Genotype-by-sequencing (GBS) methods have revolutionized the field of molecular ecology, but their application in molecular phylogenetics remains somewhat limited. In addition, most phylogenetic studies based on large GBS data sets have relied on analyses of concatenated data rather than species tree methods that explicitly account for genealogical stochasticity among loci. We explored the utility of “double-digest” restriction site-associated DNA sequencing (ddRAD-seq) for phylogenetic analyses of the Lagonosticta firefinches (family Estrildidae) and the Vidua brood parasitic finches (family Viduidae). As expected, the number of homologous loci shared among samples was negatively correlated with genetic distance due to the accumulation of restriction site polymorphisms. Nonetheless, for each genus, we obtained data sets of ∼3000 loci shared in common among all samples, including a more distantly related outgroup taxon. For all samples combined, we obtained >1000 homologous loci despite ∼20 my divergence between estrildid and parasitic finches. In addition to nucleotide polymorphisms, the ddRAD-seq data yielded large sets of indel and locus presence–absence polymorphisms, all of which had higher consistency indices than mtDNA sequence data in the context of concatenated parsimony analyses. Species tree methods, using individual gene trees or single nucleotide polymorphisms as input, generated results broadly consistent with analyses of concatenated data, particularly for Lagonosticta, which appears to have a well resolved, bifurcating history. Results for Vidua were also generally consistent across methods and data sets, although nodal support and results from different species tree methods were more variable. Lower gene tree congruence in Vidua is likely the result of its unique evolutionary history, which includes rapid speciation by host shift and occasional hybridization and introgression due to incomplete reproductive isolation. We conclude that ddRAD-seq is a cost-effective method for generating robust phylogenetic data sets, particularly for analyses of closely related species and genera.
Rapid diversification is often associated with morphological or ecological adaptations that allow organisms to radiate into novel niches. Neotropical Adelpha butterflies, which comprise over 200 species and subspecies, are characterized by extraordinary breadth in host plant use and wing colour patterns compared to their closest relatives. To examine the relationship between phenotypic and species diversification, we reconstructed the phylogenetic history of Adelpha and its temperate sister genus Limenitis using genomewide restriction-site-associated DNA (RAD) sequencing. Despite a declining fraction of shared markers with increasing evolutionary distance, the RAD-Seq data consistently generated well-supported trees using a variety of phylogenetic methods. These well-resolved phylogenies allow the identification of an ecologically important relationship with a toxic host plant family, as well as the confirmation of widespread, convergent wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that evolutionary innovations in both larvae and adults have permitted the colonization of novel host plants and fuelled adaptive diversification within this large butterfly radiation.
Speciation is a continuous and dynamic process, and studying organisms during the early stages of this process can aid in identifying speciation mechanisms. The mallard (Anas platyrhynchos) and Mexican duck (A. [p.] diazi) are two recently diverged taxa with a history of hybridization and controversial taxonomy. To understand their evolutionary history, we conducted genomic scans to characterize patterns of genetic diversity and divergence across the mitochondrial DNA (mtDNA) control region, 3523 autosomal loci and 172 Z-linked sex chromosome loci. Between the two taxa, Z-linked loci (ΦST = 0.088) were 5.2 times more differentiated than autosomal DNA (ΦST = 0.017) but comparable to mtDNA (ΦST = 0.092). This elevated Z differentiation deviated from neutral expectations inferred from simulated data that incorporated demographic history and differences in effective population sizes between marker types. Furthermore, 3% of Z-linked loci, compared to <0.1% of autosomal loci, were detected as outlier loci under divergent selection with elevated relative (ΦST ) and absolute (dXY ) estimates of divergence. In contrast, the ratio of Z-linked and autosomal differentiation among the seven Mexican duck sampling locations was close to 1:1 (ΦST = 0.018 for both markers). We conclude that between mallards and Mexican ducks, divergence at autosomal markers is largely neutral, whereas greater divergence on the Z chromosome (or some portions thereof) is likely the product of selection that has been important in speciation. Our results contribute to a growing body of literature indicating elevated divergence on the Z chromosome and its likely importance in avian speciation.
Obligate brood parasites, which typically lack reliable interactions with conspecifics early in life, acquire species recognition cues by mechanisms other than imprinting on parents and siblings. The African indigobirds (Vidua spp.) are exceptional among brood parasites in that learning and mimicry of host vocalizations play an integral role in the social behavior of the parasites. Male indigobirds have impressive vocal repertoires featuring chatter calls, host mimicry, and complex non-mimicry songs. While chatter calls are similar, sympatric indigobirds are distinguished both by their mimicry of different hosts and by unique repertoires of non-mimicry songs. A previous playback experiment showed that male indigobirds respond differentially to natural singing (i.e. playbacks including host mimicry, chatter, and complex non-mimicry) of sympatric conspecifics and heterospecifics, but the relative role of host mimicry and complex non-mimicry in species recognition remains unknown. We addressed this question in a playback experiment that tested the response of focal males to 3 treatments: sympatric conspecific, allopatric conspecific (same host mimicry combined with unfamiliar complex non-mimicry songs), and sympatric heterospecific (different host mimicry and different, but familiar, complex non-mimicry). Three behavioral responses (number of hops, latency of response, and number of chatter calls) were similar in the 2 conspecific treatments but differed in comparison with the heterospecific treatment. Our study provides evidence that host mimicry is an important cue in species recognition among territorial male indigobirds and suggests that it may contribute to species cohesion when juveniles or adults disperse beyond the boundaries of their local dialect neighborhood. This study enhances our understanding of avian recognition systems by showing that cues obtained through heterospecific imprinting can be important for species recognition in brood parasitic birds.
A growing variety of “genotype-by-sequencing” (GBS) methods use restriction enzymes and high throughput DNA sequencing to generate data for a subset of genomic loci, allowing the simultaneous discovery and genotyping of thousands of polymorphisms in a set of multiplexed samples. We evaluated a “double-digest” restriction-site associated DNA sequencing (ddRAD-seq) protocol by 1) comparing results for a zebra finch (Taeniopygia guttata) sample with in silico predictions from the zebra finch reference genome; 2) assessing data quality for a population sample of indigobirds (Vidua spp.); and 3) testing for consistent recovery of loci across multiple samples and sequencing runs. Comparison with insilico predictions revealed that 1) over 90% of predicted, single-copy loci in our targeted size range (178–328 bp) were recovered; 2) short restriction fragments (38–178 bp) were carried through the size selection step and sequenced at appreciable depth, generating unexpected but nonetheless useful data; 3) amplification bias favored shorter, GC-rich fragments, contributing to among locus variation in sequencing depth that was strongly correlated across samples; 4) our use of restriction enzymes with a GC-rich recognition sequence resulted in an up to four-fold overrepresentation of GC-rich portions of the genome; and 5) star activity (i.e., non-specific cutting) resulted in thousands of “extra” loci sequenced at low depth. Results for three species of indigobirds show that a common set of thousands of loci can be consistently recovered across both individual samples and sequencing runs. In a run with 46 samples, we genotyped 5,996 loci in all individuals and 9,833 loci in 42 or more individuals, resulting in <1% missing data for the larger data set. We compare our approach to similar methods and discuss the range of factors (fragment library preparation, natural genetic variation, bioinformatics) influencing the recovery of a consistent set of loci among samples.
HapSTRs combine information from a microsatellite (or simple tandem repeat, STR) with one or more single nucleotide polymorphisms in the DNA sequence immediately flanking the STR. These loci may offer increased power for the estima- tion of demographic parameters, but also present some challenges for data collection and analysis. We describe a process for inferring HapSTR alleles, including the flanking haplotypes, STR alleles and their phase relative to each other, directly from DNA sequence electropherograms of PCR products from heterozygous individuals. Our approach eliminates the need for more costly and time-consuming processes, such as cloning or acrylamide gel electrophoresis to separate alleles prior to sequencing.
The emberizid genera Aimophila and Pipilo represent longstanding taxonomic conundrums. Each is comprised of sub- clades whose members appear to share diagnostic morphological and behavioral characters; however, relationships among sub-clades within each of these genera remain unclear, and numerous authors have suggested that either one or both of these genera may be polyphyletic. We addressed this taxonomic problem by sequencing and analyzing complete mitochondrial cytochrome-b and NADH dehydrogenase subunit 2 genes for all members of Aimophila and Pipilo along with 33 species representing 17 additional emberizid genera. Our maximum likelihood and Bayesian analyses indicate that both Aimophila and Pipilo are polyphyletic. Aimophila is divided into a minimum of three distinct groups. The forms notosticta, ruficeps, and rufescens are part of a well-supported clade that includes all members of Melozone and some members of Pipilo. Aimophila quinquestriata is placed within Amphispiza, and the remaining members of Aimophila are placed within a clade that includes all members of Arremonops and some members of Ammodramus. Within Pipilo, the ‘‘rufous-sided’’ and ‘‘brown’’ towhee groups do not form sister groups. Rather, the former are most closely related to the tropical genus Atlapetes whereas the latter are placed nearest Melozone and some Aimophila. Our analyses reject traditional taxonomic arrangements for both genera, and we present suggestions for a revised taxonomy for all members of Aimophila and Pipilo. These results provide further evidence of discordance among phylogenetic hypotheses based on morphological and molecular characters for groups of birds with generally conserved morphology.
The Great-tailed Grackle (Quiscalus mexicanus) and Boat-tailed Grackle (Q. major) are sister species that have expanded their ranges during historical times. This expansion has created an area of sympatry between these species in Texas and Louisiana, and between distinctive Great-tailed Grackle subspecies in the southwestern United States and northern Mexico. We investigated the evolutionary histories of both species using mitochondrial DNA sequence data and modern phylogenetic methods. Our results reveal genetic structure within Great-tailed, but not Boat-tailed Grackles. Great-tailed Grackles are separated into two clades, but range expansion in the north has led to secondary contact between them. Boat-tailed Grackles are monophyletic and are embedded within the Great-tailed Grackle assemblage, rendering the latter paraphyletic. These results reveal a complex phylogeographic pattern caused by recent range expansion and secondary contact of once allopatric units.
We report bilateral gynandromorphy in a White-ruffed Manakin (Corapipo altera) collected near Santa Fe, Panama in 2004. The specimen had an oviduct and ovary on the left side and a single testis on the right. The plumage was phenotypically female on the right side and male on the left. The weight and genetic affinity of the specimen were characteristically female. Both Z and W chromosomes were detected in genetic samples from multiple tissue types and toe pads from both feet. This report is a novel record of gynandromorphy in a suboscine passerine.
We report bilateral gynandromorphy in a White-ruffed Manakin (Corapipo altera) collected near Santa Fé, Panamá in 2004. The specimen had an oviduct and ovary on the left side and a single testis on the right. The plumage was phenotypically female on the right side and male on the left. The weight and genetic affinity of the specimen were characteristically female. Both Z and W chromosomes were detected in genetic samples from multiple tissue types and toe pads from both feet. This report is a novel record of gynandromorphy in a suboscine passerine.