James M. Maley

HZAR: hybrid zone analysis using an R software package

We present a new software package (hzar) that provides functions for fitting molecular genetic and morphological data from hybrid zones to classic equilibrium cline models using the Metropolis–Hastings Markov chain Monte Carlo (MCMC) algorithm. The software applies likelihood functions appropriate for different types of data, including diploid and haploid genetic markers and quantitative morphological traits. The modular design allows flexibility in fitting cline models of varying complexity. To facilitate hypothesis testing, an autofit function is included that allows automated model selection from a set of nested cline models. Cline parameter values, such as cline centre and cline width, are estimated and may be compared statistically across clines. The package is written in the R language and is available through the Comprehensive R Archive Network (CRAN; http://cran.r-project.org/). Here, we describe hzar and demonstrate its use with a sample data set from a well‐studied hybrid zone in western Panama between white‐collared (Manacus candei) and golden‐collared manakins (M. vitellinus). Comparisons of our results with previously published results for this hybrid zone validate the hzar software. We extend analysis of this hybrid zone by fitting additional models to molecular data where appropriate.

Next-generation sequencing reveals phylogeographic structure and a species tree for recent bird divergences

Next generation sequencing (NGS) technologies are revolutionizing many biological disciplines but have been slow to take root in phylogeography. This is partly due to the difficulty of using NGS to sequence orthologous DNA fragments for many individuals at low cost. We explore cases of recent divergence in four phylogenetically diverse avian systems using a method for quick and cost-effective generation of primary DNA sequence data using pyrosequencing. NGS data were processed using an analytical pipeline that reduces many reads into two called alleles per locus per individual. Using single nucleotide polymorphisms (SNPs) mined from the loci, we detected population differentiation in each of the four bird systems, including: a case of ecological speciation in rails (Rallus); a rapid postglacial radiation in the genus Junco; recent in situ speciation among hummingbirds (Trochilus) in Jamaica; and subspecies of white-crowned sparrows (Zonotrichia leucophrys) along the Pacific coast. The number of recovered loci aligning closely to chromosomal locations on the zebra finch (Taeniopygia guttata) genome was highly correlated to the size of the chromosome, suggesting that loci are randomly distributed throughout the genome. Using eight loci found in Zonotrichia and Junco lineages, we were also able to generate a species tree of these sparrow sister genera, demonstrating the potential of this method for generating data amenable to coalescent-based analysis. We discuss improvements that should enhance the methods utility for primary data generation.

Similarity thresholds used in DNA sequence assembly from short reads can reduce the comparability of population histories across species

Comparing inferences among datasets generated using short read sequencing may provide insight into the concerted impacts of divergence, gene flow and selection across organisms, but comparisons are complicated by biases introduced during dataset assembly. Sequence similarity thresholds allow the de novo assembly of short reads into clusters of alleles representing different loci, but the resulting datasets are sensitive to both the similarity threshold used and to the variation naturally present in the organism under study. Thresholds that require high sequence similarity among reads for assembly (stringent thresholds) as well as highly variable species may result in datasets in which divergent alleles are lost or divided into separate loci (‘over-splitting’), whereas liberal thresholds increase the risk of paralogous loci being combined into a single locus (‘under-splitting’). Comparisons among datasets or species are therefore potentially biased if different similarity thresholds are applied or if the species differ in levels of within-lineage genetic variation. We examine the impact of a range of similarity thresholds on assembly of empirical short read datasets from populations of four different non-model bird lineages (species or species pairs) with different levels of genetic divergence. We find that, in all species, stringent similarity thresholds result in fewer alleles per locus than more liberal thresholds, which appears to be the result of high levels of over-splitting. The frequency of putative under-splitting, conversely, is low at all thresholds. Inferred genetic distances between individuals, gene tree depths, and estimates of the ancestral mutation-scaled effective population size (θ) differ depending upon the similarity threshold applied. Relative differences in inferences across species differ even when the same threshold is applied, but may be dramatically different when datasets assembled under different thresholds are compared. These differences not only complicate comparisons across species, but also preclude the application of standard mutation rates for parameter calibration. We suggest some best practices for assembling short read data to maximize comparability, such as using more liberal thresholds and examining the impact of different thresholds on each dataset.

Paraphyly of Cinclodes fuscus (Aves: Passeriformes: Furnariidae): implications for taxonomy and biogeography.

The Andes are a hotspot of global avian diversity, but studies on the historical diversification of Andean birds remain relatively scarce. Evolutionary studies on avian lineages with Andean-Patagonian distributions have focused on reconstructing species-level phylogenies, whereas no detailed phylogeographic studies on widespread species have been conducted. Here, we describe phylogeographic patterns in the Bar-winged Cinclodes (Cinclodes fuscus), a widespread and common species of ovenbird (Furnariidae) that breeds from Tierra del Fuego to the northern Andes. Traditionally, C. fuscus has been considered a single species composed of nine subspecies, but its long and narrow range suggests the possibility of considerable genetic variation among populations. Sequences of two mitochondrial genes revealed three discrete and geographically coherent groups of C. fuscus, occupying the southern, central, and northern Andes. Surprisingly, phylogenetic analyses indicated that these groups were more closely related to other species of Cinclodes than to each other. Relationships of the southern and northern C. fuscus clades to other species of Cinclodes were straightforward; in combination with available information on plumage, behavioral, and vocal variation, this suggests that each should be recognized as a distinct biological species. The central Andean group was paraphyletic with respect to C. oustaleti, and relationships among these taxa and C. olrogi were poorly resolved. We suggest that the central Andean C. fuscus should also be considered a different species, pending new information to clarify species limits in this group. These new phylogenetic data, along with recently developed methods, allowed us to review the biogeography of the genus, confirming southern South America and the central Andes as important areas for the diversification of these birds.

Speciation in Western Scrub-Jays, Haldane’s rule, and genetic clines in secondary contact

BackgroundHaldane’s Rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtDNA between species where females are heterogametic. Therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. We studied introgression of microsatellites and mtDNA across a secondary contact zone between coastal and interior lineages of Western Scrub-Jays (Aphelocoma californica) to test for a signature of Haldane’s Rule: a narrower cline of introgression in mtDNA compared to nuclear markers.ResultsOur initial phylogeographic analysis revealed that there is only one major area of contact between coastal and interior lineages and identified five genetic clusters with strong spatial structuring: Pacific Slope, Interior US, Edwards Plateau (Texas), Northern Mexico, and Southern Mexico. Consistent with predictions from Haldane’s Rule, mtDNA showed a narrower cline than nuclear markers across a transect through the hybrid zone. This result is not being driven by female-biased dispersal because neutral diffusion analysis, which included estimates of sex-specific dispersal rates, also showed less diffusion of mtDNA. Lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control.ConclusionsThis study adds to a growing list of studies that support predictions of Haldane’s Rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtDNA types cannot be ruled out. That Haldane’s Rule appears to be operating in this system suggests a measure of reproductive isolation between the Pacific Slope and interior lineages. Based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: A. californica (Pacific Slope); A. woodhouseii (Interior US plus Edwards Plateau plus Northern Mexico); A. sumichrasti (Southern Mexico). The distinctive Edwards Plateau population in Texas, which was monophyletic in mtDNA except for one individual, should be studied in greater detail given habitat threat.

Interpreting negative results with taxonomic and conservation implications: Another look at the distinctness of coastal California Gnatcatchers

ABSTRACT A recent study by Zink et al. (2013) raises questions about how to interpret negative results in studies when the distinctness of a species of conservation concern is in question. Zink et al. found no evidence for genetic or ecological distinctness of the coastal California Gnatcatcher (Polioptila californica californica). We discuss why the genetic markers they chose were not well suited to the question of distinctness and how they overinterpreted negative results in their genetic and ecological analyses. We reanalyze their genetic data and find evidence that several genetic loci show significant differentiation in the coastal California Gnatcatchers. We provide recommendations for best practices in determining distinctness in phenotype, genetics, and ecology for California Gnatcatchers and other populations of conservation concern.

MITOCHONDRIAL AND NEXT-GENERATION SEQUENCE DATA USED TO INFER PHYLOGENETIC RELATIONSHIPS AND SPECIES LIMITS IN THE CLAPPER/ KING RAIL COMPLEX

Abstract. The family Rallidae is a distinct, species-rich group of birds, many of which are exceptional at long-distance colonization. Six of the ten species in the genus Rallus are distributed in the Americas. Among these, R. longirostris and R. elegans have an interwoven taxonomic history that reflects their weak phenotypic differentiation, the multiplicity of allopatric, morphologically distinct populations, a long zone of secondary contact in the eastern U.S. along which hybridization occurs, and apparent ecological “replacement” of each other in marshes of varying salinity. We used mitochondrial and nuclear gene sequences, the latter generated from next-generation sequencing, to infer phylogenetic relationships in the complex from a sample of 70 individuals collected throughout their distribution. Average levels of mitochondrial and nuclear divergence were relatively low (<2%) both within and between the species. The complex is composed of three distinct biogeographic groups: (1) eastern North America and the Caribbean, (2) South America, and (3) western North America, including Mexico. Our results indicate that R. elegans as currently recognized is paraphyletic, with birds of the highlands of Mexico sister to R. longirostris of California. Rallus elegans of eastern North America and Cuba is sister to R. longirostris from eastern North America and the Caribbean. This paraphyly, along with the reproductive isolation of the ecologically divergent R. elegans and R. longirostris in the eastern United States, supports splitting the complex into five morphologically and genetically distinct species.

USE OF JUVENAL PLUMAGE IN DIAGNOSING SPECIES LIMITS: AN EXAMPLE USING BUNTINGS IN THE GENUS PLECTROPHENAX

Abstract Species limits in the genus Plectrophenax have been difficult to assess. McKay’s Buntings (Plectrophenax hyperboreus) are very similar both morphologically and behaviorally to Snow Buntings (P. nivalis). However, their breeding ranges are allopatric, and there is limited evidence of gene flow. The juvenal plumage of McKay’s Buntings has never been described as different from that of Snow Buntings. Comparison of a series of McKay’s Buntings in juvenal plumage with a series of Snow Buntings in juvenal plumage showed clear differences between the two forms. We used color spectrophotometry to quantify the differences between the two taxa in two areas of the body that appeared to be consistently different, the throat and back. The relative magnitude of the difference between McKay’s and Snow buntings was greater than homologous differences between two subspecies of Snow Bunting (P. n. nivalis and P. n. townsendi). Four out of six variables were significantly different between McKay’s and Snow buntings, whereas none of the variables were significantly different between the two subspecies of Snow Bunting. Bonferroni corrected t-tests of sexual dimorphism and regression of the variables against year of collection showed that these factors were not associated with these differences. Discriminant analysis accurately separated 100% of the specimens into their respective groups. These differences are notable given the evolutionarily conservative nature of juvenal plumage. Our results support continued recognition of McKay’s Bunting as a species and reconfirm the use of juvenal plumage to help determine species limits. Uso del Plumaje Juvenil para Diagnosticar los Límites entre Especies: un Ejemplo en el Género Plectrophenax

EVIDENCE fROM ThE GENETICS Of LANDbIRDS fOR A fORESTED PLEISTOCENE GLACIAL REfUGIUM IN ThE hAIDA GwAII AREA

Abstract. Pleistocene refugia likely contributed to the modern biodiversity of northern areas. Using the mitochondrial DNA cytochrome-b gene, we compared 11 forest-dwelling bird species from Haida Gwaii (formerly the Queen Charlotte Islands) with populations from Alaska, Washington, and other locations in the United States. If Haida Gwaii was an unglaciated refugium, its modern populations should feature a high number of endemic lineages and divergence times that predate the end of the last glacial maximum, ca. 13,000–19,000 years before present (ybp). Furthermore, the genetic diversity of these populations should be higher than that in areas colonized after the glacial retreat. Four of the species examined from Haida Gwaii showed old divergences and a high percentage of endemic lineages: the Northern Saw-whet Owl (Aegolius acadicus), Hairy Woodpecker (Picoides villosus), Stellers Jay (Cyanocitta stelleri), and Pine Grosbeak (Pinícola enucleator); all four have endemic subspecies on these islands. The Pacific Wren (Troglodytes pacificus) and Song Sparrow (Melospiza melodia) showed genetic trends associated with populations in refugia, including high genetic diversity on Haida Gwaii. Estimated divergence dates of these six species were fairly uniform (∼20,000–30,000 ybp), being greatest for the Hairy Woodpecker (>70,000 ybp) and Pine Grosbeak (>120,000 ybp). There was an association between apparent occurrence in a refugium and a sedentary lifehistory strategy and a trend for endemic subspecies (4 of 6) also to show this association. Our findings suggest that the Haida Gwaii area hosted a forested refugium during the cycles of climatic change in the late Pleistocene.

Bridging multilocus species delimitation and DNA barcoding through target enrichment of UCEs: A case study with Mexican highland frogs

Recently, molecular studies have uncovered significant cryptic diversity in the Mexican Highlands, leading to the description of many new endemic species. DNA approaches to this kind of species discovery have included both mitochondrial DNA (mtDNA) sequencing and multilocus genomic methods. While these marker types have often been pitted against one another, there are benefits to deploying them together, as linked mtDNA data can provide the bridge between uncovering lineages through rigorous multilocus genomic analysis and identifying lineages through comparison to existing mtDNA databases. Here, we apply one class of multilocus genomic marker, ultraconserved elements (UCEs), and linked mtDNA data to a species complex of frogs (Sarcohyla bistincta) found in the Mexican Highlands. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species and captured mitochondrial genomes for most samples. Genetic analyses based on both whole loci and SNPs agree there are numerous distinct and divergent lineages within S. bistincta. The SNP-based species tree provides the most conservative estimate of 8 well-supported lineages in three major clades. Having linked mtDNA data allowed us to tap into the large number of mtDNA sequences available on GenBank and identify one of these lineages as an already-described species, S. pentheter. One identified clade (containing 2 of the 8 lineages) was 10% divergent in mtDNA and paraphyletic with other S. bistincta, making this clade a clear candidate for species status. Phylogenies from UCEs and mtDNA mostly agreed in their topologies, but differed in that mtDNA suggested a more complex evolutionary history perhaps influenced by gene flow between some neighboring lineages. Our study demonstrates that the Mexican Highlands still hold substantial undescribed diversity. Combining multilocus genomic data with linked mtDNA data is a useful approach for identifying potential new species and associating them with already described taxa, which is especially important in groups with undescribed subadult phenotypes, where geographic ranges are unclear, or where phenotypes are conserved.Species delimitation has been divided by two approaches: DNA barcoding that focuses on standardization of the genetic marker and multilocus methods that place a premium on genomic coverage and conceptual rigor in modeling the divergence process. Most multilocus methods fail as barcodes, however, because few assay the same marker set and are therefore not readily comparable across studies and databases. We introduce ultraconserved elements (UCEs) as potential genomic barcodes that allow rigorous species delimitation and a bridge to DNA barcoding database to allow both rigorous species delimitation and standardized identification of delimited taxa. UCEs query thousands of loci across the nuclear genome in way that is replicable across broad taxonomic groups (i.e., vertebrates). We apply UCEs to species delimitation in a species complex of frogs found in the Mexican Highlands. Sarcohyla contains 24 described species, many of which are critically endangered and known only from their type localities. Evidence suggests that one broadly distributed member of the genus, S. bistincta, might contain multiple species. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species. We also captured mitochondrial genomes for most samples as off-target bycatch of the UCE enrichment process. Phylogenies from UCEs and mtDNA agreed in many ways, but differed in that mtDNA suggested a more complex evolutionary history perhaps influenced by reticulate processes. The species delimitation method we used identified eight putative species (which we call lineages pending further study) within S. bistincta. Being able to compare linked mtDNA data to existing sequences on Genbank allowed us to identify one of these lineages nested within S. bistincta as an already-described species, S. pentheter. Another lineage nested within S. bistincta is currently being described as a new species (referred to here as sp. nov.). The remaining six lineages fell into two non-sister clades, one containing the core S. bistincta mostly in Oaxaca and Guerrero, and another in the Transvolcanic Belt. The latter clade, at 10% divergence in mtDNA and paraphyletic with respect to other S. bistincta, is a clear candidate for species status. Our study demonstrates not only that UCEs can be used as effective genomic DNA barcodes, but that combining multilocus genomic data with mtDNA is a powerful approach for both delimiting species and identifying them in poorly described and phenotypically challenging groups.