BROAD SCALE SAMPLING
The two major groups within the Swainson's thrush have been referred to previously as coastal and inland (Ruegg and Smith 2002; Kelly et al. 2005), russet- and olive-backed (Evans Mack and Yong 2000), and the ustulatus and swainsoni subspecies (Evans Mack and Yong 2000) on the basis of plumage color (Bond 1963; Phillips 1991) and genetic differences (Ruegg and Smith 2002; Ruegg et al. 2006b). Throughout this article I use the terms coastal and inland to refer to the subspecies. To examine the broad scale distribution of coastal and inland mtDNA clades, I combined my sampling efforts with those of 19 bird banding stations to sample 460 total individuals (Table 1; Fig. 1B). Genetic samples consisted of the calamus of one rectrix or approximately 100 μl of blood collected by brachial vein puncture and preserved in lysis buffer (Seutin et al. 1991).
Table 1. Locations of sampling localities and numbers of individuals belonging to the coastal and inland mtDNA clades.
| 1||Sunshine Coast (0)||BC||49.50||−123.75||34|| 2|
| 2||*Squamish (33)||BC||49.91||−123.29||37|| 7|
| 3||Whistler (51)||BC||50.09||−123.04|| 8||12|
| 4||Shadow Lake (63)||BC||50.22||−122.88||12||14|
| 5||Pemberton (70)||BC||50.31||−122.80|| 9||31|
| 6||Lillooet (116)||BC||50.53||−122.13|| 0||31|
| 7||Kamloops (215)||BC||50.50||−120.36|| 1||19|
| 8||*Yukon Flats||AK||66.38||−148.10|| 0||15|
| 9||*Tongass National Forest||AK||58.42||−136.55|| 5|| 5|
|10||*Queen Charlotte Island||BC||53.00||−132.00||20|| 0|
|13||Mt. Baker National Forest||WA||48.05||−121.50||10|| 0|
|14||Pierce County, Fort Lewis||WA||47.06||−122.58|| 7|| 0|
|15||Wenatchee National Forest||WA||46.95||−121.31|| 5|| 4|
|16||*Flathead National Forest||MT||47.93||−113.00|| 0||20|
|17||*Umatilla National Forest||OR||45.83||−117.95|| 0||20|
|19||*Siuslaw National Forest||OR||44.33||−123.00||20|| 0|
|20||Willamette National Forest #1||OR||44.20||−122.00||12|| 0|
|21||Willamette National Forest #2||OR||43.32||−122.85|| 2|| 1|
|22||Camas Valley||OR||42.94||−123.69|| 8|| 0|
|23||Siskyou National Forest||OR||42.15||−123.42||11|| 1|
|24||Humboldt||CA||41.25||−123.70|| 9|| 0|
|25||*Migratory Bird Refuge||UT||41.50||−112.37|| 0||11|
|26||Tahoe National Forest||CA||39.62||−120.53|| 0|| 2|
|28||Mono Lake||CA||37.97||−119.11|| 0|| 1|
|29||Sequoia National Park||CA||36.78||−118.58|| 2|| 0|
MORPHOLOGY AND PLUMAGE ACROSS THE HYBRID ZONE
To characterize the hybrid zone, I established a southwest-to-northeast transect through a narrow valley transecting the Coast Mountains in southern British Columbia (Fig. 1C). This is essentially perpendicular to the direction of contact between the subspecies as defined morphologically (Fig. 1A), and for the purpose of cline analysis, it was assumed to represent the steepest transition between the two forms. Fieldwork was conducted during the month of June in the years 2000–2005. At each of seven locations (locations 1–7, Table 1; Fig. 1C), I captured 20–44 individuals using mist nets (Ralph et al. 1993). Each individual was measured, banded, and sampled for genetic analysis. Photographs of wing shape and plumage color for a subset of individuals from across the hybrid zone can be found in the University of California Museum of Vertebrate Zoology's digital collection.
To exclude the effects of sexual size dimorphism and to limit the possibility of inadvertently including migrants or hatch year birds, only adult breeding males known to be singing on their territories were used in the morphological analysis. I measured six morphological characters following the methods of Baldwin et al. (1931): wing cord (length of unflattened wing from bend of wing to longest primary); tail (point of insertion of central rectrices to tip of longest rectrix); tarsus length (the length between the intertarsal joint and the distal end of the last leg scale before the toes emerge); culmen length (from the anterior edge of the nare to the tip of the bill); bill depth (at its base); and bill width (at its base).
I quantified plumage coloration in live birds using an X-Rite digital swatchbook spectrophotometer (X-Rite Inc., Grandville, MI). The digital swatchbook provides an unbiased, repeatable method of quantifying plumage coloration in the field (Hill 1998). Plumage color was measured as the average of four spectral readings taken from the central back region. Each spectral reading consisted of the percent transmission at 10-nm intervals from 390 nm to 700 nm. I used Principle Components Analysis (PCA; SPSS Inc., Chicago, IL) to analyze the morphological and plumage color data.
Previous phylogenetic analysis of mtDNA control region sequences identified two reciprocally monophyletic haplotype groups corresponding to coastal and inland clades (Ruegg and Smith 2002). To assign individuals from across the Pacific Northwest to coastal and inland groups, including those from the hybrid zone transect, I screened 455 individuals using a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method (see Ruegg and Smith 2002).
To assess the degree of hybridization between coastal and inland forms within the hybrid zone, I screened amplified fragment length polymorphisms (AFLPs) following a modified version of the methods used in Vos et al. (1995). To limit the possibility of false negatives, only high quality extractions (260/280 ratio between 1.8 and 2.0) with a starting concentration greater than 20 ng/μL were used in the AFLP analysis. Approximately 250 ng of total genomic DNA was digested with 1 unit of MseI and 5 units of EcoRI and ligated with 60 Weiss units of T4 DNA ligase (New England Biolabs Inc., Ipswich, MA), 1.0 μL of 50 μM E-adaptor (5′-CTCGTAGACTGCGTACC-3′ and 3′-CATCTGACGCATGGTTAA-3′), and 1.0 μL of 5 μM M-adaptor (5′-GACGATGACTCCTGAG-3′ and 3′-TACTCAGGACTCAT-5′) (Vos et al. 1995). The restriction-ligation reaction was incubated at room temperature (25°C) for 12–18 h and subsequently diluted with H2O to a volume of 100 μL, for a final DNA concentration of 2.5 ng/μL. Products were stored at −20°C for up to six months. The preselective amplification followed the methods of Bensch et al. (2002). In short, 10 μL of the diluted ligation-digestion product was combined with 1.9 μL of H20, 0.06 μL of 100 μM E-primer with an additional T (5′-GACTGCGTACCAATTCT-3′), 0.06 μL of 100 μM M-primer with an additional C (5′-GATGAGTCCTGAGTAAC-3′), 4.0 μL of 1 mM dNTPs, 1.9 μL of 25 mM MgCl2, 2.0 μL of 10× PCR buffer, and 0.08 μL of 5 u/μL Taq DNA polymerase (Roche Inc., Nutley, NJ). The thermocycle for preamplification was as follows: 94°C for 2 min, followed by 20 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 60 sec, and a final extension at 72°C for 10 min. Eight microliter of product was visualized on a 1% agarose gel and the remaining 12 μL was diluted in 100 μL of H2O and stored at −20°C. For selective amplification, 2.5 μL of diluted preamplification product was added to 2.2 μL of H20, 0.6 μL of 10 μM fluorescently labeled selective amplification E-primer (FAM, blue), 0.6 μL of 10 μM selective amplification M-primer, 2 μl of 1 mM dNTPs, 0.6 μL of 25 mM MgCl2, 1 μL of 10× PCR buffer, and 0.08 μl of 5 u/μL Taq DNA polymerase. The selective amplification reactions were incubated according to the following protocol: 2 min denaturation at 94°C, followed by 12 cycles of 94°C for 30 sec, 65°C for 30 sec (increasing by 0.7 with each cycle), 72°C for 60 sec, 23 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 60 sec, and a final extension at 72°C. Selective amplification products were diluted with two parts TE buffer, combined with one volume formamide, 0.25 μL blue dextran, and 0.50 μl GeneScan-500 (Perkin Elmer Inc., Waltham, MA) ROX-labeled (red) size standard, and run on a 5% polyacrylamide gel. Fluorescently labeled products were visualized by running on an ABI 377 for 2.5 h.
To identify repeatable and polymorphic loci, I prescreened 15 selective primer combinations in 20 individuals (10 coastal, 10 inland), with one individual from each of these groups run five times. The initial screening panel was composed of individuals from a diversity of coastal and inland locations (coastal populations 8 and 1, inland populations 7, 9, and 10; Table 1; Fig. 1) to ensure that the markers were informative on a broader geographic scale. The ABI Gene Scan files were visualized using Genographer software (http://hordeum.oscs.montana.edu/genographer/). All visible bands from 50 to 350 base pairs were scored in the initial screening of primer combinations. Most AFLP bands were monomorphic or present at low frequency. To select robust and repeatable polymorphic markers for screening in all individuals across the hybrid zone, I considered only AFLP bands that occurred in 25–75% of individuals and were repeatable within an individual. Of the 15 primer combinations screened, nine primer pairs yielded polymorphic markers meeting the above criteria, resulting in a total of 15 markers that were scored in 154 individuals across the hybrid zone.
To determine the frequency of coastal, inland, and hybrid individuals across the hybrid zone, AFLP data were scored as present or absent for each band size and the data were analyzed using NewHybrids software, version 1.1 beta 3 (Anderson and Thompson 2002). Using a Markov chain Monte Carlo (MCMC) scheme incorporating allele frequency uncertainty, NewHybrids computes, for each individual, the posterior probability of belonging to pure coastal, pure inland, F1, F2, coastal backcross, and inland backcross categories. These categories are defined in terms of the expected proportions (E0, E1, and E2) of loci at which an individual carries 0, 1, or 2 gene copies of coastal origin. For example, a coastal backcross bird has E0= 0, E1= 0.5, and E2= 0.5; an F1 has E0= 0, E1= 1.0, and E2= 0.0. The model underlying NewHybrids includes the population of origin of each gene copy as a latent variable, allowing inference even with markers having nondiagnostic allele frequency differences. An AFLP marker is modeled as a diallelic locus with alleles, a and A, that are unobserved latent variables that get integrated out during MCMC. The observed datum at each AFLP locus is the presence or absence of a band, which is determined by the alleles carried at the locus: that is, aA and AA yield bands, whereas aa does not (Eric C. Anderson, NOAA Fisheries, pers. comm.).
Numerous, randomly started MCMC runs confirmed consistent convergence. Final results were obtained with 10,000 sweeps of burn-in and 50,000 sweeps of sample collection. Summing E1+ 2E2 over all hybrid and parental categories, weighted by the average posterior probability for each category across all individuals at each sampling site, yielded an estimate of the average proportion of genes of coastal origin at each sampling site. This proportion was used in the following cline analysis to estimate the change in nuclear genetic frequency across the hybrid zone. It should be noted that given the number of divergent AFLP markers available, second- or third-generation (or more advanced) backcrosses may have high posterior probability for the “parental” categories. This does not, however, greatly affect the identification of recent hybrids (F1s, F2s, and first-generation backcrosses) or the estimation of the proportion of coastal alleles in each population. Therefore throughout the remainder of the article, the term “parental” refers to anything greater than or equal to a second-generation backcross, whereas the term “hybrid” refers to F1s, F2s, and first-generation backcrosses.
CLINE COINCIDENCE AND WIDTH
I fitted maximum-likelihood (ML) clines for morphological characters (body size estimated as PC1 from the morphological analysis, and plumage color estimated as PC2 from the analysis of spectral data) and molecular characters (proportion of coastal mtDNA and proportion of coastal alleles based on the AFLP analyses) using the tanh and exponential cline fitting model of Szymura and Barton (1986) as implemented in the ANALYSE software package (Barton and Baird 1996). A tanh cline is defined as y = (1 + tanh (2(x – c)/w))/2, where x is the distance from the center of the cline, c is the position of the center of the cline, and w is the width of the cline, defined as the inverse of the maximum slope (Szymura and Barton 1986). I assumed a simple single locus tanh cline and did not explore the more complex stepped cline due to limitations in my sampling at the tails of the clines (Barton and Gale 1993). The tanh cline one-dimensional model was first developed for one locus with two alleles ranging in frequency from 0 to 1, but has subsequently been applied to assignment scores for nondiagnostic loci and morphological traits (Bridle et al. 2001; Phillips et al. 2004; Takami and Suzuki 2005). To apply the model to quantitative characters, morphology and plumage population means were scaled to values between 0 and 1 following the methods of Takami and Suzuki (2005). Pmax and Pmin (the maximum and minimum gene frequency values in the tail ends of a cline) were set at 0 and 1 and not allowed to vary. The distribution of sampling localities was most appropriate for a one-dimensional cline analysis—sampling started at the edge of the Pacific Coast of British Columbia and continued in a northeasterly through a narrow valley within the Coast Mountains (Fig. 1C). Based on the broad scale assessment of the distribution of coastal and inland groups (see Results), this orientation represents a good approximation of the direction of steepest character change.
I evaluated cline center coincidence and cline width concordance using the ML cline fitting procedures described by Phillips et al. (2004). In short, the likelihood surface of each character was explored stepwise along the axes for both center position c and width w simultaneously. The ML confidence intervals were estimated at the points where the log likelihood dropped two units below the maximum. Cline center coincidence and cline width concordance were assessed using a likelihood ratio test, in which values of the ML cline center and width calculated assuming coincidence and concordance were compared with values of ML centers and widths calculated assuming no coincidence and no concordance (Phillips et al. 2004). A significant difference between the ML values of the consensus cline width and center and the ML values generated for each individual cline suggests clines are not concordant.
ESTIMATION OF DISPERSAL DISTANCE
The RMS dispersal is the distance along a single dimension from where an individual was born to where it lays its first clutch (reviewed in Moore and Dolbeer 1989). I estimated RMS dispersal distance using U.S. Fish and Wildlife Service (USFWS) recovery records for Swainson's thrushes and the methods described by Moore and Dolbeer (1989). Bird banding localities are recorded in 10-min latitude–longitude blocks and therefore any movement within the 10-min block was considered no dispersal. Due to the fact that short-distance movements factor less into the overall calculation of RMS dispersal than do long-distance movements (Moore and Dolbeer 1989), the potential bias toward underestimating dispersal distance was considered negligible. Only nestling and hatch year birds banded and recaptured in the months of June, July, and August, when Swainson's thrushes are most likely near the nesting site and least likely to be migrating (Evans Mack and Yong 2000), were considered in the analysis. An estimate of RMS dispersal was calculated as the square root of the sum of the individual dispersal distances squared, divided by the number of observations (Moore and Buchanan 1985).
ENVIRONMENTAL GRADIENT AND DENSITY ANALYSIS
To compare the position of the hybrid zone to variation in climate, I used DIVA-GIS software (Hijmans et al. 2004). I extracted 10 climate variables (summarized as means for the years 1950–2000) from each sampling point based on an interpolated climate surface created by Worldclim, a global climate database with a spatial resolution of ∼ 1 km (Hijmans et al. 2005). Ten variables were selected from a group of 16 environmental variables because they were the least correlated (r < 0.80): (1) seasonal variation in temperature (coefficient of variation across all months); (2) mean temperature of the driest quarter; (3) annual precipitation; (4) seasonal variation in precipitation (coefficient of variation across all months); (5) precipitation of the coldest quarter; (6) precipitation of the warmest quarter; (7) precipitation of the driest quarter; (8) annual mean temperature; (9) mean temperature of the wettest quarter; and (10) mean temperature of the warmest quarter. The data were reduced into two vectors using PCA, and the PC1 score for each population was plotted to illustrate overall climatic variation across the hybrid zone.
To estimate density across the hybrid zone, I conducted transects following the methods of Ralph and Scott (1981). In short, density was approximated as the number of birds seen or heard singing or calling while walking a 100-m transect in 10 min. To reduce the potential for variation in the data given weather, time of day, and time of year, all transects were conducted on clear weather days, at peak singing h (6:30–9:00 p.m.), and during the beginning of the breeding season in British Columbia (June 1–June 29) (Campbell et al. 1997). All transects were conducted at sampling locations where Swainson's thrushes are known to occur and care was taken to conduct transects in the highest quality habitat available (usually near a lake or a stream within designated Forest Service land). A total of 13 transects were completed. In some locations, especially in the center of the hybrid zone in which personal observation suggested that density was low, two or three replicate transects were completed at multiple sites within a sampling locality on the same evening and the data were pooled to attain a mean density with standard errors. Coastal transects were conducted approximately two to three weeks before inland transects to account for variation in the start of the breeding season within coastal and inland habitat types (Campbell et al. 1997).