; 3) preservar áreas centrais de várzea na bacia Amazônica das quais diversas outras espécies dependem. Estratégias de conservação não deveriam somente preservar a atual diversidade genética, mas também os processos evolutivos que têm gerado os padrões observados.

Conservation strategies for Arapaima gigas (Schinz, 1822) and the Amazonian várzea ecosystem.

In the present study we report a spatial autocorrelation analysis of molecular data obtained for Arapaima gigas, and the implication of this study for conservation and management. Arapaima is an important, but critically over-exploited giant food fish of the Amazonian várzea. Analysis of 14 variable microsatellite loci and 2,347 bp of mtDNA from 126 individuals sampled in seven localities within the Amazon basin suggests that Arapaima forms a continuous population with extensive genetic exchange among localities. Weak effect of isolation-by-distance is observed in microsatellite data, but not in mtDNA data. Spatial autocorrelation analysis of genetic and geographic data suggests that genetic exchange is significantly restricted at distances greater than 2,500 km. We recommend implementing a source-sink metapopulation management and conservation model by proposing replicate high quality várzea reserves in the upper, central, and lower Amazon basin. This conservation strategy would: 1) preserve all of the current genetic diversity of Arapaima; 2) create a set of reserves to supply immigrants for locally depleted populations; 3) preserve core várzea areas in the Amazon basin on which many other species depend. We stress that conservation strategies should not only preserve current genetic diversity, but also the evolutionary processes which have generated the observed patterns.

The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes.

The mitochondrial cytochrome b (cyt-b) gene is widely used in systematic studies to resolve divergences at many taxonomic levels. The present study focuses mainly on the utility of cyt-b as a molecular marker for inferring phylogenetic relationship at various levels within the fish family Cichlidae. A total of 78 taxa were used in the present analysis, representing all the major groups in the family Cichlidae (72 taxa) and other families from the suborders Labroidei and Percoidei. Gene trees obtained from cyt-b are compared to a published total evidence tree derived from previous studies. Minimum evolution trees based on cyt-b data resulted in topologies congruent with all previous analyses. Parsimony analyses downweighting transitions relative to transversions (ts1:tv4) or excluding transitions at third codon positions resulted in more robust bootstrap support for recognized clades than unweighted parsimony. Relative rate tests detected significantly long branches for some taxa (LB taxa) which were composed mainly by dwarf Neotropical cichlids. An improvement of the phylogenetic signal, as shown by the four-cluster likelihood mapping analysis, and higher bootstrap values were obtained by excluding LB taxa. Despite some limitations of cyt-b as a phylogenetic marker, this gene either alone or in combination with other data sets yields a tree that is in agreement with the well-established phylogeny of cichlid fish.

Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes.

We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids.

Mitochondrial DNA phylogeny of the family Cichlidae: monophyly and fast molecular evolution of the neotropical assemblage.

A mitochondrial DNA (mtDNA) phylogeny of cichlid fish is presented for the most taxonomically inclusive data set compiled to date (64 taxa). 16S rDNA data establish with confidence relationships among major lineages of cichlids, with a general pattern congruent with previous morphological studies and less inclusive molecular phylogenies based on nuclear genes. Cichlids from Madagascar and India are the most basal groups of the family Cichlidae and sister to African-Neotropical cichlids. The cichlid phylogeny suggests drift-vicariance events, consistent with the fragmentation of Gondwana, to explain current biogeographic distributions. Important phylogenetic findings include the placement of the controversial genus Heterochromis basal among African cichlids, the South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage, and the close relationship of the Neotropical genera Cichla with Astronotus rather than with the crenicichlines. Based on a large number of South American genera, the Neotropical cichlids are defined as a monophyletic assemblage and shown to harbor significantly higher levels of genetic variation than their African counterparts. Relative rate tests suggest that Neotropical cichlids have experienced accelerated rates of molecular evolution. But these high evolutionary rates were significantly higher among geophagine cichlids.

Biochemical adjustments to hypoxia by Amazon cichlids

The isozyme distribution of cichlid lactate dehydrogenase (LDH) is related to species environmental preferences. Cichlasoma amazonarum. occurs in different environments and presents LDH tissue distribution patterns that correlate with oxygen tension at the capture location. Cichlasoma amazonarum was exposed to long term severe hypoxia (51 days at 36.4 +/- 5.9 mmHg), tissue LDH isozyme distribution was analyzed by electrophoresis and enzyme activities were measured by monitoring the oxidation of NADH as pyruvate was reduced to lactate. The exposure of Cichlasoma amazonarum to long-term severe hypoxia resulted in changes in the tissue distribution of LDH isozymes. The major changes in response to hypoxia occurred in heart, liver and brain: isozyme A4 was activated in heart and brain, whereas isozyme B4 was activated in liver. The most significant quantitative change occurred in brain LDH of hypoxia-exposed animals which adopted muscle type kinetics, reflecting a new LDH isozyme distribution. LDH activity was significantly reduced (P<0.05) in animals exposed to hypoxia (N = 8), suggesting an overall LDH suppression. Pyruvate inhibition decreased in all hypoxia-exposed tissues. Thus, the ability of Cichlasoma amazonarum to regulate LDH tissue expression according to oxygen availability allows the animal to survive chronic hypoxic environments. This phenotypic plasticity may occur in other hypoxia-tolerant fish species.

Biochemical adjustments to hypoxia by Amazon cichlids.

The isozyme distribution of cichlid lactate dehydrogenase (LDH) is related to species environmental preferences. Cichlasoma amazonarum occurs in different environments and presents LDH tissue distribution patterns that correlate with oxygen tension at the capture location. Cichlasoma amazonarum was exposed to long-term severe hypoxia (51 days at 36.4 +/- 5.9 mmHg), tissue LDH isozyme distribution was analyzed by electrophoresis and enzyme activities were measured by monitoring the oxidation of NADH as pyruvate was reduced to lactate. The exposure of Cichlasoma amazonarum to long-term severe hypoxia resulted in changes in the tissue distribution of LDH isozymes. The major changes in response to hypoxia occurred in heart, liver and brain: isozyme A4 was activated in heart and brain, whereas isozyme B4 was activated in liver. The most significant quantitative change occurred in brain LDH of hypoxia-exposed animals which adopted muscle type kinetics, reflecting a new LDH isozyme distribution. LDH activity was significantly reduced (P < 0.05) in animals exposed to hypoxia (N = 8), suggesting an overall LDH suppression. Pyruvate inhibition decreased in all hypoxia-exposed tissues. Thus, the ability of Cichlasoma amazonarum to regulate LDH tissue expression according to oxygen availability allows the animal to survive chronic hypoxic environments. This phenotypic plasticity may occur in other hypoxia-tolerant fish species.

Effects of temperature on leucocytes of Colossoma macropomum and Hoplosternum littorale (Pisces).

1. The effects of different thermal regimens on qualitative and quantitative characteristics of leucocytes were evaluated in two fish species of the Amazon region. 2. The proportion of circulating types of leucocytes changed significantly in Colossoma macropomum (tambaqui) but not in Hoplosternum littorale (tamoatá) exposed for four-week terms to 20, 25, 30, 35, and 40 degrees C. 3. The proportion of circulating lymphocytes decreased significantly in tambaquis exposed to 30, 35, and 40 degrees C. No changes in lymphocyte proportions were observed in tamoatás. 4. Neutrophils were almost absent in tambaquis, except in animals exposed to 40 degrees C. No significant changes in circulating neutrophils were observed in tamoatás. 5. The circulating leucocytes of Colossoma macropomum and Hoplosternum littorale are affected in different ways by temperature changes, suggesting species-specific adjustments to this parameter.

Effects of temperature on leucocytes of Colossoma macropomum and Hoplosternum littorale (Pisces)

1. The effects of different thermal regimens on qualitative and quantitative characteristics of leucocytes were evaluated in two fish species of the Amazon region. 2. The proportion of circulating types of leucocytes changed significantly in Colossoma macropomum (tambaqui) but not in Hoplosternum littorale (tamoatá) exposed for four-week terms to 20, 25, 30, 35, and 40 degrees C. 3. The proportion of circulating lymphocytes decreased significantly in tambaquis exposed to 30, 35, and 40 degrees C. No changes in lymphocyte proportions were observed in tamoatás. 4. Neutrophils were almost absent in tambaquis, except in animals exposed to 40 degrees C. No significant changes in circulating neutrophils were observed in tamoatás. 5. The circulating leucocytes of Colossoma macropomum and Hoplosternum littorale are affected in different ways by temperature changes, suggesting species-specific adjustments to this parameter.