Comparative genomics of two Empidonax flycatchers reveal candidate genes for bird song production.

Whole-genome-level comparisons of sister taxa that vary in phenotype against a background of high genomic similarity can be used to identify the genomic regions that might underlie their phenotypic differences. In wild birds, this exploratory approach has detected markers associated with plumage coloration, beak and wing morphology, and complex behavioral traits like migration. Here, we use genomic comparisons of two closely related suboscine flycatchers (Empidonax difficilis and E. occidentalis) and their hybrids to search for candidate genes underlying their variation in innate vocal signals. We sequenced the genomes of 20 flycatchers that sang one of two species-specific pure song types and 14 putative hybrid individuals with intermediate song types. In the resulting genomic comparisons, we found six areas of high differentiation that may be associated with variation in nonlearned songs. These narrow regions of genomic differentiation contain a total of 67 described genes, of which three have been previously associated with forms of language impairment and dyslexia in humans and 18 are known to be differentially expressed in the song nuclei regions of the avian brain compared with adjacent parts of the avian brain. This "natural experiment" therefore may help identify loci associated with song differences that merit further study across bird lineages with both learned and innate vocalizations.

Mechanisms involved in the production of differently colored feathers in the structurally colored swallow tanager (Tersina viridis; Aves: Thraupidae).

Non-iridescent, structural coloration in birds originates from the feather's internal nanostructure (the spongy matrix) but melanin pigments and the barb's cortex can affect the resulting color. Here, we explore how this nanostructure is combined with other elements in differently colored plumage patches within a bird. We investigated the association between light reflectance and the morphology of feathers from the back and belly plumage patches of male swallow tanagers (Tersina viridis), which look greenish-blue and white, respectively. Both plumage patches have a reflectance peak around 550 nm but the reflectance spectrum is much less saturated in the belly. The barbs of both types of feathers have similar spongy matrices at their tips, rendering their reflectance spectra alike. However, the color of the belly feather barbs changes from light green at their tips to white closer to the rachis. These barbs lack pigments and their morphology changes considerably throughout. Toward the rachis, the barb is almost hollow, with a reduced area occupied by spongy matrix, and has a flattened shape. By contrast, the blue back feathers' barbs have melanin underneath the spongy matrix resulting in a much more saturated coloration. The color of these barbs is also even along the barbs' length. Our results suggest that the color differences between the white and greenish-blue plumage are mostly due to the differential deposition of melanin and a reduction of the spongy matrix near the rachis of the belly feather barbs and not a result of changes in the characteristics of the spongy matrix.

A test of the riverine barrier hypothesis in the largest subtropical river basin in the Neotropics.

The riverine barrier hypothesis proposes that large rivers represent geographical barriers to gene flow for terrestrial organisms, leading to population differentiation and ultimately allopatric speciation. Here we assess for the first time if the subtropical Paraná-Paraguay River system in the Del Plata basin, second in size among South American drainages, acts as a barrier to gene flow for birds. We analysed the degree of mitochondrial and nuclear genomic differentiation in seven species with known subspecies divided by the Paraná-Paraguay River axis. Only one species showed genetic differentiation concordant with the current river channel, but another five species have an east/west genetic split broadly coincident with the Paraná River's dynamic palaeochannel, suggesting this fluvial axis has had a past role in shaping present-day genetic structure. Moreover, dating analyses show that these splits have been asynchronous, with species responding differently to the riverine barrier. Comparisons informed by the geological history of the Paraná River and its influence on the ecological and climatic differences among ecoregions in the study area further bolster the finding that responses to this geographical barrier have been species-specific.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence.

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%-9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail.

Continental-scale analysis reveals deep diversification within the polytypic Red-crowned Ant Tanager (Habia rubica, Cardinalidae).

We explored the phylogeographic patterns of intraspecific diversity in the Red-crowned Ant Tanager (Habia rubica) throughout its continent-wide distribution, in order to understand its evolutionary history and the role of evolutionary drivers that are considered to promote avian diversification in the Neotropics. We sampled 100 individuals of H. rubica from Mexico to Argentina covering the main areas of its disjunct distribution. We inferred phylogenetic relationships through Bayesian and maximum parsimony methodologies based on mitochondrial and nuclear markers, and complemented genetic analyses with the assessment of coloration and behavioral differentiation. We found four deeply divergent phylogroups within H. rubica: two South American lineages and two Mexican and Middle American lineages. The divergence event between the northern and southern phylogroups was dated to c. 5.0 Ma, seemingly related to the final uplift of the Northern Andes. Subsequently, the two South American phylogroups split c. 3.5 Ma possibly due to the development of the open vegetation corridor that currently isolates the Amazonian and Atlantic forests. Diversification throughout Mexico and Middle America, following dispersion across the Isthmus of Panama, was presumably more recent and coincident with Pleistocene climatic fluctuations and habitat fragmentations. The analyses of vocalizations and plumage coloration showed significant differences among main lineages that were consistent with the phylogenetic evidence. Our findings suggest that the evolutionary history of H. rubica has been shaped by an assortment of diversification drivers at different temporal and spatial scales resulting in deeply divergent lineages that we recommend should be treated as different species.