Matthew D. Carling

Haldane's Rule in an Avian System: Using Cline Theory and Divergence Population Genetics to Test for Differential Introgression of Mitochondrial, Autosomal, and Sex-Linked Loci Across the Passerina Bunting Hybrid Zone

Abstract Using cline fitting and divergence population genetics, we tested a prediction of Haldanes rule: autosomal alleles should introgress more than z-linked alleles or mitochondrial haplotypes across the Passerina amoena/Passerina cyanea (Aves: Cardinalidae) hybrid zone. We screened 222 individuals collected along a transect in the Great Plains of North America that spans the contact zone for mitochondrial (two genes), autosomal (four loci) and z-linked (two loci) markers. Maximum-likelihood cline widths estimated from the mitochondrial (223 km) and z-linked (309 km) datasets were significantly narrower on average than the autosomal cline widths (466 km). We also found that mean coalescent-based estimates of introgression were larger for the autosomal loci (0.63 genes/generation, scaled to the mutation rate μ) than for both the mitochondrial (0.27) and z-linked loci (0.59). These patterns are consistent with Haldanes rule, but the among-locus variation also suggests many independently segregating loci are required to investigate introgression patterns across the genome. These results provide the first comprehensive comparison of mitochondrial, sex-linked, and autosomal loci across an avian hybrid zone and add to the body of evidence suggesting that sex chromosomes play an important role in the formation and maintenance of reproductive isolation between closely related species.

Gene Sampling Strategies for Multi-Locus Population Estimates of Genetic Diversity (θ)

Background Theoretical work suggests that data from multiple nuclear loci provide better estimates of population genetic parameters than do single loci, but just how many loci are needed and how much sequence is required from each has been little explored. Methodology/Principle Findings To investigate how much data is required to estimate the population genetic parameter θ (4Neμ) accurately under ideal circumstances, we simulated datasets of DNA sequences under three values of θ per site (0.1, 0.01, 0.001), varying in both the total number of base pairs sequenced per individual and the number of equal-length loci. From these datasets we estimated θ using the maximum likelihood coalescent framework implemented in the computer program Migrate. Our results corroborated the theoretical expectation that increasing the number of loci impacted the accuracy of the estimate more than increasing the sequence length at single loci. However, when the value of θ was low (0.001), the per-locus sequence length was also important for estimating θ accurately, something that has not been emphasized in previous work. Conclusions/Significance Accurate estimation of θ required data from at least 25 independently evolving loci. Beyond this, there was little added benefit in terms of decreasing the squared coefficient of variation of the coalescent estimates relative to the extra effort required to sample more loci.

HISTORICAL DIVERGENCE AND GENE FLOW: COALESCENT ANALYSES OF MITOCHONDRIAL, AUTOSOMAL AND SEX‐LINKED LOCI IN PASSERINA BUNTINGS

Quantifying the role of gene flow during the divergence of closely related species is crucial to understanding the process of speciation. We collected DNA sequence data from 20 loci (one mitochondrial, 13 autosomal, and six sex‐linked) for population samples of Lazuli Buntings (Passerina amoena) and Indigo Buntings (Passerina cyanea) (Aves: Cardinalidae) to test explicitly between a strict allopatric speciation model and a model in which divergence occurred despite postdivergence gene flow. Likelihood ratio tests of coalescent‐based population genetic parameter estimates indicated a strong signal of postdivergence gene flow and a strict allopatric speciation model was rejected. Analyses of partitioned datasets (mitochondrial, autosomal, and sex‐linked) suggest the overall gene flow patterns are driven primarily by autosomal gene flow, as there is no evidence of mitochondrial gene flow and we were unable to reject an allopatric speciation model for the sex‐linked data. This pattern is consistent with either a parapatric divergence model or repeated periods of allopatry with gene flow occurring via secondary contact. These results are consistent with the low fitness of female avian hybrids under Haldanes rule and demonstrate that sex‐linked loci likely are important in the initial generation of reproductive isolation, not just its maintenance.

Speciation in Passerina buntings: introgression patterns of sex‐linked loci identify a candidate gene region for reproductive isolation

Sex‐chromosomes are thought to play an important role in speciation, but few studies of non‐model organisms have investigated the relative influence of multiple sex‐linked markers on reproductive isolation. We collected 222 individuals along a geographical transect spanning the hybrid zone between Passerina amoena and P. cyanea (Aves: Cardinalidae). Using maximum‐likelihood cline fitting methods, we estimated locus‐specific introgression rates for 10 z‐linked markers. Although the cline width estimates ranged from 2.8 to 584 km, eight of 10 loci had cline widths between 224 and 271 km. We also used coalescent‐based estimates of locus‐specific divergence times between P. amoena and P. cyanea to test a recently proposed hypothesis of an inverse relationship between divergence time and cline width but did not find a significant association. The narrow width (2.8 km) of the cline estimated from the VLDLR9 locus indicates strong selection retarding introgression of alleles at this locus across the hybrid zone. Interestingly, a mutation in the very low density lipoprotein receptor (VLDLR) gene, in which VLDLR9 is an intron, is known to reduce the egg‐laying ability of some chickens, suggesting a possible link between this gene region and reproductive isolation between P. amoena and P. cyanea. These results underscore the importance of sampling multiple loci to investigate introgression patterns across a chromosome or genome and support previous findings of the importance of sex‐linked genes in speciation.

Integrating Evolutionary and Functional Tests of Adaptive Hypotheses: A Case Study of Altitudinal Differentiation in Hemoglobin Function in an Andean Sparrow, Zonotrichia capensis

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the α(A)-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the α(D)-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative α(A)-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling.

Spatio‐temporal changes in the genetic structure of the Passerina bunting hybrid zone

Although theoretical models predict that the structure of a hybrid zone can change under a variety of scenarios, only a few empirical studies of hybrid zones have unequivocally demonstrated zone movement. These studies are rare because few data sets exist that include repeated, temporally spaced, samples of the same hybrid zone. We analysed mitochondrial DNA haplotype data from samples separated by 40–45 years from across the Passerina amoena (Lazuli Bunting) and Passerina cyanea (Indigo Bunting) hybrid zone to investigate whether the genetic structure of this zone has changed during that interval. Both cline and generalized linear mixed modelling analyses uncovered a significant narrowing and a substantial westward shift of the Passerina bunting hybrid zone, clearly illustrating hybrid zone movement. The cause of the change may be due to a combination of ecological, demographic and behavioural factors. Our results predict that the width of the hybrid zone will continue to narrow over time, a finding consistent with reinforcement theory.

The influence of sampling design on species tree inference: a new relationship for the New World chickadees (Aves: Poecile).

In this study, we explore the long‐standing issue of how many loci are needed to infer accurate phylogenetic relationships, and whether loci with particular attributes (e.g., parsimony informativeness, variability, gene tree resolution) outperform others. To do so, we use an empirical data set consisting of the seven species of chickadees (Aves: Paridae), an analytically tractable, recently diverged group, and well‐studied ecologically but lacking a nuclear phylogeny. We estimate relationships using 40 nuclear loci and mitochondrial DNA using four coalescent‐based species tree inference methods (BEST, *BEAST, STEM, STELLS). Collectively, our analyses contrast with previous studies and support a sister relationship between the Black‐capped and Carolina Chickadee, two superficially similar species that hybridize along a long zone of contact. Gene flow is a potential source of conflict between nuclear and mitochondrial gene trees, yet we find a significant, albeit low, signal of gene flow. Our results suggest that relatively few loci with high information content may be sufficient for estimating an accurate species tree, but that substantially more loci are necessary for accurate parameter estimation. We provide an empirical reference point for researchers designing sampling protocols with the purpose of inferring phylogenies and population parameters of closely related taxa.

Spatial Patterns of Avian Malaria Prevalence in Zonotrichia capensis on the Western Slope of the Peruvian Andes

Abstract Environmental heterogeneity largely dictates the spatial distributions of parasites and therefore the susceptibility to infection of host populations. We surveyed avian malaria infections in Rufous-collared sparrows (Zonotrichia capensis) across replicated altitudinal and latitudinal transects along the western slope of the Peruvian Andes to assess geographic patterns of prevalence. We found malaria infection prevalence peaked at midelevations along all 3 altitudinal transects (x̄ ≈ 2,733 m), with highest overall prevalence observed in the northern transect. We observed low levels of malarial parasite diversity, with 94% of infected birds carrying a single Haemoproteus (subgenus Parahaemoproteus) strain. The remaining infected birds harbored either a single alternate Haemoproteus or 1 of 2 Plasmodium strains. Our data suggest that temperature and precipitation are the primary drivers of the spatial patterns in avian malaria prevalence along the western slope of the Andes.

Reconstructing the phylogeny of "Buarremon" brush-finches and near relatives (Aves, Emberizidae) from individual gene trees.

Gene trees are often assumed to be equivalent to species trees, but processes such as incomplete lineage sorting can generate incongruence among gene topologies and analyzing multilocus data in concatenated matrices can be prone to systematic errors. Accordingly, a variety of new methods have been developed to estimate species trees using multilocus data sets. Here, we apply some of these methods to reconstruct the phylogeny of Buarremon and near relatives, a group in which phylogenetic analyses of mitochondrial DNA sequences produced results that were inconsistent with relationships implied by a taxonomy based on variation in external phenotype. Gene genealogies obtained for seven loci (one mitochondrial, six nuclear) were varied, with some supporting and some rejecting the monophyly of Buarremon. Overall, our species-tree analyses tended to support a monophyletic Buarremon, but due to lack of congruence between methodologies, resolution of the phylogeny of this group remains uncertain. More generally, our study indicates that the number of individuals sampled can have an important effect on phylogenetic reconstruction, that the use of seven markers does not guarantee obtaining a strongly-supported species tree, and that methods for species-tree reconstruction can produce different results using the same data; these are important considerations for researchers using these new phylogenetic approaches in other systems.

Willet be one species or two? A genomic view of the evolutionary history of Tringa semipalmata

ABSTRACT The Willet (Tringa semipalmata; Scolopacidae) is composed of 2 morphologically and vocally distinct subspecies with broadly disjunct breeding distributions in North America. Nominate T. s. semipalmata breeds in coastal salt and brackish marshes along the Atlantic and Gulf coasts of eastern North America and the West Indies, and T. s. inornata breeds in wet grasslands and prairies in the northwestern interior of North America. To assess divergence and test for hybridization between the 2 subspecies, we sampled breeding and wintering populations and collected morphological data, mitochondrial DNA sequences from the ND2 locus, and nuclear DNA sequences from genomic libraries enriched for ultraconserved elements (UCEs). Mitochondrial haplotypes were reciprocally monophyletic between the 2 subspecies and indicated divergence approximately 700,000 yr ago. The UCE dataset included 4,635 loci containing 19,322 single nucleotide polymorphisms (SNPs), and, based on these data, individuals clustered by subspecies with no evidence of admixture between them and no substructure within subspecies. We identified 42 nuclear loci that contained SNPs fixed for alternate alleles between the 2 subspecies. Of the 42 loci with fixed differences, a statistically disproportional 17 were Z-linked, indicating a role for sexual selection in the divergence of the 2 subspecies. Genetic, morphological, ecological, and behavioral differences suggest that the 2 Willet subspecies may merit treatment as separate species. Further studies are needed to determine the presence of pre- or post-mating reproductive isolation.