A role of asynchrony of seasons in explaining genetic differentiation in a Neotropical toad.

The process of diversification can be studied at the phylogeographic level by attempting to identify the environmental features that promote and maintain population divergence. Here we investigate diversification in Rhinella granulosa, a Neotropical toad from northeastern Brazil, by testing a range of hypotheses that encompass different putative mechanisms reducing gene flow among populations. We sequenced single nucleotide polymorphisms and examined individual predictions related to the role of geographic barriers (rivers), ecological gradients, historical habitat stability, and spatial variation in climate seasonality, also known as the asynchrony of seasons hypothesis. This hypothesis postulates that temporal asynchrony of wet and dry seasons over short distances causes parapatric populations to become isolated by time. After determining genetic structure, inferring past distributions, ranking demographic models, and estimating the power of monthly climatic variables, our results identified two populations that are not associated with geographic barriers, biome gradients, or historical refugia. Instead, they are predicted by spatial variation in monthly rainfall and minimum temperature, consistent with the asynchrony of seasons hypothesis, supported also by our comparative framework using multiple matrix regression and linear mixed effects modeling. Due to the toad's life history, climate likely mediates gene flow directly, with genetic differentiation being provoked by neutral mechanisms related to climate driven population isolation, and/or by natural selection against migrants from populations with different breeding times. The asynchrony of seasons hypothesis is seldom considered in phylogeographic studies, but our results indicate that it should be tested in systems where breeding is tightly coupled with climate.

Non-invasive sampling in Itatiaia National Park, Brazil: wild mammal parasite detection.

BACKGROUND: Non-invasive sampling through faecal collection is one of the most cost-effective alternatives for monitoring of free-living wild mammals, as it provides information on animal taxonomy as well as the dynamics of the gastrointestinal parasites that potentially infect these animals. In this context, this study aimed to perform an epidemiological survey of gastrointestinal parasites using non-invasive faecal samples from carnivores and artiodactyls identified by stool macroscopy, guard hair morphology and DNA sequencing in Itatiaia National Park. Between 2017 and 2018, faeces from carnivores and artiodactyls were collected along trails in the park. The host species were identified through macroscopic and trichological examinations and molecular biology. To investigate the parasites, the Faust, Lutz and modified Ritchie and Sheather techniques and enzyme immunoassays to detect Cryptosporidium sp. antigens were used. RESULTS: A total of 244 stool samples were collected. The species identified were Chrysocyon brachyurus, Leopardus guttulus, Canis familiaris, Cerdocyon thous, Puma yagouaroundi, Leopardus pardalis, Puma concolor and Sus scrofa. There were 81.1% samples that were positive for parasites distributed mainly in the high part of the park. Helminths, especially eggs of the family Ascarididae, were more frequently detected in carnivore faeces (70.9%). Protozoa, especially Cryptosporidium sp., represented the highest frequency of infection in artiodactyl faeces (87.1%). This zoonotic protozoon was detected in eight mammalian species, including in a wild boar. High values of structural richness and Shannon and Simpson diversity indices were observed for the parasites, especially in the faeces of C. brachyurus. Significant differences in parasite diversity were observed between wild and domestic animals, such as C. brachyurus and C. familiaris, respectively, and between taxonomically distant species, such as C. brachyurus and S. scrofa. The highest values for parasite similarity were found among the species that frequented similar areas of the park, such as C. brachyurus and L. guttulus. CONCLUSIONS: The animals and parasite infections were identified through the combination of three techniques. High frequency parasite structures were diagnosed. Zoonotic protozoa were found and mainly occurred in samples from introduced species.

Low coverage of species constrains the use of DNA barcoding to assess mosquito biodiversity.

Mosquitoes (Culicidae) represent the main vector insects globally, and they also inhabit many of the terrestrial and aquatic habitats of the world. DNA barcoding and metabarcoding are now widely used in both research and routine practices involving mosquitoes. However, these methodologies rely on information available in databases consisting of barcode sequences representing taxonomically identified voucher specimens. In this study, we assess the availability of public data for mosquitoes in the main online databases, focusing specifically on the two most widely used DNA barcoding markers in Culicidae: COI and ITS2. In addition, we test hypotheses on possible factors affecting species coverage (i.e., the percentage of species covered in the online databases) for COI in different countries and the occurrence of the DNA barcode gap for COI. Our findings showed differences in the data publicly available in the repositories, with a taxonomic or species coverage of 28.4-30.11% for COI in BOLD + GenBank, and 12.32% for ITS2 in GenBank. Afrotropical, Australian and Oriental biogeographic regions had the lowest coverages, while Nearctic, Palearctic and Oceanian had the highest. The Neotropical region had an intermediate coverage. In general, countries with a higher diversity of mosquitoes and higher numbers of medically important species had lower coverage. Moreover, countries with a higher number of endemic species tended to have a higher coverage. Although our DNA barcode gap analyses suggested that the species boundaries need to be revised in half of the mosquito species available in the databases, additional data must be gathered to confirm these results and to allow explaining the occurrence of the DNA barcode gap. We hope this study can help guide regional species inventories of mosquitoes and the completion of a publicly available reference library of DNA barcodes for all mosquito species.

Genetic diversity in ex situ populations of the endangered Leontopithecus chrysomelas and implications for its conservation.

Leontopithecus chrysomelas, the Golden-headed Lion Tamarin (GHLT), is an endangered and endemic Neotropical primate from the Atlantic Forest of Brazil that has suffered a reduction of its habitat and population size in the wild. Ex situ populations have been established as a relevant alternative to safeguard the species and retain its genetic diversity and evolutionary potential. This study evaluated the genetic diversity and structure of the two main Brazilian captive populations of GHLT, which have been under human care at the Primatology Center of Rio de Janeiro (CPRJ) and the Zoological Park Foundation of São Paulo (FPZSP). Our results revealed levels of genetic diversity overall comparable to those observed for other Leontopithecus species and for ex situ and in situ populations of GHLT previously studied. Bayesian and principal coordinate analyses showed a moderate differentiation between CPRJ and FPZSP populations. Both populations presented observed heterozygosity values higher than expected heterozygosity values for most of the microsatellites used in this study, suggesting that the management has been efficient in avoiding an increase in homozygosity. However, simulations point to a significant loss of genetic diversity in the next 100 years, mainly in the FPZSP population. Such data are relevant for further decision-making on the metapopulation management of L. chrysomelas in captive conditions and for integrating in situ and ex situ conservation plans.

Moderate Genetic Diversity and Demographic Reduction in the Threatened Giant Anteater, Myrmecophaga tridactyla.

In general, large mammal species with highly specialized feeding behavior and solitary habits are expected to suffer genetic consequences from habitat loss and fragmentation. To test this hypothesis, we analyzed the genetic diversity distribution of the threatened giant anteater inhabiting a human-modified landscape. We used 10 microsatellite loci to assess the genetic diversity and population structure of 107 giant anteaters sampled in the Brazilian Central-Western region. No genetic population structuring was observed in this region suggesting no gene flow restriction within the studied area. On the other hand, the moderate level of genetic diversity (Ho = 0.54), recent bottleneck detected and inbreeding (Fis, 0.13; p ≤ 0.001) signatures suggest potential impacts on the genetic variation of this Xenarthra. Additionally, a previous demographic reduction was suggested. Thus, considering the increased human-promoted impacts across the entire area of distribution of the giant anteater, our results can illustrate the potential effects of these disturbances on the genetic variation, allowing us to request the long-term conservation of this emblematic species.

Next-Generation Sequencing of the Complete Mitochondrial Genome of the Endangered Species Black Lion Tamarin Leontopithecus chrysopygus (Primates) and Mitogenomic Phylogeny Focusing on the Callitrichidae Family.

We describe the complete mitochondrial genome sequence of the Black Lion Tamarin, an endangered primate species endemic to the Atlantic Rainforest of Brazil. We assembled the Leontopithecus chrysopygus mitogenome, through analysis of 523M base pairs (bp) of short reads produced by next-generation sequencing (NGS) on the Illumina Platform, and investigated the presence of nuclear mitochondrial pseudogenes and heteroplasmic sites. Additionally, we conducted phylogenetic analyses using all complete mitogenomes available for primates until June 2017. The single circular mitogenome of BLT showed organization and arrangement that are typical for other vertebrate species, with a total of 16618 bp, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region (D-loop region). Our full phylogenetic tree is based on the most comprehensive mitogenomic dataset for Callitrichidae species to date, adding new data for the Leontopithecus genus, and discussing previous studies performed on primates. Moreover, the mitochondrial genome reported here consists of a robust mitogenome with 3000X coverage, which certainly will be useful for further phylogenetic and evolutionary analyses of Callitrichidae and higher taxa.

Contributions to the cytogenetics of the Neotropical fish fauna.

Brazilian fish cytogenetics started as early as the seventies in three pioneering research groups, located at the Universidade Estadual Paulista (UNESP, Botucatu, SP), Universidade Federal de São Carlos (UFSCar, São Carlos, SP) and Universidade de São Paulo (USP, São Paulo, SP). Investigations that have been conducted in these groups led to the discovery of a huge chromosomal and genomic biodiversity among Neotropical fishes. Besides, they also provided the expansion of this research area, with the genesis of several other South American research groups, in view of a number of dissertations and doctoral theses developed over years. The current authors were encouraged to make their thesis catalog accessible from a public source, in order to share informations on the taxa and subject matter analyzed. Some of the key contributions to evolutionary fish cytogenetics are also being highligthed.

Patterns of orchid bee species diversity and turnover among forested plateaus of central Amazonia.

The knowledge of spatial pattern and geographic beta-diversity is of great importance for biodiversity conservation and interpreting ecological information. Tropical forests, especially the Amazon Rainforest, are well known for their high species richness and low similarity in species composition between sites, both at local and regional scales. We aimed to determine the effect and relative importance of area, isolation and climate on species richness and turnover in orchid bee assemblages among plateaus in central Brazilian Amazonia. Variance partitioning techniques were applied to assess the relative effects of spatial and environmental variables on bee species richness, phylogeny and composition. We hypothesized that greater abundance and richness of orchid bees would be found on larger plateaus, with a set of core species occurring on all of them. We also hypothesized that smaller plateaus would possess lower phylogenetic diversity. We found 55 bee species distributed along the nine sampling sites (plateaus) with 17 of them being singletons. There was a significant decrease in species richness with decreasing size of plateaus, and a significant decrease in the similarity in species composition with greater distance and climatic variation among sampling sites. Phylogenetic diversity varied among the sampling sites but was directly related to species richness. Although not significantly related to plateau area, smaller or larger PDFaith were observed in the smallest and the largest plateaus, respectively.

Genetic multipartitions based on D-Loop sequences and chromosomal patterns in brown chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic.

Connectivity levels among Brazilian reef fish fauna populations have attracted growing interest, mainly between mainland shores and oceanic islands. The Pomacentridae, whose phylogeographic patterns are largely unknown in the Atlantic, are a family of dominant fish in reef regions. We present data on the variability and population structure of damselfish Chromis multilineata in different areas along the northeast coast of Brazil and in the waters around the oceanic islands of Fernando de Noronha (FNA) and Saint Peter and Saint Paul Archipelago (SPSPA) through analysis of the HVR1 mtDNA sequence of the control region. The remote SPSPA exhibits the highest level of genetic divergence among populations. Conventional and molecular cytogenetic analysis showed similar karyotype patterns (2n=48 acrocentrics) between these insular areas. Our estimates reveal three genetically different population groups of C. multilineata on the Brazilian coast. The level of genetic structure is higher than previous data suggested, indicating complex panel of interactions between the oceanic island and coastal populations of Brazil.

Informative microsatellites for genetic population studies of black-faced lion tamarins (Leontopithecus caissara).

Leontopithecus caissara is a critically endangered primate species from the Brazilian Atlantic Forest. Nineteen microsatellite loci, previously developed for congeneric species, were tested with 34 L. caissara individuals from Superagüi Island. Of the 19 loci, 17 (89.4%) produced robust alleles, nine (47.4%) of these proved to be polymorphic, with a total of 23 alleles and an average of 2.56 alleles per locus. Expected and observed heterozygosity averaged 0.483 and 0.561, respectively. The exclusion power for identifying the first parent of an arbitrary offspring was 0.315 over all loci. The results thus indicate both the usefulness and limitations of these nine microsatellite loci in the genetic analysis of L. caissara, as well as their potentiality for genetic investigation in other congeneric species.

Early replication banding in Leporinus species (Osteichthyes, Characiformes) bearing differentiated sex chromosomes (ZW).

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2'-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.

Population substructuring in a migratory freshwater fish Prochilodus argenteus (Characiformes, Prochilodontidae) from the São Francisco River.

The construction of hydroelectric dams, pollution of rivers and other environmental changes are responsible for the disappearance of many natural fish stocks. The purpose of this work was to analyze the fish Prochilodus argenteus inhabiting the region of the Três Marias dam in the São Francisco River (Brazil) collected in two sites having distinct environmental characteristics. Three novel homologous and one known cross-specific microsatellites were used to assess genetic variation within and between the two collection sites (namely A and B) in order to confirm the occurrence of population substructuring previously suggested using RAPD markers. A higher number of exclusive alleles and a greater genetic variability in region B strongly reinforce the co-existence of different reproductive units in this area. F(ST) estimates showed a significant population differentiation between the two sites, indicating the possible existence of distinct gene pools. Considering the economic importance of this fishery resource in the São Francisco River, these findings could provide very important information for fisheries management, aquaculture and conservation of the stocks of this species.

A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA.

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.

Assessing genetic diversity of Brazilian reef fishes by chromosomal and DNA markers.

Little is known on genetics of Brazilian coral reef fish and most of this information is limited to chromosome characterization of major representative species. The diploid chromosome number in marine fish varies from 2n= 22-26 to 2n = 240-260. Despite of this apparent diversity, most studied marine species have a diploid complement with 48 acrocentric chromosomes. This latter trend is mostly observed among Perciformes, an important major taxon of coral reef fishes. Studies in the families Pomacentridae, Pomacanthidae and Chaetodontidae, for example, have shown a common karyotype pattern entirely formed by 48 uniarmed chromosomes. However, rare numerical and structural chromosome polymorphisms and cryptic chromosome rearrangements involving heterochromatin segments and/or nucleolar organizing sites have been reported among such fishes. Although new chromosome forms can contribute to the establishment of genetically isolated populations, their role in reef fish speciation at marine realm still is an open question. More recently, genomic DNA analyses using RAPD and microsatellites, and sequencing and RFLP of mitochondrial DNA have increasingly been used in Atlantic reef fish species. Genetic homogeneity over wide geographical ranges has been reported for different fish groups, in contrast to several cases of population substructuring related to environmental constraints or evolutionary history. Amazonas outflow and upwelling on the Southeastern coast of Brazil are believed to be strong barriers to dispersal of some reef species. Moreover, it is suggested that the pattern of speciation and population structure at South Atlantic is quite distinctive from Pacific Ocean, even when comparing closely related taxa. Further genetic studies are strongly encouraged in Brazilian reef fishes in order to provide a reliable scenario of the genetic structure in this important and diverse fish group.

Chromosomal diversification of reef fishes from genus Centropyge (Perciformes, Pomacanthidae).

The genus Centropyge is remarkable for species richness, composing a highly specialized fish group amongst members from family Pomacanthidae. However, cytogenetical reports are nearly absent in these animals. New data are provided from karyotypical studies carried out on Centropyge aurantonotus from the Brazilian coast of the Atlantic Ocean and C. ferrugatus from the Philippines Sea of the Indo-Pacific Ocean. Both species present 2n = 48 but karyotypes are differentiated by fundamental number. C. aurantonotus has a great number of biarmed chromosomes (4 m + 14 sm + 16 st + 4 a), while C. ferrugatus presents only acrocentric chromosomes. Single nucleolar organizer regions (NORs) are located at interstitial position of an acrocentric pair in C. ferrugatus and on short arms of a subtelocentric pair in C. aurantonotus, as confirmed by fluorescent in situ hybridization (FISH) with 18S rDNA probes. Heterochromatin is distributed over NOR and centromeric regions in both species, but additional GC-rich heterochromatic blocks on short arms of up to eight chromosomal pairs can be detected in C. aurantonotus. 5S rDNA segments were located interstitially on two chromosomal pairs in C. ferrugatus and on nine pairs in C. aurantonotus, mostly equivalent to heterochromatic blocks on short arms of biarmed chromosomes. C. ferrugatus can be considered a species in which basal chromosomal features proposed for modern Teleosteans were conserved. The derived karyotype pattern of C. aurantonotus seems to be determined by pericentric inversions and heterochromatin addition which probably determined the notorious dispersion of 5S rRNA (pseudo)genes. It is demonstrated that, even within a group generally characterized by cytogenetical homogeneity as the family Pomacanthidae, diversified karyotypes can be found.

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes, Prochilodontidae).

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the São Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n = 54 and FN = 108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.