A comparative phylogenomic analysis of birds reveals heterogeneous differentiation processes among Neotropical savannas.

The main objective of this study was to evaluate biogeographical hypotheses of diversification and connection between isolated savannas north (Amazonian savannas) and south (Cerrado core) of the Amazon River. To achieve this, we used genomic markers (genotyping-by-sequencing) to evaluate the genetic structure, population phylogenetic relationships and historical range shifts of four Neotropical passerines with peri-Atlantic distributions: the narrow-billed woodcreeper (Lepidocolaptes angustirostris), the plain-crested elaenia (Elaenia cristata), the grassland sparrow (Ammodramus humeralis) and the white-banded tanager (Neothraupis fasciata). Population genetic analyses indicated that landscape (e.g., geographic distance, landscape resistance and percentage of tree cover) and climate metrics explained divergence among populations in most species, but without indicating a differential role between current and historical factors. Our results did not fully support the hypothesis that isolated populations in Amazonian savannas have been recently derived from the Cerrado core domain. Intraspecific phylogenies and gene flow analyses supported multiple routes of connection between the Cerrado and Amazonian savannas, rejecting the hypothesis that the Atlantic corridor explains the peri-Atlantic distribution. Our results reveal that the biogeographical history of the region is complex and cannot be explained by simple vicariant models.

Avian host composition, local speciation and dispersal drive the regional assembly of avian malaria parasites in South American birds.

Identifying the ecological factors that shape parasite distributions remains a central goal in disease ecology. These factors include dispersal capability, environmental filters and geographic distance. Using 520 haemosporidian parasite genetic lineages recovered from 7,534 birds sampled across tropical and temperate South America, we tested (a) the latitudinal diversity gradient hypothesis and (b) the distance-decay relationship (decreasing proportion of shared species between communities with increasing geographic distance) for this host-parasite system. We then inferred the biogeographic processes influencing the diversity and distributions of this cosmopolitan group of parasites across South America. We found support for a latitudinal gradient in diversity for avian haemosporidian parasites, potentially mediated through higher avian host diversity towards the equator. Parasite similarity was correlated with climate similarity, geographic distance and host composition. Local diversification in Amazonian lineages followed by dispersal was the most frequent biogeographic events reconstructed for haemosporidian parasites. Combining macroecological patterns and biogeographic processes, our study reveals that haemosporidian parasites are capable of circumventing geographic barriers and dispersing across biomes, although constrained by environmental filtering. The contemporary diversity and distributions of haemosporidian parasites are mainly driven by historical (speciation) and ecological (dispersal) processes, whereas the parasite community assembly is largely governed by host composition and to a lesser extent by environmental conditions.

Phylogeographic variation within the Buff-browed Foliage-gleaner (Aves: Furnariidae: Syndactyla rufosuperciliata) supports an Andean-Atlantic forests connection via the Cerrado.

We evaluated whether the Andean and the Atlantic forests acted as refugia during the Quaternary, and tested biogeographic hypotheses about the regions involved in the connectivity between those biomes (through the Chaco or the Cerrado). To achieve these goals we selected the Buff-browed Foliage-gleaner Syndactyla rufosuperciliata (Aves, Furnariidae) as a study system, a taxon distributed between the Andean and Atlantic forest. We first explored the historical connectivity between regions through niche modeling. We subsequently used DNA sequences (n = 71 individuals) and genomic analyses (ddRADseq, n = 33 individuals) to evaluate population genetic structure and gene flow within this species. Finally, we performed population model selection using Approximate Bayesian Computation. Our findings indicate that the Andean and the Atlantic forests acted as refugia, and that the populations of the focal species from both regions contacted through the Cerrado region, thus suggesting that the historical dynamics of Andean and Atlantic forests are important for the evolution of forest birds in the region. The results are in agreement with studies of other organisms and may indicate a more general pattern of connectivity among biomes in the Neotropics. Finally, we recommend recognizing both the Andean and the Altantic forests lineages of S. rufosuperciliata as independent species.

Polymorphic microsatellite loci and partial mitogenome for the Chestnut-bellied Seed-finch Sporophila angolensis (Aves, Passeriformes) using next generation sequencing.

Brazil is one of the major contributors to international trade in wildlife and species of the bird genus Sporophila are currently under threat due to illegal trade. Microsatellite loci and mitochondrial DNA constitute important molecular markers for population genetics studies and parentage analyses, and hold great potential to help authorities manage illegal trafficking and control commercial breeders. We describe and characterize 19 polymorphic microsatellite loci and recover part of the mitochondrial genome for Sporophila angolensis using massive parallel sequencing with the Illumina platform. DNA sequencing resulted in a dataset with 2,379,295 paired reads, of which 392 were mapped to the mitogenome of S. maximiliani, resulting in a partial mitogenome of 16,785 bp for S. angolensis. The microsatellite search identified a total of 4737 loci, from which 27 primer pairs were tested on 24 individuals of unknown geographic origin. Nineteen of the 27 loci were successfully amplified and exhibited high levels of genetic variation, with a mean of 11.2 alleles per locus, a mean observed heterozygosity of 0.588 and a mean expected heterozygosity of 0.852. About half of the loci showed significant evidence for the presence of a null allele and significant deviation from Hardy-Weinberg equilibrium; the remaining eight loci had high paternity exclusion probabilities and low identity probabilities. The high levels of polymorphism for these loci, as well as their high paternity exclusion probability and low identity probability, indicate that they hold potential for parentage analyses and population genetics studies of S. angolensis.

Development and characterization of 19 polymorphic microsatellite loci from the Red-cowled Cardinal (Paroaria dominicana, Passeriformes, Aves) using next-generation sequencing.

The Red-cowled Cardinal (Paroaria dominicana) is an endemic passerine of the Caatinga biome in Brazil, and is one of the most traded passerines in the country. Illegal trade can have serious impacts on wild populations, such as reduced population sizes, the introduction of the species to areas outside their historical range or mixing individuals from different populations. Microsatellites constitute an important tool for population genetics and forensics studies, and hold great potential to help authorities manage illegal trafficking and inspect commercial breeders. We developed new microsatellite loci using massive parallel sequencing and characterized them in 23 seized Red-cowled Cardinals with unknown geographic origin. The DNA sequencing generated 2,068,684 paired-reads of which we identified 10,322 tri- to hexanucleotide loci. We selected 30 loci for amplification and polymorphism tests, of which 21 successfully amplified and 19 were polymorphic. The number of alleles ranged from 7 to 18 and the mean expected heterozygosity was 0.863. Six loci deviated from Hardy-Weinberg equilibrium probably due to null alleles and/or the Wahlund effect. Polymorphic loci in Hardy-Weinberg equilibrium showed low identity probability and high paternity exclusion probability. Our results indicate that this new set of microsatellite loci constitutes an important tool for both population genetic and forensic studies, with ultimate potential for assisting authorities in managing animal victims of illegal trafficking and the inspection of commercial breeders of the Red-cowled Cardinal.

Development and characterization of novel microsatellite loci for the Blue-fronted Amazon (Amazona aestiva, Psittaciformes, Aves) and cross-species amplification for other two threatened Amazona species.

Parrots are among the most threatened avian groups of the world, with illegal pet trade being a major threat to some Amazon (Amazona genus) and macaw (Ara and Anodorhynchus genera) species. Population genetic studies and effective control of commercial breeders are important actions for the conservation of these parrot species; however, few microsatellite loci are available for most Amazona species to date. In this study, 25 polymorphic microsatellite loci were identified in silico and characterized for the Blue-fronted Amazon [Amazona aestiva (Aa)]. Loci were tested in 24 Blue-fronted Amazons from wild population from central Brazil with cross-species amplified in two individuals of Amazona vinacea (Av) and Amazona pretrei (Ap) from northeastern Minas Gerais and Rio Grande do Sul, in southeastern and south of Brazil, respectively. The number of alleles per locus for Aa ranged from 5 to 24 with an average of 13.1. Twenty-four and 25 loci were successfully amplified for Av and Ap, respectively. The observed and expected heterozygosities for Aa ranged from 0.27 to 1.00 and from 0.35 to 0.94, with averages of 0.75 and 0.85, respectively. Nine loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (likely due to null alleles) and no significant linkage disequilibrium between loci pairs was detected. The combined paternity exclusion probability was very high, and the probability of identity was extremely low. This new set of microsatellite loci will be useful for analyzing population genetic structure and making conservation and management decisions, as well as for parentage analysis and the control of commercial breeding of Aa and potentially other Amazona species.

In silico identification and characterization of novel microsatellite loci for the Blue-and-yellow Macaw Ara ararauna (Linnaeus, 1758) (Psittaciformes, Psittacidae).

The illegal trade is a major threat to many bird species, and parrots are common victims of this activity. Domestic and international pet markets are interested on different parrot species, such as the Blue-and-yellow Macaw (Ara ararauna). This South American macaw is not globally threatened, but is under protection from over-exploitation. This study aimed to identify and characterize novel microsatellite loci for population and parentage analysis of A. ararauna. Scaffold sequences of Ara macao available in the NCBI database were used for microsatellite searches using MsatCommander software. We tested a total of 28 loci, from which 25 were polymorphic, one was monomorphic, and two did not generated amplification products. For polymorphic loci, the mean number of alleles was 8.24 (4 - 15 alleles per locus), the observed heterozygosity ranged from 0.333 to 0.917, and the expected heterozygosity from 0.353 to 0.890. The paternity exclusion probability and identity probability were highly discriminatory. Thus, these novel microsatellite markers can be useful for population assignment and paternity tests, helping the authorities to manage macaws from the illegal trafficking and control commercial breeders.

Mitogenomes of two neotropical bird species and the multiple independent origin of mitochondrial gene orders in Passeriformes.

At least four mitogenome arrangements occur in Passeriformes and differences among them are derived from an initial tandem duplication involving a segment containing the control region (CR), followed by loss or reduction of some parts of this segment. However, it is still unclear how often duplication events have occurred in this bird order. In this study, the mitogenomes from two species of Neotropical passerines (Sicalis olivascens and Lepidocolaptes angustirostris) with different gene arrangements were first determined. We also estimated how often duplication events occurred in Passeriformes and if the two CR copies demonstrate a pattern of concerted evolution in Sylvioidea. One tissue sample for each species was used to obtain the mitogenomes as a byproduct using next generation sequencing. The evolutionary history of mitogenome rearrangements was reconstructed mapping these characters onto a mitogenome Bayesian phylogenetic tree of Passeriformes. Finally, we performed a Bayesian analysis for both CRs from some Sylvioidea species in order to evaluate the evolutionary process involving these two copies. Both mitogenomes described comprise 2 rRNAs, 22 tRNAs, 13 protein-codon genes and the CR. However, S. olivascens has 16,768 bp showing the ancestral avian arrangement, while L. angustirostris has 16,973 bp and the remnant CR2 arrangement. Both species showed the expected gene order compared to their closest relatives. The ancestral state reconstruction suggesting at least six independent duplication events followed by partial deletions or loss of one copy in some lineages. Our results also provide evidence that both CRs in some Sylvioidea species seem to be maintained in an apparently functional state, perhaps by concerted evolution, and that this mechanism may be important for the evolution of the bird mitogenome.

Genetic diversity of the pampas deer (Ozotoceros bezoarticus) population in the Brazilian Pantanal assessed by combining fresh fecal DNA analysis and a set of heterologous microsatellite loci.

The pampas deer (Ozotoceros bezoarticus) is close to being classified as 'globally threatened', with the largest population occurring in the Brazilian Pantanal. Since capture is stressful to these animals, non-invasive sampling methods such as the use of feces can provide reliable sources of DNA. The aim of this study was to use fecal samples to evaluate the genetic variability of the Brazilian Pantanal population of pampas deer. Six heterologous microsatellite markers were used to screen 142 stool specimens. Seventy-four deer were identified, of which 50 adults were used to determine the genetic characteristics of the population. The Pantanal population showed high genetic diversity (mean number of alleles per locus = 11.5, expected heterozygosity = 0.75). This is the first investigation to characterize a South American deer species using fecal DNA and demonstrates the usefulness and efficiency of this approach, as well as the feasibility of obtaining information that could not have been easily obtained by traditional DNA sampling. Our findings suggest that management strategies for this species may be much more effective if applied now when the population still shows high genetic variability.

Characterization of the complete mitochondrial genome and a set of polymorphic microsatellite markers through next-generation sequencing for the brown brocket deer Mazama gouazoubira.

The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America.

DNA sequence analysis to guide the release of blue-and-yellow macaws (Ara ararauna, Psittaciformes, Aves) from the illegal trade back into the wild.

The illegal wildlife trade is one of the major threats to Brazil's biodiversity. Approximately 80 % of illegally captured animals are birds, and 4 % of those are parrots. Although many seized birds do not survive, those that are recovered may be returned to the wild. The release of seized individuals into the wild should be conducted with caution, as local populations may suffer adverse effects if genetically different individuals are introduced. In this study, we evaluated the genetic relationships between 13 illegally captured blue-and-yellow macaws selected for release in northeastern Goiás, Brazil, and previously studied Brazilian macaw populations. We identified the seized macaws that were genetically similar to those from northwestern Goiás and that were therefore most suitable for release in that area. The genetic relationship was evaluated by sequence analysis of 403 bp of mitochondrial DNA control region. Relationships between mitochondrial haplotypes were computed via a median-joining network. Only six of the seized macaws were closely related to the macaws of northeastern Goiás, indicating that those macaws were potential candidates for release in that area. However, the release of these birds should follow all technical recommendations required by the Brazilian environmental authorities.

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (Rhynchotus rufescens, Tinamiformes, Aves) and cross-amplification in other tinamous.

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2-12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy-Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10(-5)), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.

Isolation and characterization of microsatellite loci for Neothraupis fasciata, (Emberizidae, Passeriformes) with widely cross amplification in neotropical passerines.

We described the development and characterization of 16 microsatellite loci for the white-banded tanager (Neothraupis fasciata), a Neotropical passerine from Cerrado (Brazilian savanna). Based on 24 N. fasciata from a single population, we detected moderate to high number of alleles per locus (2-18 alelles) with expected heterozygosities ranging from 0.12 to 0.92. The high combined paternity exclusion probability (0.999) and low combined genetic identity (1.72 x 10(-10)) show that the battery of loci is suitable for parentage and population analyses. Some of these markers also showed amplification in 8 species from 4 Neotropical passerine families, indicating the potential of these markers to address similar questions in these species.

Late Pleistocene climatic changes promoted demographic expansion and population reconnection of a Neotropical savanna-adapted bird, Neothraupis fasciata (Aves: Thraupidae).

We performed phylogeographic and genetic structure analyses of Neothraupis fasciata joined with species distribution modelling to evaluate whether: (1) the distribution of genetic variability shows a pattern expected by the isolation-by-distance model; (2) the influence of the Pleistocene climate changes on species distribution; and (3) climate/climatic stability (hypothesis of climatic stability) as a predictor of population genetic diversity. Based on two molecular datasets (ND2 and FIB-5), the isolation-by-distance hypothesis was not supported. The mitochondrial haplotype network indicated the existence of historically isolated populations at the southern range of the species distribution, and recent population expansion was identified by both neutrality tests and extended Bayesian skyline plot analysis. Thus, the climatic changes during the Pleistocene might have promoted the reconnection of the partially isolated southern populations, which may have persisted in the plateaus during the cycles of savanna contractions. Subsequently, this species (re)colonized northern areas of the species present distribution, following the continuous vegetation on the São Francisco and Central plateaus about 60 kyr, and also reached the Amazonian savannas likely via the central corridor. Thus, our results indicated that the intrinsic relationship between the relief heterogeneity (plateaus and depressions) and the climatic fluctuations, mainly in the Pleistocene, promoted population reconnection and demographic expansion of N. fasciata.

Biogeography of speciation of two sister species of neotropical amazona (Aves, Psittaciformes) based on mitochondrial sequence data.

Coalescent theory provides powerful models for population genetic inference and is now increasingly important in estimates of divergence times and speciation research. We use molecular data and methods based on coalescent theory to investigate whether genetic evidence supports the hypothesis of A. pretrei and A. tucumana as separate species and whether genetic data allow us to assess which allopatric model seems to better explain the diversification process in these taxa. We sampled 13 A. tucumana from two provinces in northern Argentina and 28 A. pretrei from nine localities of Rio Grande do Sul, Brazil. A 491 bp segment of the mitochondrial gene cytochrome c oxidase I was evaluated using the haplotype network and phylogenetic methods. The divergence time and other demographic quantities were estimated using the isolation and migration model based on coalescent theory. The network and phylogenetic reconstructions showed similar results, supporting reciprocal monophyly for these two taxa. The divergence time of lineage separation was estimated to be approximately 1.3 million years ago, which corresponds to the lower Pleistocene. Our results enforce the current taxonomic status for these two Amazon species. They also support that A. pretrei and A. tucumana diverged with little or no gene flow approximately 1.3 million years ago, most likely after the establishment of a small population in the Southern Yungas forest by dispersion of a few founders from the A. pretrei ancestral population. This process may have been favored by habitat corridors formed in hot and humid periods of the Quaternary. Considering that these two species are considered threatened, the results were evaluated for their implications for the conservation of these two species.

Genetic diversity of the pampas deer (Ozotoceros bezoarticus) population in the Brazilian Pantanal assessed by combining fresh fecal DNA analysis and a set of heterologous microsatellite loci

Abstract The pampas deer (Ozotoceros bezoarticus) is close to being classified as 'globally threatened', with the largest population occurring in the Brazilian Pantanal. Since capture is stressful to these animals, non-invasive sampling methods such as the use of feces can provide reliable sources of DNA. The aim of this study was to use fecal samples to evaluate the genetic variability of the Brazilian Pantanal population of pampas deer. Six heterologous microsatellite markers were used to screen 142 stool specimens. Seventy-four deer were identified, of which 50 adults were used to determine the genetic characteristics of the population. The Pantanal population showed high genetic diversity (mean number of alleles per locus = 11.5, expected heterozygosity = 0.75). This is the first investigation to characterize a South American deer species using fecal DNA and demonstrates the usefulness and efficiency of this approach, as well as the feasibility of obtaining information that could not have been easily obtained by traditional DNA sampling. Our findings suggest that management strategies for this species may be much more effective if applied now when the population still shows high genetic variability.

Genetic evaluation of the mating system in the blue-and-yellow macaw (Ara ararauna, Aves, Psittacidae) by DNA fingerprinting.

More than 90% of birds are socially monogamous, although genetic studies indicate that many are often not sexually monogamous. In the present study, DNA fingerprinting was used to estimate the genetic relationships between nestlings belonging to the same broods to evaluate the mating system in the socially monogamous macaw, Ara ararauna. We found that in 10 of 11 broods investigated, the nestlings showed genetic similarity levels congruent with values expected among full-sibs, suggesting that they shared the same parents. However, in one brood, the low genetic similarity observed between nestlings could be a result of intraspecific brood parasitism, intraspecific nest competition or extra-pair paternity. These results, along with available behavioral and life-history data, imply that the blue-and-yellow macaw is not only socially, but also genetically monogamous. However, the occurrence of eventual cases of extra-pair paternity cannot be excluded.

Comparative analysis of microsatellite variability in five macaw species (Psittaciformes, Psittacidae): Application for conservation.

Cross-amplification was tested and variability in microsatellite primers (designed for Neotropical parrots) compared, in five macaw species, viz., three endangered blue macaws (Cyanopsitta spixii [extinct in the wild], Anodorhynchus leari [endangered] and Anodorhynchus hyacinthinus [vulnerable]), and two unthreatened red macaws (Ara chloropterus and Ara macao). Among the primers tested, 84.6% successfully amplified products in C. spixii, 83.3% in A. leari, 76.4% in A. hyacinthinus, 78.6% in A. chloropterus and 71.4% in A. macao. The mean expected heterozygosity estimated for each species, and based on loci analyzed in all the five, ranged from 0.33 (A. hyacinthinus) to 0.85 (A. macao). As expected, the results revealed lower levels of genetic variability in threatened macaw species than in unthreatened. The low combined probability of genetic identity and the moderate to high potential for paternity exclusion, indicate the utility of the microsatellite loci set selected for each macaw species in kinship and population studies, thus constituting an aid in planning in-situ and ex-situ conservation.

Genetic evaluation of the mating system in the blue-and-yellow macaw (Ara ararauna, Aves, Psittacidae) by DNA fingerprinting

More than 90 percent of birds are socially monogamous, although genetic studies indicate that many are often not sexually monogamous. In the present study, DNA fingerprinting was used to estimate the genetic relationships between nestlings belonging to the same broods to evaluate the mating system in the socially monogamous macaw, Ara ararauna. We found that in 10 of 11 broods investigated, the nestlings showed genetic similarity levels congruent with values expected among full-sibs, suggesting that they shared the same parents. However, in one brood, the low genetic similarity observed between nestlings could be a result of intraspecific brood parasitism, intraspecific nest competition or extra-pair paternity. These results, along with available behavioral and life-history data, imply that the blue-and-yellow macaw is not only socially, but also genetically monogamous. However, the occurrence of eventual cases of extra-pair paternity cannot be excluded.

Comparative analysis of microsatellite variability in five macaw species (Psittaciformes, Psittacidae): application for conservation

Cross-amplification was tested and variability in microsatellite primers (designed for Neotropical parrots) compared, in five macaw species, viz., three endangered blue macaws (Cyanopsitta spixii [extinct in the wild], Anodorhynchus leari [endangered] and Anodorhynchus hyacinthinus [vulnerable]), and two unthreatened red macaws (Ara chloropterus and Ara macao). Among the primers tested, 84.6 percent successfully amplified products in C. spixii, 83.3 percent in A. leari, 76.4 percent in A. hyacinthinus, 78.6 percent in A. chloropterus and 71.4 percent in A. macao. The mean expected heterozygosity estimated for each species, and based on loci analyzed in all the five, ranged from 0.33 (A. hyacinthinus) to 0.85 (A. macao). As expected, the results revealed lower levels of genetic variability in threatened macaw species than in unthreatened. The low combined probability of genetic identity and the moderate to high potential for paternity exclusion, indicate the utility of the microsatellite loci set selected for each macaw species in kinship and population studies, thus constituting an aid in planning in-situ and ex-situ conservation.