Cryptic Speciation in the Warbling Vireo (Vireo Gilvus) Scott F

AmericanOrnithology.org

Volume 138, 2021, pp. 1–16 DOI: 10.1093/ornithology/ukaa071 RESEARCH ARTICLE Cryptic speciation in the Warbling Vireo (Vireo gilvus) Scott F. Lovell,a,*, M. Ross Lein, and Sean M. Rogers

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada a Current address: Department of Biology, St. Mary’s University, Calgary, Alberta, Canada Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 * Corresponding author: [email protected] Submission Date: April 20, 2020; Editorial Acceptance Date: October 8, 2020; Published January 20, 2021

ABSTRACT Eastern (Vireo gilvus gilvus) and western (V. g. swainsoni) forms of the Warbling Vireo have essentially allopatric breeding ranges across north-central North America, but come into contact in central Alberta, Canada. In 1986, Jon Barlow presented preliminary morphological and song evidence suggesting that the Warbling Vireo complex might comprise more than one valid species. However, to date, Barlow’s suggestion is supported by only limited DNA evidence, demonstration of molt and migration differences between the taxa, and anecdotal accounts of differences in song, morphology, plumage, and ecology. We analyzed variation in both mitochondrial and nuclear DNA in birds from Alberta and surrounding areas to determine the levels of genetic differentiation and hybridization occurring in the contact zone, and whether the two taxa warrant recognition as separate biological species. Our analyses reveal that Warbling Vireos in Alberta and the surrounding areas are separated into two well- defined, genetically differentiated, and monophyletic clades corresponding to previously recognized taxonomic groups. The two taxa come into contact in a narrow (~85 km) zone in Barrhead County, northwest of Edmonton, Alberta. They show evidence of limited hybridization. The distinct genetic differences are maintained in the contact zone, where individuals of the two taxa may occupy neighboring territories. Differences in spring arrival dates, molt schedules, and migration routes indicate that a migratory divide may play an important role in reproductive isolation. We suggest that the two taxa are distinct cryptic species: an eastern form, Vireo gilvus, and a western form, Vireo swainsoni. Keywords: hybridization, Pleistocene, reproductive isolation, speciation, Vireo gilvus, Warbling Vireo

LAY SUMMARY • Two subspecies of Warbling Vireo meet in a narrow contact zone in central Alberta, Canada. • Analysis of mitochondrial and nuclear DNA found significant genetic differentiation between the two taxa, suggesting a divergence in the early Pleistocene. • Hybrids between the two taxa were uncommon in the contact zone. • These findings suggest that the two taxa are reproductively isolated and, therefore, should be recognized as “good” biological species.

Especiación críptica en Vireo gilvus RESUMEN Vireo gilvus presenta dos formas, V. g. gilvus y V. g. swainsoni, que tienen esencialmente rangos reproductivos alopátricos a través del norte y centro de Norteamérica, pero que entran en contacto en el centro de Alberta, Canadá. En 1986, Jon Barlow presentó evidencia preliminar morfológica y de canto sugiriendo que el complejo de V. gilvus podría incluir más de una especie válida. Sin embargo, a la fecha la sugerencia de Barlow está apoyada solo por evidencia limitada de ADN, la demostración de diferencias de muda y migración entre los taxones, y relatos anecdóticos de diferencias en canto, morfología, plumaje y ecología. Analizamos variaciones en ADN mitocondrial y nuclear en las aves de Alberta y las áreas circundantes para determinar los niveles de diferenciación genética e hibridación que se presentan en la zona de contacto, y si los dos taxones justifican el reconocimiento como especies biológicas separadas. Nuestros análisis revelan que V. gilvus en Alberta y las áreas circundantes está separado en dos clados monofiléticos bien definidos, genéticamente diferenciados, correspondiéndose con los grupos taxonómicos previamente reconocidos. Los dos taxones se ponen en contacto en una zona estrecha (~85 km) en el Condado de Barrhead, noroeste de Edmonton, Alberta. Ellos muestran evidencia de hibridación limitada. Las diferencias genéticas distintivas se mantienen en la zona de contacto, donde los individuos de los dos taxones pueden ocupar territorios vecinos. Las diferencias en las fechas de arribo en primavera, en

los esquemas de muda y en las rutas migratorias indican que una división migratoria puede jugar un rol importante en el aislamiento reproductivo. Sugerimos que los dos taxones son dos especies crípticas distintas: una forma este, Vireo gilvus, y una forma oeste, Vireo swainsoni. Palabras clave: aislamiento reproductivo, especiación, hibridación, Pleistoceno, Vireo gilvus

INTRODUCTION 1989). They can persist through evolutionary time (e.g., Arnold 1997, Baxter et al. 1997), or rapidly result in the The climatic cycles of the Pleistocene Epoch (2.6–11 kya) collapse of one or both parental species (e.g., Whitmore had profound influences on the avian biota of temperate 1983, Echelle and Connor 1989). Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 North America and Europe (Rand 1948, Johnson and While the scenarios of taxa hybridizing in secondary Cicero 2004, Weir and Schluter 2004, Saitoh et al. 2010). contact zones are well documented in the literature In particular, the ranges of species inhabiting the boreal (Barton and Hewitt 1985, Abbott et al. 2013), the evolu- and montane forests at higher latitudes shifted in location, tionary processes involved, the maintenance of the hybrid and contracted and expanded in response to the repeated zones, the potential outcomes, and the importance of these advance and retreat of continental glaciers. Significantly, zones are still the subjects of much debate (Barton 2013, glacial advances split some wide-ranging species into two Fraïsse et al. 2014, Gompert et al. 2017). or more disjunct populations isolated in separate refugia Hybrid zones often exhibit clines for an array of south of the ice sheets (Hubbard 1973, Weir and Schluter characters that are similar in their location (concordant) 2004, Lovette 2005). Such populations subsequently had and width (coincident), and the transition from one pa- independent evolutionary trajectories while in allopatry, rental taxon to the other is usually abrupt, occurring over potentially developing significant genetic and phenotypic a short distance (Moore 1977). Cline theory (Haldane differences in response to varying selective pressures in 1948, Slatkin 1973, Endler 1977, Barton and Gale 1993) their novel environments and lack of gene flow among provides a conceptual and mathematical framework to populations in different refugia (Pielou 1991, Avise 2000, understand the maintenance of clines and the spread of Weir and Schluter 2004, Toews and Irwin 2008, Irwin traits or genes across hybrid zones (Endler 1977, Barton et al. 2009). 1979, Barton and Hewitt 1985, Barton and Gale 1993). Today, we observe the outcome of range expansions Three main hypotheses have been proposed to explain following the most recent glacial recession (~11,000 yr the maintenance of hybrid zones and the resulting clines. ago). Formerly isolated taxa in secondary contact dem- The neutral diffusion hypothesis (Endler 1977, Barton onstrate various evolutionary outcomes ranging from and Gale 1993) predicts that, in the absence of selection free interbreeding to complete reproductive isolation or a barrier to reproduction, cline width will be propor- (Avise and Walker 1998, Johnson and Cicero 2004, Weir tional to the product of the root-mean-square (RMS) and Schluter 2004, Abbott et al. 2013). These cases pro- dispersal distance per generation and the square root of vide opportunities to examine the processes of evolution, the number of generations since secondary contact. The speciation, selection, and genetic interactions between bounded hybrid superiority hypothesis (Moore 1977, populations (Mayr 1963, Mallet 1995, Coyne and Orr 2004, Moore and Price 1993) states that hybrid zones occur in Price 2008, Abbott et al. 2013). However, uncertainty re- ecotonal areas and are maintained by selection favoring mains regarding the importance of the most recent glacial hybrid individuals and against parental individuals in cycles in generating pairs of divergent populations, relative the hybrid zone. The width of the hybrid zone under this to that of earlier cycles (e.g., Klicka and Zink 1997, Johnson hypothesis is dictated by the width of the ecotone. The and Cicero 2004, Weir and Schluter 2004, Lovette 2005). tension zone or dynamic-equilibrium hypothesis (Barton The impact of the Pleistocene glacial cycles is par- and Hewitt 1985) assumes that hybrids have reduced fit- ticularly noticeable along the Front Range of the Rocky ness, and that a stable hybrid zone persists because the Mountains in Alberta, where the boreal forest meets the reduced fitness of hybrids is balanced by constant move- montane coniferous forest. Numerous pairs of avian taxa ment of “pure” parental taxa into the zone. Therefore, a have secondary contact zones between montane and bo- tension zone exists as a dynamic equilibrium between real representatives in this region (e.g., Hubbard 1969, immigration of parental forms into the hybrid zone and Toews and Irwin 2008, Irwin et al. 2009, Natola and Burg natural selection against hybrids. Tension zones are 2018). Secondary contact frequently results in hybrid zones characterized by the steepness of clines reflecting the when genetically distinct populations meet and inter- intensity of selection against hybrids, clines of multiple breed, producing offspring of mixed ancestry (Barton and characters being concordant and coincident, and signif- Hewitt 1989, Hewitt 1989). These zones can vary from a icant linkage disequilibria at the center of the tension few meters to several hundred kilometers in width (Hewitt zone. Most hybrid zones are believed to be maintained as

tension zones (Barton and Hewitt 1985, 1989, Barton and Gale 1993, Barton 2001). The Warbling Vireo complex comprises 4 (American Ornithologists’ Union 1957), 5 (Phillips 1991), or 6 (Blake 1968) subspecific taxa (excluding taxa currently assigned to Vireo leucophrys). It has been suggested that the complex represents a superspecies and that the gilvus and swainsoni groups represent distinct species (Sibley and Monroe 1990, American Ornithologists’ Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 Union 1998). The gilvus group (including a single subspecies, V. g. gilvus) breeds in deciduous forests across eastern North America from central Alberta south to southeastern Colorado and east to Nova Scotia and south to Virginia and Mississippi. The swainsoni group (including V. g. swainsoni, V. g. victoriae, and V. g. leucopolius [American Ornithologists’ Union 1957]) breeds in deciduous and mixed forests across western North America from southeastern Alaska southeast to central Alberta and south to California, New Mexico, and the Sierra Madre Occidental of Mexico. These two groups meet in a con- FIGURE 1. Locations of Warbling Vireo populations in Alberta tact zone in central Alberta. Limited published informa- and adjacent regions sampled in this study. Inset map shows the tion and anecdotal evidence indicate that the two forms position of the main map in North America. differ in a number of aspects. Vireo g. gilvus is larger than V. g. swainsoni, greener on the back and more yellowish METHODS on the flanks (Semenchuk 1992, Lovell 2010). Limited analyses of mitochondrial DNA show differences of 3.0– Tissue Sampling 3.5% between the taxa (Murray et al. 1994, Hebert et al. We obtained 213 tissue samples (191 used for mtDNA 2004). Songs of the two forms are recognizably different to analyses and 145 used for nuclear DNA analyses) from the ear, with swainsoni having “more breaks and peaks in field-collected birds and museum specimens collected its song, giving it a choppy character” (W. B. McGillivray during the breeding season (20 May–15 July). Most in Semenchuk 1992) or “burrier or ‘less musical’ overall samples were from Alberta, but additional samples were than the relatively clear and ‘musical’ song of [gilvus]” from British Columbia, Saskatchewan, Montana, and (Floyd 2014). In southwestern Alberta swainsoni birds ar- Ontario (Figure 1, Supplementary Material Table S1). rive on their breeding grounds ~2 weeks earlier than do We combined small samples from adjacent locations gilvus birds (J. B. McGillivray in Semenchuk 1992; Lovell into 23 populations (Table 1) of sufficient size for anal- 2010). This probably reflects the differences in molt ysis. Collecting during 2005–2007 concentrated along schedules and migration routes of the two forms (Voelker a transect of 10 populations in central Alberta spanning and Rohwer 1998). The two taxa also differ in their re- the purported contact zone between gilvus and swainsoni sponse to cowbird parasitism. Sealy et al. (2000) provide (Populations 1–10 in Figure 1). Samples of liver, heart, and evidence that gilvus is an egg rejecter while swainsoni pectoral tissue were preserved in either dry ice (CO2) or accepts parasite eggs. liquid nitrogen (N2) in the field and were subsequently To address the suggestion that there may be more than stored in an ultra-cold freezer (–80°C). one species of Warbling Vireo, we examined genetic variation within and between the two forms in a contact Mitochondrial DNA Sequencing zone in central Alberta. Our study addressed three main We isolated genomic DNA from 191 tissue samples questions. First, do the observed patterns of variation (Supplementary Material Table S1) using an AutoGenprep in mtDNA and nuclear DNA correspond to currently 965CE DNA extractor and the mouse tail (tissue) DNA recognized taxonomic limits? Second, to what extent protocol (AutoGen, Holliston, Massachusetts, USA). do the two taxa exhibit genetic introgression in the con- Genomic DNA extracts were quantified using a NanoDrop tact zone and is there evidence of reproductive isolation? (ND-1000) spectrophotometer and diluted to 50 ng μL–1 Third, are differences between the two taxa consistent working stock solutions. We used the polymerase chain re- with a late Pleistocene divergence, or did this separation action (PCR) to amplify a 1,078-base pair (bp) portion of the occur at an earlier time? cytochrome b (Cyt b) gene using the two external primer

TABLE 1. Summary of populations of Warbling Vireos sampled in this study. Populations 1–10 comprise the transect across the contact zone. Population numbers correspond to maps in Figures 1 and 3. Population Transect mtDNA % mtDNA % Hybrid index b AFLP (Population number) Latitude Longitude distance (km) Ancestry a gilvus swainsoni mtDNA (n) (mean ± SD) (n) NW Alberta (1) 56.26° –117.61° 0 swainsonic 11.1 88.9 9 0.01 ± 0.01 8 Swan Hills (2) 55.04° –115.04° 211 swainsonic 6.7 93.3 15 0.02 ± 0.02 16 Freeman River (3) 54.42° –115.03° 262 swainsoni 0 100 9 0.10 ± 0.27 8 NW Barrhead (4) 54.19° –114.87° 289 Mixed 60 40 5 0.33 ± 0.46 8 Peanut Lake (5) 54.04° –114.34° 323 Mixed 73.3 26.7 15 0.82 ± 0.35 15 SE Barrhead (6) 53.95° –114.17° 338 Mixed 83.3 16.7 6 0.67 ± 0.44 8 Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 Cooking Lake (7) 53.45° –112.77° 440 gilvus 100 0 9 0.99 ± 0.01 8 Beaverhill Lake (8) 53.38° –112.53° 457 gilvus c 83.3 16.7 6 0.97 ± 0.02 3 Camrose (9) 53.02° –113.17° 444 gilvus 100 0 8 0.99 ± 0.01 7 Pine Lake (10) 51.93° –114.36° 527 gilvus 100 0 10 0.95 ± 0.14 10 Bellis (11) 54.15° –112.15° n/a gilvusc 75 25 4 n/a 0 Skaro Lake(12) 53.93° –112.72° n/a gilvus 100 0 4 n/a 0 Alder Flats(13) 52.93° –115.01° n/a swainsoni 0 100 9 0.07 ± 0.15 6 Rocky Mtn House(14) 52.37° –114.92° n/a swainsonic 25 75 4 n/a 0 Forestry Trunk Rd (15) 51.92° –115.33° n/a swainsoni 0 100 9 0.01 ± 0.00 12 Drumheller(16) 51.41° –112.64° n/a gilvus 100 0 1 n/a 0 Kananaskis (17) 51.31° –115.02° n/a swainsoni 0 100 11 0.01 ± 0.01 16 Suffield (18) 50.20° –111.17° n/a gilvus 100 0 3 0.95 ± 0.09 3 Cypress Hills (19) 49.57° –110.13° n/a swainsoni 0 100 5 n/a 0 Castle River (20) 49.55° –114.37° n/a swainsoni 0 100 28 0.01 ± 0.00 16 Saskatchewan (21) 52.78° –108.28° n/a gilvus 100 0 2 n/a 0 British Columbia (22) 49.30° –115.12° n/a swainsonic 10 90 10 n/a 0 Montana (23) 48.41° –114.34° n/a swainsoni 0 100 8 0.01 1 a Ancestry based on dominant mtDNA phenotype in population. b Hybrid index is the probability that an individual was assigned to the gilvus cluster by STRUCTURE.. c One individual had an mtDNA haplotype characteristic of the other taxon. pairs: L1450 (5′-GCCTACCTAGGARCATTYGCCC-3′) We performed cycle sequencing reactions on 12 μL and H15944 (5′-TTCTAGTACWCCTGCRATRGGG-3′), aliquots comprised of 0.5 μL of BigDye (Applied and two internal primer pairs: L15281 Biosystems [ABI], Foster City, California, USA), 2 μL of (5′-CYGCYTTYGTAGGRTACGTCC-3′) and H15385 BigDye Buffer (5X), 3 μL of primer, 0.5 μL of distilled ′ ′ (5 -TACTGAGAATCCKCCTCAGGC-3 ). These primers H2O, and 6 μL (20–40 ng) of the purified PCR product. correspond approximately to positions 1450, 15944, 15281, Sequencing reactions were purified with Sephadex and and 15385, respectively, of the chicken mtDNA genome run on an ABI 3730xl automated sequencer. The resulting (Desjardins and Morais 1990). DNA fragments were assembled from forward and reverse We carried out PCR amplification in a 10 μL reac- strands using Sequencher 4.8 (Gene Codes Corporation, tion volume containing ~50 ng of genomic DNA, 10 μM Ann Arbor, Michigan, USA). All sequences were translated of each external primer, and 5 μL of 2X Diamond Mix and compared to the Gallus Cyt b sequence (Desjardins (contains buffer, deoxyribonucleotide triphosphates, and Morais 1990) to confirm the correct reading frame

MgCL2, and Taq polymerase) (Bioline, Taunton, and to check for the presence of stop codons. This resulted Massachusetts, USA). We used an MJ Research PTC-200 in a sequence of 1,078 bp for each individual. DNA Engine Thermal Cycler (Waltham, Massachusetts, USA) to perform PCR amplifications under the fol- AFLP Genotypes lowing conditions: 95°C for 3 min followed by 25 cycles To estimate genetic differentiation and to assess hybridi- each of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min zation between the two taxa across the contact zone, we and concluding with an extension period of 10 min isolated nuclear DNA from tissues of 145 Warbling Vireos at 72°C. We purified PCR products using Exosap-IT from 16 of the 23 populations used in the mtDNA anal- (USB Corporation, Cleveland, Ohio, USA), with 1.5 μL ysis (Table 1) using standard phenol–chloroform methods of diluted Exosap-IT (1:9 dilution with distilled H2O) (Sambrook and Russell 2001). We used a modified AFLP added to 10 μL of PCR product, then placed on a thermal Plant Mapping Kit (Applied Biosystems) protocol. Selective cycler at 37°C for 30 min and 80°C for 15 min. amplification of each DNA sample was done with three

primer combinations: EcoRIACT-MseICAA, EcoRIAGG- et al. 1985). We assumed a generation time of 1 yr (Gardali MseICTA, and EcoRIAGC-MseICTT. We assigned and Ballard 2000) and used mutation rates of 1.6% per myr individuals randomly to PCR plate wells and included neg- (Fleischer et al. 1998) and 2.1% per myr (Weir and Schluter ative and positive controls on each plate. Electrophoresis 2008). consisted of pooling PCR products with an internal size We tested whether the mtDNA data were consistent standard (LIZ 500 bp, Applied Biosystems) and loading with a hypothesis of neutral evolution under a constant onto the Applied Biosystems 3500xl Automated Sequencer. population size using ARLEQUIN 3.5 (Excoffier and Allelic sizes (in base pairs) in resulting electropherograms Lischer 2010) to calculate Tajima’s (1989) D statistic and were determined by reference to the internal sizing standard Fu’s (1997) Fs statistic. Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 in the software GENEMAPPER v3.7 (Applied Biosystems), We examined the geographic structure of the genetic with background filtering detection threshold of 100 fluo- variation using an analysis of molecular variance (AMOVA, rescence units to avoid artifacts. AFLP peaks were scored Excoffier et al. 1992) implemented in ARLEQUIN. In in 1 bp bins as dominant loci (i.e., present = AA or Aa, ab- the AMOVA, Φ-statistics (analogous to Wright’s [1949] sent = aa) following standard AFLP practices for increased F-statistic) were used to examine the contribution of mo- reproducibility (e.g., Rogers et al. 2001, Kingston et al. lecular variance at three levels: (1) between the two clades 2012). (defined by results of the phylogenetic analysis and the minimum spanning network) (Φct); (2) among populations Mitochondrial DNA Data Analysis within each taxon (assignment of populations into subspe- We constructed multiple alignments using CLUSTALW cific categories based on the predominant mtDNA clade at (Higgins and Sharp 1988) and imported the aligned each population) (Φsc); and (3) among individuals within sequences into PAUP* 4.0 (Swofford 2003). We used un- the population (Φst). The null distribution was generated corrected distances to produce a neighbor-joining (NJ) by calculating the variance parameters on each of 10,000 tree, and assessed node confidence using 1,000 NJ boot- resampled replicate data sets. strap replicates. We used MODELTEST 3.07 (Posada and We tested for association between matrices of genetic

Crandall 1998), with no outgroup included, to determine distances (pairwise FST values from ARLEQUIN) and ge- the best-fit maximum-likelihood (ML) model of sequence ographic distances among sample populations (pairwise evolution. ML and maximum parsimony (MP) phyloge- geographic distances from Geographic Distance Matrix netic analyses using PAUP* identified major clades and Generator (http://biodiversityinformatics.amnh.org/ relationships among haplotypes. MP analysis used heu- open_source/gdmg) using a Mantel test (Mantel 1967). ristic search with 10 random addition repetitions and Separate Mantel tests were run with 30,000 iterations tree-bisection-reconnection branch swapping. We rooted first for all populations, and then for only contact zone Warbling Vireo trees using a V. leucophrys sequence populations, using Isolation by Distance (IBD) 1.52 (GenBank AF081960) as an outgroup. This taxon is closely (Bohonak 2002). related to those of interest (Cicero and Johnson 1998). Because of the large number of haplotypes present in the AFLP Data Analysis data set, we used a parsimony ratchet (PAUPRat) (Sikes AFLP genotyping characterized 796 polymorphic AFLP and Lewis 2001) to aid in searching MP tree space. We markers. None of these were diagnostic, exhibiting dom- constructed minimum spanning networks of haplotype inant marker frequency differentials between the two relationships using PopART 1.7 (Leigh and Bryant 2015), taxa ranging from 0.1 to 90.6%. To assess the possibility using the Templeton, Crandall, and Sing method of con- of admixture between taxa in the contact zone, we set a struction (Clement et al. 2000) which estimates genealogies minimum dominant marker frequency differential (FD) using 95% statistical parsimony probabilities as defined by threshold of 20% between parental taxa for loci in our Templeton et al. (1992). We constructed networks for all data set, reducing our data set to 67 informative AFLP haplotypes, and for a subset of haplotypes that were found loci. We calculated assignment probabilities of each indi- in 2 or more individuals. vidual in the data set to a number of clusters (K) ranging

We calculated the corrected genetic distance (dA) from 1 to 5 using STRUCTURE 2.3.4 (Pritchard et al. between clades using the number of nucleotide changes 2000). Each run consisted of a 100,000-step burn-in with between haplotypes, taking into account intragroup poly- 500,000 additional cycles, and the parameter set ran for 10 morphism. This analysis used MEGA 4.0 (Tamura et al. iterations for each value of K. To deal with a statistical ar-

2007), where dA = dxy – ((dx + dy)/2) in which dx and dy tifact produced by STRUCTURE that results in estimates are within-group distances for each clade, respectively, and of higher likelihoods and variance with larger values of K dxy is the uncorrected average genetic distance (calculated (which can make determining the true number of clusters in MEGA) between all samples within each clade (Wilson in a data set problematic), we identified the K value

with the largest ∆K using STRUCTURE HARVESTER of the exponential decay curve (tail) parameters [distance (Evanno et al. 2005). We used the probability (Q value) from the cline center to the tail (δ) and tail slope (τ)]. These that STRUCTURE assigned an individual to the gilvus models included neither tail, right tail only, left tail only, cluster as a hybrid index (HI). We then used these HI mirrored tails, or both tails separately. Finally, the models scores to assign individuals to one of three categories: varied as to whether they did or did not estimate allele pure swainsoni (Q ≤ 0.1), pure gilvus (Q ≥ 0.9), or hy- frequencies at the cline ends (pmin and pmax). We compared brid ancestry (0.1 > Q < 0.9) (Baldassarre et al. 2014). the 15 models using AIC corrected for small sample sizes

To assess whether the threshold of frequency differen- (AICc), selecting the model with the lowest AICc as the tial impacted estimates of admixture, in particular lower best-fitting model (Derryberry et al. 2014). Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 estimates that include more minor allele frequencies into To determine whether clines for individual AFLP the data set, we varied FD from 5% (273 loci) to 30% (45 markers and the mtDNA cline were concordant with the loci) and recalculated assignment probabilities and HI HI cline, we compared the 2 log-likelihood unit support assignment. limits for the cline center of the best-fitting model and that We generated estimates of genetic differentiation at all of the HI cline. In addition, we compared the difference

67 loci using two methods. First, AFLP-SURV (Vekemans in AICc scores between the chosen model and the same

2002) was used to calculate FST, the fraction of variance in model with the cline center constrained to that of the HI allele frequencies that is explained by the difference be- cline (Baldassarre et al. 2014, Wielstra et al. 2017). tween the two taxa. To avoid a potentially-biased estimation of allele frequencies from dominant markers (Lynch Testing Pleistocene Hypotheses and Milligan 1994, Zhivotovsky 1999), allele frequencies Estimations of effective population sizes (Ne), for both were estimated using a Bayesian approach (with uniform gilvus and swainsoni, were required to test null hypotheses prior distribution of allele frequencies) assuming Hardy- regarding Pleistocene divergence of the taxa. We

Weinberg equilibrium within each taxon. AFLP-SURV estimated Ne using a coalescent approach implemented in used these allele frequencies to calculate an overall FST. MIGRATE-N 3.4.2 (Beerli 2006) to calculate the param-

To facilitate comparison to previous studies, we also eter θ, where θ = 2Neµ. We used a conservative mutation –8 calculated marker-based FST using ARLEQUIN (Excoffier rate (µ) of 1.00 × 10 per site per year for the Cyt b region et al. 2005). Because AFLP markers are dominant markers (Weir and Schluter 2008). Search parameters in MIGRATE this is not directly comparable to FST values calculated consisted of 10 short chains of 100,000 steps followed by from co-dominant markers such as microsatellites. While two long chains of 20,000,000 steps. Chains were sampled ARLEQUIN was not designed to use dominant makers, it every 100 steps following a burn-in of 20,000 steps, and is often used for this purpose (Svensson et al. 2004, Bensch default settings were used for the initial estimation of θ. and Åkesson 2005, Helbig et al. 2005, Irwin et al. 2005, MIGRATE was run twice to ensure convergence using dif-

Toews and Irwin 2008). This marker-based FST was used ferent random seeds as starting points. to measure pairwise differentiation between gilvus and We tested 6 a priori hypotheses in relation to the number swainsoni. of refugia and divergence times for Warbling Vireos during We used principal components analysis (PCA) to sum- the Pleistocene (Pielou 1991, Newton 2003, Carstens et al. marize patterns of variation in the AFLP presence/absence 2005, Steele and Storfer 2006). First, we tested a single re- matrix for a subset of the original 145 individuals for which fugium hypothesis using the Ne from the point estimate all markers were amplified (n = 80) (Irwin et al. 2005, Toews of gilvus. Second, we tested a single refugium hypothesis and Irwin 2008), using R (R Core Team 2018). using the Ne from the point estimate of swainsoni. The re- maining 4 hypotheses predict a two-refugia situation, but Cline Analysis differ in the estimated time of divergence: at the Pliocene- We fit frequencies for the proportion of gilvus mtDNA Pleistocene boundary (2.6 mya); in early-Pleistocene (1.8 haplotypes, the mean HI, and mean frequency of each AFLP mya); at mid-Pleistocene (0.8 mya); and at the end of the marker in each transect population to a series of geographic Pleistocene (11 kya). cline models (Szymura and Barton 1986, Gay et al. 2008) We used MESQUITE 2.75 (Maddison and Maddison using the Metropolis-Hastings Markov Chain Monte Carlo 2004) to conduct coalescent simulations of the mtDNA algorithm employed in the R package HZAR (Derryberry data set for each of the six hypotheses for potential et al. 2014). For each analysis, we ran 15 separate models Pleistocene refugia. Two types of coalescent simulations that varied in the number of cline shape parameters were performed. In the first, 1,000 coalescent genealogies estimated. All models estimated cline center (the distance were generated under each historical scenario and the from sampling location 1, c) and width (maximum slope−1, distributions of the minimum number of sorting events w), but could estimate additionally different combinations required to explain the population subdivision (S) (Slatkin

phylogeny with one (gilvus) or two (swainsoni) common haplotypes, and with other haplotypes differing from these common haplotypes by one to three substitutions (Figure 2). The minimum spanning network for all 78 haplotypes shows the same pattern but with more minor branching and reticulations within the two clades (Supplementary Material Figure S2). Average raw sequence divergence between gilvus and FIGURE 2. Minimum-spanning network of unique haplotypes with a frequency >1 for the Warbling Vireo complex. The sizes of swainsoni clades was 4.0% (3.6% when corrected for Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 the circles correspond to the relative frequency of each haplotype intra-population polymorphism). These values corre- and hash marks represent single nucleotide substitutions. The spond to uncorrected divergence times of 2,500,000 yr ago small black circles represent intermediate haplotypes that (2,250,000 corrected) using mutation rates of 1.6% Myr–1 were absent in the dataset. The two clades are separated by 35 (Fleischer et al. 1998) and 1,900,000 yr ago (1,714,000 diagnostic substitutions. corrected) using 2.1% Myr–1 (Weir and Schluter 2008). The average raw sequence divergence between samples within each clade was low, 0.30% within the gilvus clade and 0.40% and Maddison 1989) were recorded. In the second type, within the swainsoni clade. the S value from our ML genealogy (ingroup only) was The hypothesis of neutral evolution of population size compared with the S values generated for each hypothesis was rejected. Tajimas’s test of selective neutrality was sig- to evaluate model fit in MESQUITE. The same model of nificant for gilvus (D = –2.29, P = 0.0005 and swainsoni evolution used to calculate our ML tree topology was used (D = –2.12, P = 0.002). Fu’s test also rejected selective for the coalescent simulations. neutrality in both cases (gilvus: Fs = –26.84, P = 0.0001;

swainsoni: Fs = –26.61, P = 0.0001). RESULTS A pairwise FST value of 0.93 (P < 0.001) was estimated between gilvus and swainsoni clades, indicating significant ge- Phylogenetic Analysis of mtDNA netic differentiation. The AMOVA confirmed that 94% of We found 78 unique haplotype sequences among the the variance could be explained by the deep divergence be- 191 individuals (Supplementary Material Table S1), tween these two Warbling Vireo clades, with 6% of variance with 53 confined to single individuals. The unique explained by within-clade variation. There was a significant haplotypes produced 104 variable sites distributed het- association between genetic distance (FST values) and geo- erogeneously along the sequence, with 61 sites being graphic distance among all populations (Mantel test; r = 0.15; parsimony-informative. MODELTEST determined that P = 0.02). Additionally, there was significant association be- the best-fit model was the Kimura (1981) model with tween FST values for the transect populations and geographic unequal frequencies with an invariant site parameter distance along the transect (Mantel test; r = 0.47; P = 0.002). (K81uf+I; substitution rate matrix R[A-C] = 1.00, R[A- Of the 24 populations sampled, nine contained both G] = 15.90, R[A-T] = 0.18, R[C-G] = 0.18, R[C-T] = 15.90, eastern and western haplotypes (Figure 3A). Six of these R[G-T] = 1.00; the proportion of invariant sites = 0.83, populations [NW Alberta (1), Swan Hills (2), NW Barrhead A = 0.30, C = 0.34, G = 0.13, T = 0.22). This model of (4), Peanut Lake (5), SE Barrhead (6), and Beaverhill Lake sequence evolution produced a single most-likely ML (8)] were included in the contact zone transect and are tree (–ln L = 2608.51). MP analysis using the parsimony discussed below. The other three mixed populations [BC ratchet produced 10 equally parsimonious trees that were (22), Rocky Mountain House (16), and Bellis (13)] are out- 186 steps in length. The MP, ML, and rooted NJ trees in- side the contact zone and each contained only a single indicated two well-supported clades. The trees produced dividual with the less frequent haplotype at the location. from the three tree-building methods differed from one another only in the placement of haplotypes within the Analysis of AFLP Markers major clades. Therefore, we show only the NJ tree from the We generated AFLP profiles for a total of 145 samples MODELTEST results (Supplementary Material Figure S1). (Supplementary Material Table S1). STRUCTURE anal- The two major clades had strong bootstrap support ysis estimated that the most likely number of populations (99%), but the branches leading to haplotypes within each involved was 2 (ln P(X|K) = –5975.41, ΔK = 2 (687.38)). clade lacked structure and had minimal bootstrap sup- Q values indicated that 136 of 145 individuals were ei- port (Supplementary Material Figure S1). The minimum- ther pure swainsoni (Q ≤ 0.1) or pure gilvus (Q ≥ 0.9) spanning network of haplotypes with a frequency of >1 (Supplementary Material Table S1). Nine individuals had Q shows that the two main clades are separated by 35 di- values suggesting mixed ancestry (0.1 > Q < 0.9) (Table 2). agnostic mutations (Figure 2). Each clade has a star-like Additionally, four birds demonstrated a mismatch between

Ornithology 138:1–16 © 2021 American Ornithological Society 8 Warbling Vireo speciation S. F. Lovell, M. R. Lein, and S. M. Rogers Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021

FIGURE 3. (A) Relative frequencies of individuals with swainsoni (blue) and gilvus (red) mtDNA haplotypes in populations sampled in Alberta and adjacent regions. (B) Relative frequencies (based on Q values from STRUCTURE analysis of AFLP profiles) of swainsoni individuals (Q <0.1, blue), gilvus individuals (Q >0.9, red), and “hybrids” (0.1> Q <0.9, green) in populations sampled in Alberta and adjacent regions. White symbols are populations lacking AFLP data. Panels on the sides of each map are enlarged views of symbols for mixed populations. Population names correspond to the map in Figure 1 and sample sizes are given in Table 3. Habitat map modified from 2005 Natural Regions and Subregions of Alberta, Alberta Sustainable Resource Development (https://open.alberta.ca/opendata/ gda-2f36921e-41e3-4cd8-813e-3333ea3c5983).

TABLE 2. Hybrid individuals (with Q values from STRUCTURE TABLE 3. Individuals with mismatches between mtDNA between 0.1 and 0.9) and the populations from which they were haplotype and AFLP Q value assignment from STRUCTURE, and collected. Population numbers correspond to the map in Figure 1. the populations from which they were collected. Population Accession number Q value Population (Pop’n. No.) numbers correspond to the map in Figure 1. RAM Z99.11.8 0.380 Alder Flats (13) Accession mtDNA Population number haplotype Q value (Pop’n. No.) RAM Z87.23.54 0.508 SE Barrhead (6)a RAM Z07.8.19 0.524 Peanut Lake (5)a RAM Z99.10.31 EAWV 0.012 NW Alberta (1) RAM Z07.8.15 0.558 Pine Lake (10) RAM Z07.8.4 EAWV 0.078 Swan Hills (2) RAM Z06.6.23 0.657 NW Barrhead (4)a RAM Z07.8.16 WEWV 0.995 Peanut Lake (5)a RAM Z85.19.16 0.773 Freeman River (3) RAM Z07.8.24 WEWV 0.998 Peanut Lake (5) a RAM Z94.12.99 0.844 CFB Suffield (18) a Indicates individuals from the contact zone. RAM Z07.8.40 0.873 SE Barrhead (6)a RAM Z06.6.13 0.899 Peanut Lake (5)a a Indicates individuals from the contact zone. A PCA of the 67 AFLP profiles generated 20 principal components with eigenvalues >1, explaining 76% of the variation in the data set. Only PC1 differed significantly be- Q value assignment and mtDNA haplotype (Table 3). tween gilvus and swainsoni (t-test with hybrid individuals

Two individuals had swainsoni Q values (≤ 0.08) but had [n = 7] excluded; t78 = –19.4; P < 0.001), and explained 25% gilvus mtDNA haplotypes, while the other two individuals of the variation. A plot of PC1 versus PC2 (Figure 4) shows had gilvus Q values (≥0.99) but had swainsoni mtDNA a clear separation of gilvus and swainsoni individuals (as haplotypes. assigned by mtDNA haplotype). The differences observed in AFLP markers between Six transect populations had both western and eastern gilvus and swainsoni can be further quantified using mtDNA haplotypes (Table 1, Figure 5). STRUCTURE

F-statistics. First, FST based on allele frequencies (using analyses using individuals from the 10 transect populations AFLP-SURV) was 0.21 (excluding hybrid individuals). demonstrated a clear geographic pattern for all 67 AFLP

Second, FST based on marker frequencies in ARLEQUIN markers analyzed (Figures 3B and 6). Individuals from was 0.37. Both FST values are significantly different from populations at NW Alberta (1), Swan Hills (2), and Freeman zero (P < 0.001). River (3) had low probabilities of belonging to the gilvus

FIGURE 6. Clines for relative frequency of gilvus mtDNA Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 haplotypes (black), mean hybrid index value (red), and frequencies of 67 concordant AFLP loci (gray) in populations FIGURE 4. AFLP principal component values of swainsoni across the transect through the contact zone. Transect distances individuals (blue circles), gilvus individuals (red circles), hybrid are measured from Population 1 (NW Alberta). Hybrid index is the individuals (green squares), and individuals with mismatches probability that an individual was assigned to the gilvus cluster between mtDNA and AFLP hybrid index (yellow triangles). by STRUCTURE. Individuals were classified based on the Q values generated by STRUCTURE: swainsoni (Q ≤ 0.1), gilvus (Q ≥ 0.9), or hybrid (0.1 > best-fitting model for the HI cline (Supplementary Material Q < 0.9). Table S2). The estimated values for centers (mean ± SD: 319 ± 70 km) and widths (219 ± 205 km) among clines for individual AFLP markers were highly variable (Figure 6). The

most frequent best-fitting model included fixed pmin and pmax at 0 and 1, respectively (28 of 67 AFLP loci) (Supplementary Material Table S2). Centers for the mtDNA cline and for each of the clines for the 67 AFLP markers do not deviate significantly from that of the HI cline (Figure 6), based on the criteria of overlapping confidence intervals and differences in AICc scores between the cline unconstrained, and the cline constrained, to the same center as the HI cline. FIGURE 5. Population assignments from STRUCTURE. Each bar represents the probability of assignment of an individual Testing Pleistocene Hypotheses corresponding to swainsoni (blue) and gilvus (red). Population numbers refer to the map in Figure 1. Populations 1–10 comprise Prior to testing divergence during the Pleistocene, we the transect through the contact zone. Population 23 (Montana) used MIGRATE-N to estimate effective population size contained a single individual and is not included. (Ne) for the total sample (Ne Total = 2,805,000), for gilvus

only, (Ne gilvus = 1,225,000), and for swainsoni only (Ne

swainsoni = 1,510,000). We also estimated genetic diversity (θ) clade (mean ± SD: 0.04 ± 0.14), whereas 97% of individuals for the total sample (θ Total = 0.0561; 95% CI: 0.0434–0.0727), sampled from the Cooking Lake (7), Beaverhill Lake (8), for gilvus only (θ gilvus = 0.0245; 95% CI: 0.0167–0.0361), and Camrose (9), and Pine Lake (10) populations had high for swainsoni only (θ swainsoni = 0.0302; 95% CI: 0.0212– 0.0433). probabilities of belonging to the gilvus clade (≥ 0.97 ± 0.08). We calculated that a single sorting event (S = 1) (Slatkin and Populations between these two groups (NW Barrhead [4], Maddison 1989) was required for our ML genealogy. The co- Peanut Lake [5], and SE Barrhead [6]) had an intermediate alescent simulations rejected both hypotheses of a single re- mean Q value (0.66 ± 0.44). However, only 5 individuals had fugium (both S = 0; P < 0.001). We also rejected two-refugia Q values characteristic of hybrids (Table 2); most individuals hypotheses for separation dates in the mid-Pleistocene (0.8 (26 of 31) in these populations had Q values characteristic of mya; S = 4.58; P < 0.001), and in the late Pleistocene (11 kya; one of the parental taxa (Supplementary Material Table S1). S = 28.48; P < 0.001). We could not reject the two-refugia The center of the mtDNA cline was located at 302 km hypotheses for separation dates early in the Pleistocene on the transect, placing it between the NW Barrhead (4) (1.8 mya; S = 2.47; P > 0.99), or at the Pliocene–Pleistocene and Peanut Lake (5) populations. The cline width was boundary (2.6 mya; S = 1.81; P > 0.99). estimated to be 69 km (Figure 6). The best-fitting model for the mtDNA data was the fixed pmin/pmax, indicating no introgression outside the contact zone (Supplementary DISCUSSION Material Table S2). The HI cline had a center that was located at 304 km and The eastern (V. g. gilvus) and the western (V. g. swainsoni) a width of 85 km (Figure 6). The fixed pmin/pmax was also the forms of the Warbling Vireo have largely allopatric breeding

ranges across north-central North America, but come into to the isolated population of swainsoni in the Cypress Hills contact in central Alberta, Canada. Barlow et al. (1986) (19). We found no evidence of hybridization in populations presented preliminary morphological and song evidence more distant from areas of demonstrated or probable con- suggesting that the Warbling Vireo complex might include tact. This suggests modest levels of hybridization within more than one valid species. However, this suggestion has the contact zone, but little introgression outside the con- been supported only by limited DNA evidence (Johnson tact zone (Figures 4 and 5). et al. 1988, Murray et al. 1994, Hebert et al. 2004), a dem- In addition to this evidence of minimal introgression onstration of migration and molt differences between the between the two taxa, four individuals showed a mis- taxa (Voelker and Rohwer 1998), and suggested differences match between mtDNA haplotype and Q value assign- Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 in morphology, plumage, and ecology (Semenchuk 1992, ment (Table 3). These represent cases of hybridization a Murray et al. 1994). number of generations previously, followed by continued Our phylogenetic analysis revealed that Warbling Vireos backcrossing of females (carrying the mtDNA of one in Alberta and the surrounding areas are separated into taxon) with males of the other taxon. Two of these birds two well-defined, genetically-differentiated (Figures 2, 4, are from the contact zone, but two are from populations in and 5), and monophyletic (Supplementary Material Figure northwestern Alberta. The latter cases may have been the S1) clades corresponding to previously-recognized tax- result of female hybrids dispersing from the contact zone onomic groups (American Ornithologists’ Union 1998). into populations in which there were only swainsoni males.

The mtDNA analyses showed a significant FST of 0.93 and The clines for mtDNA and the HI show an abrupt a corrected sequence divergence of 3.6% between the taxa. change in gilvus frequencies across Barrhead County. The The nuclear DNA (AFLP) markers also demonstrate a clear majority of clines for individual AFLP markers show a low signal of divergence, with an FST estimate of 0.37 based on frequency on one side of the cline, an increase in the hy- AFLP marker frequencies. These estimates of genetic di- brid zone, and high frequency on the other side of the cline vergence are greater than or equal to those for other pairs (Figure 6). of sister taxa that diverged during the Pleistocene (Weir The expected cline width under the neutral diffusion and Schluter 2004, Helbig et al. 2005, Toews and Irwin model may be calculated using the equation in Endler 2008, Irwin et al. 2009). (1977) and Barton and Gale (1993): The statistical phylogeographic analyses suggest that the = σ√ π eastern and western clades of Warbling Vireos diverged w 2 T, in the early Pleistocene, failing to reject the hypotheses where w is the estimated cline width, σ is the RMS dis- of a two-refugia scenario early in the Pleistocene or at the persal distance per generation, and T is the number of gen- boundary between the Pliocene and Pleistocene epoch. erations since contact. Assuming 6,000 yr since contact This finding is concordant with a pattern found in many (Strong and Hills (2005), and a generation time of 1 yr and pairs of boreal and north temperate birds, placing diver- an RMS dispersal distance of 21.56 km (estimated from gence of pairs of sister taxa solely in the Pleistocene (Avise data from Gardali and Ballard 2000), the expected cline and Walker 1998, Klicka and Zink 1999, Johnson and width would be ~4,192 km. This is ~60× greater than the

Cicero 2004). Several analyses (Tajima’s D, Fu’s Fs) suggest estimated cline width of 69 km for the mtDNA cline and that both taxa have undergone a substantial period of pop- 50× greater than the estimated cline width of 84 km for ulation growth and/or selective sweeps, indicative of taxa the HI cline. Additionally, the mean width of all individual coming back into secondary contact following a period of AFLP clines was ~220 km which is ~19 times narrower allopatric isolation (Hudson 1990, Avise 2000). than the estimated cline under neutral diffusion. To sup- The two taxa come into contact in central Alberta port the estimated mean cline widths (mtDNA and HI) in a narrow zone centered in Barrhead County (sample under the neutral diffusion model, dispersal would have localities 4–6; Figures 1 and 3). The AFLP markers and to be close to 0.40 km per generation, one of the smallest subsequent Q value assignments demonstrate that nine of estimates of dispersal for a migratory songbird (Ruegg 145 individuals examined were hybrids between the two 2008). taxa. Five hybrids are from the contact zone (Table 2). Two The bounded hybrid superiority hypothesis predicts others came from transect populations (Freeman River [3], that hybrid zones form on ecotones and are maintained Pine Lake [10]) near the contact zone. A third was from by selection favoring hybrids within the ecotones because Alder Flats (13), near the transition from montane forest they are better adapted to the environmental conditions to the aspen parkland (Figure 3) and possibly another area within the ecotone than are individuals of the parental of contact between the two taxa. The hybrid from Suffield taxa (Moore 1977). Additionally, under the bounded hy- (18) is distant from the contact zone. However, this spec- brid superiority hypothesis, cline widths will vary with the imen is from riverine forest in a grassland area that is close width of the ecological gradient, as seen in the Northern

Flicker (Colaptes auratus) and Glaucous-winged Gull– Alaska, northern British Columbia, and southwestern Western Gull (Larus glaucescens – L. occidentalis) contact Northwest Territories. Wapple (2019) suggests that birds zones (Moore and Price 1993, Bell 1997). The Warbling from northern Saskatchewan are gilvus but provides no ev- Vireo contact zone does appear to follow along an eco- idence to support this conclusion. Additional studies are tone (see below). However, the lack of a large number of needed to determine whether our findings are character- genomic hybrids in the center of the zone, suggests that istic of other areas of contact between the two groups. selection against hybrids is occurring, suggesting that the An outlier population with swainsoni haplotypes bounded hybrid superiority hypothesis not applicable in occurs in the Cypress Hills (19) (Figure 3A). However, this this situation. is not surprising given the terrain and geological history Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 The narrowness of cline widths suggests that selec- of the area. The Cypress Hills of southeastern Alberta tion is maintaining the hybrid zone (Barton and Hewitt and southwestern Saskatchewan support an island of 1985, Barton and Gale 1993). The pattern observed in the montane forest surrounded by shortgrass prairie. The Warbling Vireo contact zone supports the hypothesis of a Cypress Hills have disjunct populations of 14 montane tension zone because clines for all loci are relatively steep, bird species separated from the main ranges by 250–300 and concordant in terms of the location of the cline center. km (Thompson and Kuijt 1976, Lein 1979). It is likely that Both parental and hybrid genotypes are found in the cline the conifer forests became established on the Cypress center, suggesting a hybrid sink (Barton and Hewitt 1985). Hills by colonization from migrating belts of coniferous The other predictions (significant linkage disequilibria at forest following the receding Laurentide ice sheet at the the center of the cline and reduced hybrid fitness compared end of the Wisconsin glaciation. These coniferous forests to parental taxa) were not tested in this study and would and associated biota were then isolated by the expan- require large samples and further testing regarding hybrid sion of grassland vegetation, ~10,000 yr ago (Thompson fitness. and Kuijt 1976, Strong and Hills 2005). The presence of The distribution of swainsoni and gilvus mtDNA a gilvus haplotype in Elko, BC (22) (Figure 3A) is not as haplotypes and ALFP profiles among populations show a easy to explain. It may represent a haplotype retained striking pattern when plotted in relation to the major hab- from the past range of gilvus (Rohwer et al. 2001, Krosby itat types in Alberta. Swainsoni haplotypes and individuals and Rohwer 2009). assigned as pure swainsoni based on Q values are found The importance of the Pleistocene and its role in avian primarily in the foothills, mountains, boreal forests, and speciation in North America remains an ongoing and in mixed coniferous forests which consist mainly of lodge- highly-debated topic (Klicka and Zink 1997, Avise and pole pine (Pinus contorta) and white spruce (Picea glauca) Walker 1998, Johnson and Cicero 2004, Weir and Schluter interspersed with trembling aspens (Populus tremuloides) 2004, Irwin et al. 2005, Lovette 2005, Zink and Klicka 2006, (Figure 3A, B). Gilvus haplotypes and individuals assigned Toews and Irwin 2008, Irwin et al. 2009). However, Weir to pure gilvus based on Q values are found in the eastern and Schluter (2004) pointed out that most of these studies part of Alberta, primarily in the aspen parklands and along have focused primarily on avian taxa in areas south of the river systems in the grasslands (Figure 5A, B). The con- the regions covered by the ice. The birds of the northern tact zone in Barrhead County is in the boreal transitional forests may have experienced higher rates of speciation zone between aspen parkland to the south and east and compared with birds with ranges farther south whose hab- mixed boreal forest to the north and west. itat was not sundered by the glaciations of the Pleistocene South of this contact zone the boundary between the (Weir and Schluter 2004). All of the pairs of boreal forest swainsoni and gilvus groups approximates the western taxa that Weir and Schluter examined demonstrated coa- edge of the Great Plains from southwestern Alberta south- lescence events that fell within the Pleistocene, while only west to west-central Texas (American Ornithologists’ 51% of the pairs south of the ice sheets had coalescence Union 1998). Birds of the swainsoni group occur in mon- times in the Pleistocene. This suggests that the taxa whose tane and foothills areas whereas gilvus birds breed in the populations became disjunct during the Pleistocene as a eastern deciduous forest and the riparian corridors of the result of glacial advances experienced a higher rate of di- Great Plains. They may come into contact in numerous versification than those taxa not directly impacted by the locations along this boundary. Floyd (2014) reported a glaciers. Our findings add another pair of taxa which meet sharp boundary, based on song differences, between the at the interface between montane and northern forests two groups at the edge of the steep foothills in Boulder in western Canada, and that diverged during the earliest County, CO. The boundary between the two forms north stages of the Pleistocene. and east of the contact zone which we studied is un- The large genetic differences between the two taxa and clear. Birds from the boreal forest of northern Alberta the apparent absence of extensive introgression outside the are swainsoni, as are those from extreme southeastern contact zone suggest that one or more factors are limiting

mating between them, or reducing the viability of hybrid The common name of Western Warbling-Vireo, which is offspring. Winker (2009) argues that genetic data alone are already in use in the 1998 American Ornithologists’ Union weak evidence of reproductive isolation and that pheno- checklist and elsewhere, would apply to this taxon. The typic evidence of isolating mechanisms is necessary. The nominate subspecies would retain the Vireo gilvus species two taxa have small but consistent differences in their name, and have the common name of Eastern Warbling- song patterns and morphology (Sibley and Monroe 1990, Vireo. We suggest that future work may determine that ad- Semenchuk 1992, Lovell 2010). However, the potential role ditional cryptic species occur within the swainsoni group. of these in pair formation is unknown. There does not ap- The distribution and validity of recognized subspecies pear to be any ecological segregation in the contact zone, in this group are poorly understood and deserve further Downloaded from https://academic.oup.com/auk/article/138/1/ukaa071/6104490 by AOS Member Access user on 03 March 2021 where males of the two taxa may occupy adjacent terri- scrutiny. tories (Barlow 1988, Lovell 2010). The ecology and beha- vior of birds in the contact zone, and especially of mated pairs of known genetic identity, clearly warrant additional SUPPLEMENTARY MATERIAL research. It appears that a migratory divide (e.g., Helbig 1991, Supplementary Material is available at Ornithology online. Ruegg 2008, Turbek et al. 2018) may be the most important factor limiting gene exchange between the two taxa. Individuals of swainsoni arrive on their breeding grounds ACKNOWLEDGMENTS in central Alberta about two weeks before individuals of gilvus (Semenchuk 1992, Lovell 2010), reducing the prob- S.F.L. thanks Mike Braun and his group at the Smithsonian ability of pairing between males and females of the two Institution (especially Jeffrey Hunt, Sarah Kingston, and groups. The two taxa differ in the scheduling of their Sarah Sonsthagen) for their hospitality and help with both prebasic molts, with gilvus completing this molt on the mtDNA sequencing and AFLP issues. We thank Mark Peck breeding grounds whereas adult swainsoni begin their (Royal Ontario Museum) and Jocelyn Hudon (Royal Alberta Museum) for providing access to their tissue collections. Andy prebasic molt on the breeding grounds, but continue it Murphy helped us to gain access to properties managed by the during migration. Adult swainsoni may interrupt their Alberta Conservation Association, the Alberta Fish and Game migration in northwestern Mexico to replace their flight Association, Ducks Unlimited, and the Nature Conservancy feathers (Voelker and Rohwer 1998). In addition, the two of Canada. We also thank Kelly Carscadden for help with the taxa use different routes during migration to their winter AFLP analyses. Graham and Elizabeth Derryberry helped to ranges. Individuals of gilvus migrate through mid-western get HZAR to run correctly. Jocelyn Hudon and Gary Erickson and eastern USA, following the coast of the Gulf of of the Royal Alberta Museum aided S.F.L. in collecting birds. Mexico to their wintering grounds from southern Mexico Darren Irwin, Kevin Winker, and two anonymous reviewers to Nicaragua, while swainsoni birds migrate through improved the manuscript with their comments. the western USA and western Mexico to their wintering Funding statement: Funding for the research was provided grounds from southern Mexico south to El Salvador by a Challenge Grant in Biodiversity (Alberta Conservation (Voelker and Rohwer 1998). Migratory patterns, including Association) to S.F.L. and Operating Grants from the Natural Sciences and Engineering Research Council of Canada to scheduling of molts, have a genetic basis (Berthold and M.R.L. (A9690) and S.M.R. (RT735287). Helbig 1992, Berthold 2001, Pulido 2007, Rohwer and Ethics statement: This research was reviewed and approved Irwin 2011). Interbreeding between populations with dif- by the Life and Environmental Sciences Animal Care ferent patterns is argued to produce offspring with sub- Committee of the University of Calgary (Protocol BI 2005– optimal combinations of genes, potentially resulting in 39) and under permits from the Canadian Wildlife Service maladaptive migratory orientation or molt schedules (CWS05-A005) and the Province of Alberta (GP-20307, and reduced fitness of hybrids (Rohwer and Irwin 2011, CN-23176, and RC-06-17). Delmore and Irwin 2014, Turbek et al. 2018). Author contributions: S.F.L. and M.R.L. conceived the idea The finding that gilvus and swainsoni are genetically dis- and formulated the research questions. S.F.L conducted the tinct in sympatry suggests that they are almost completely field and laboratory work and analyzed the data. S.F.L and isolated reproductively and should qualify as “good spe- M.R.L. wrote the article with input from S.M.R. S.M.R provided advice on methodology and lab space for some of the cies” under the biological species concept (Mayr 1963). analyses. S.F.L. and M.R.L. provided funding for the study. Therefore, we propose that the western subspecies Vireo Conflict of interest statement: We have no conflict of in- gilvus swainsoni, along with other subspecies currently terest to declare. recognized in the swainsoni group (Phillips 1991, American Data availability: Mitochondrial genetic data are deposited in Ornithologists’ Union 1998), should be promoted to the GenBank https://www.ncbi.nlm.nih.gov/genbank/. Accession species level designation of Vireo swainsoni Baird 1858. numbers: MT181848–MT181925

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