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.

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.

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.

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.

Molecular phylogenetic position of endangered Wilfredomys within Sigmodontinae (Cricetidae) based on mitochondrial and nuclear DNA sequences and comments on Wiedomyini.

Wilfredomys, a monotypic genus of endangered sigmodontine rats, was historically related to the tribe Thomasomyini or considered "incertae sedis". Given no molecular data is available for Wilfredomys, the phylogenetic position of this taxon is uncertain in relation to modern, molecular hypotheses of sigmodontine relationships. We investigate the phylogeny of Wilfredomys to provide a hypothesis of its position within Sigmodontinae based on nuclear and mitochondrial DNA sequences. Bayesian and maximum likelihood phylogenetic analyses recovered Wilfredomys oenax as sister to Wiedomys pyrrhorhinos, and Wie. cerradensis fell out sister to this clade. At the genus level, Phaenomys is sister to Wilfredomys + Wiedomys, forming a novel and well-supported sigmodontine clade. Our results suggest that tribe Wiedomyini should encompass Wilfredomys in addition to Wiedomys and Cholomys, thus the hypothesis that Wiedomys is paraphyletic should be investigated further. Another plausible classification scheme consistent with our results would be to expand Wiedomyini to encompass the clade composed of Phaenomys + Wilfredomys + Wiedomys. Last, our recovery of an "Atlantic clade" composed of lineages restricted to eastern South America supports the idea that this region has likely played an important role in sigmodontine diversification.