Subtle Edge-Of-Range Genetic Structuring in Transcontinentally Distributed North American Tree Swallows

The Condor 111(3):470–478 ¡ The Cooper Ornithological Society 2009

SUBTLE EDGE-OF-RANGE GENETIC STRUCTURING IN TRANSCONTINENTALLY DISTRIBUTED NORTH AMERICAN TREE SWALLOWS

LAURA M. STENZLER1,5, CHRISTOPHER A. MAKAREWICH1, AURÉLIE COULON1,3, DANIEL R. ARDIA4, IRBY J. LOVETTE1,2, AND DAV I D W. WINKLER2 1Fuller Evolutionary Biology Program, Laboratory of Ornithology, Cornell University, Ithaca, NY 14850 2Museum of Vertebrates and Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853 3UMR 7179 Muséum National d’Histoire Naturelle/Centre National de la Recherche Scientiﬁque, 1 avenue du Petit Château, 91800 Brunoy, France 4Department of Biology, Franklin and Marshall College, Lancaster, PA 17604

Abstract. Understanding how genetic variation in the Tree Swallow (Tachycineta bicolor) is geographically structured is informative because this broadly distributed North American bird is increasingly used as a model for studies of mating systems, life-history traits, and physiology. We explored patterns of phylogeographic differentiation across the Tree Swallow’s breeding range by using nine microsatellite loci and a mitochondrial DNA sequence marker. Contrary to this species’ high population-level variation in life-history traits and other ecologically impor- tant characteristics, we found no genetic structuring across the majority of its distribution, spanning Tennessee, New York, and Alaska, but we found that birds from California form a distinct yet subtly differentiated genetic cluster. The Tree Swallow can be characterized as a species with both continent-wide genetic panmixia and slight differentiation at one edge of its breeding distribution. This pattern of genetic variation has implications for understanding the underlying basis of geographic variation in this species’ life history and other phenotypic traits.

Key words: genetic structure, geographic variation, microsatellites, mitochondrial DNA, Tachycineta bicolor, Tree Swallow. Sutil Estructuración Genética en el Borde del Área de Distribución Transcontinental de Tachycineta bicolor Resumen. Entender la estructuración geográfica de la variación genética de Tachycineta bicolor es infor- mativo debido a que esta especie, que se distribuye ampliamente en Norteamérica, se está usando cada vez más como modelo para estudios sobre sistemas de apareamiento, caracteres de historia de vida y fisiología. Explora- mos los patrones de diferenciación filogeográfica en todo el área de distribución reproductiva de T. bicolor usando nueve loci microsatelitales y un marcador de secuencia de ADN mitocondrial. De modo contrastante a la alta variación que se observa a nivel poblacional en los caracteres de historia de vida y otras características ecológi- cas importantes, no encontramos estructuración genética en la mayor parte del área de distribución, que incluye los estados de Tennessee, Nueva York y Alaska. Sin embargo, encontramos que las aves de California forman un agrupamiento genético distintivo aunque sutilmente diferenciado. Esta golondrina puede ser caracterizada tanto como una especie con panmixia continental, como una especie con una pequeña diferenciación geográfica en un extremo de su distribución geográfica. Este patrón de variación genética tiene implicaciones importantes para el entendimiento de la variación geográfica de las características de historia de vida y otros caracteres fenotípicos de esta especie.

species is taxonomically monotypic with no described sub- INTRODUCTION species, it shows substantial geographic variation in reproduc- Owing to the hundreds of studies that have taken advantage tive effort (Dunn et al. 2000, Ardia 2005, Monroe et al. 2008), of its tractability, the Tree Swallow (Tachycineta bicolor) offspring development (McCarty 2001, Ardia 2006), and im- has been termed a “model species” for research topics span- mune responses (Ardia 2005, Ardia 2007). Little is known, ning mating systems to immunology to climate change (Jones however, about patterns of genetic differentiation or connec- 2003). Tree Swallows breed across nearly all of temperate tivity among Tree Swallow populations or about the extent to North America, and past research on them has taken place at which the observed geographic variation in phenotypic traits many locations across this broad distribution. Although the is related to underlying population structuring.

Manuscript received 6 October 2008; accepted 29 April 2009. 5E-mail: [email protected]

The Condor, Vol. 111, Number 3, pages 470–478. ISSN 0010-5422, electronic ISSN 1938-5422. 2009 by The Cooper Ornithological Society. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Rights and Permissions website, http://www.ucpressjournals.com/ reprintInfo.asp. DOI: 10.1525/cond.2009.080052 470

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Despite the paucity of information on genetic structur- As the Tree Swallow has the broad distribution and low mor- ing, the ease with which cavity-nesting Tree Swallows can be phological variation often associated with this type of recent banded and monitored during the breeding season has resulted range enlargement, we explored whether its pattern of genetic in a large and well-documented collection of information on variation in mitochondrial DNA is consistent with a history of direct dispersal distances (Hosner and Winkler 2007), which demographic expansion. To examine distribution-wide levels allows us to make a priori predictions about the likely ge- of genetic population structuring further, we obtained sam- netic population structure of the species. Tree Swallows breed ples from sites that span the range of the species north–south throughout central and northern North America and are con- and east–west, and we tested for differentiation among these tinuously distributed from Maine to Alaska and as far south populations by using both microsatellite and mitochondrial as Tennessee and southern California (Robertson et al. 1992). sequence markers. Banding recoveries indicate that Tree Swallows have substantial potential for long-distance dispersal from where they METHODS fledge or breed (Winkler et al. 2004, 2005). Recapture data SAMPLE COLLECTION from the U.S. Bird Banding Laboratory and the more spatially focused Swallow Dispersal Study allowed Hosner and Win- Tree Swallows frequently use man-made nest boxes for breed- kler (2007) to document dispersal distances of up to 2367 km, ing, which made it possible for us to collect blood from adult although the majority (85%) of known dispersals occurred birds trapped when they entered to brood or feed young. Sam- among sites separated by less than 15 km. Nevertheless, even pled individuals were banded with a U.S. Fish and Wildlife rare long-distance dispersal could be sufficient to homogenize Service band before blood was collected (100–200 µl) from allele frequencies across long distances (Wright 1931, Slat- the brachial vein into heparinized microhematocrit tubes and kin 1987). This combination of a continuous distribution with stored at room temperature in 0.5 ml of lysis buffer (0.1 M no known major physical barriers to movement, annual mi- Tris [pH 8.0], 0.1 M EDTA, 10 mM NaCl, 0.5% SDS) (White gration, and documented long-distance dispersal leads to the and Densmore 1992). The geographic coordinates of sampled potential for high genetic exchange and little or no spatial ge- boxes (or clusters of closely located boxes) were obtained with netic structure in the Tree Swallow, as was found in one of the a handheld GPS unit. No voucher specimens were collected classic first phylogeographic surveys of a broadly distributed because all swallows were from study sites used for active North American songbird, the Red-winged Blackbird (Age- monitoring of reproductive success and other ecological and laius phoeniceus), whose geographic variation in morphology behavioral variables. is greater (Ball et al. 1988). Some other North American birds We sampled birds from four sites that together span the also have little genetic structure over large portions of their majority of the species’ breeding range: sites in New York and breeding ranges (e.g., Gibbs et al. 2000, Milot et al. 2001, Zink Ontario were sampled in 1998 and 1999, sites in Alaska, Cali- et al. 2006). However, geographic patterns of genetic differ- fornia, and Tennessee in 2003. The spatial distribution of sam- entiation may arise even in highly dispersive taxa as a result of pled birds differed somewhat by site: 300 birds came from 15 evolutionary forces opposing gene flow, such as local adapta- sites in New York and one in Ontario (11–412 km apart) (Sten- tion in response to strong selection and/or genetic drift acting zler 2001), 37 birds were sampled from three sites in Alaska on small effective population sizes within locally interbreed- (3–8 km apart), 46 birds were from three sites in California ing populations (Slatkin 1987, Freeman-Gallant 1996), from (separated by up to 500 km), and 25 birds were sampled from barriers to dispersal that would have otherwise gone unrec- one site in Tennessee. ognized (e.g., Mackenzie et al. 2004, Zardi et al. 2007), and even from random sorting of neutral genetic markers across MOLECULAR METHODS geography (Irwin 2002). Overall, North American birds show We extracted total genomic DNA from blood samples by using a high degree of taxon-specific variation in phylogeographic DNeasy Tissue Kits (Qiagen, Valencia, CA) or Perfect gDNA patterning (Zink 1996), rendering it necessary to examine (Eppendorf, Westbury, NY) blood-extraction kits according patterns of historical differentiation on a case-by-case basis. to the manufacturer’s instructions, except that proteinase-K Numerous phylogeographic studies of North American addition was followed by an overnight incubation at 65n C. birds have addressed the demographic influences of late Pleis- Extracted DNA was archived at −70n C until analyzed. tocene climate changes, which must have had profound effects Individuals were genotyped via the polymerase chain re- on the distribution of many bird species (Hewitt 1996, Klicka action (PCR) at nine microsatellite loci, four of which were and Zink 1997). Although species differ in the details of their developed in species other than T. bicolor. The loci were HrU7 genetic structuring, surveys employing various molecular (Primmer et al. 1995), IBI 5–29 and IBI MP3-31 (Crossman markers have suggested that some North American birds have 1996), TBI-81, TBI-104, and TBI-106 (Stenzler 2001), Pca3 expanded their ranges in the Holocene from one or more past (Dawson et al. 2000), LTR6 (McDonald and Potts 1994), refugia (e.g., Ruegg and Smith 2002, Milá et al. 2006, 2007). and Ase29 (Richardson et al. 2000). Forward primers were

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modified at the 5` end by addition of a fluorescent label (PET, populations defined by geographic location were also calcu- 6-FAM, VIC, or NED; Applied Biosystems, Foster City, CA), lated with GENEPOP. and an unpaired sequence of GTTTCT was added to the 5` end We used FSTAT v2.9.3.2 (Goudet 1995) to test for devia- of reverse primers to ensure complete adenylation of products tions from Hardy–Weinberg equilibrium at several hierarchi- (Brownstein et al. 1996). We used a multiplexing PCR proto- cal levels, first among birds within sampling sites, then within col (Hailer et al. 2005), allowing individuals to be genotyped populations defined by geographic location after combining at all nine loci in three multiplexed PCR reactions. Multi- sampling sites within each state. This program performs an

plexed PCR reactions (10 µL) contained 10–100 ng of genomic exact test based on Wright’s FIS (Wright 1951), a measure of DNA, 0.25 units of Jumpstart Taq polymerase (Sigma-Al- the degree of inbreeding within a group and thus a reﬂection drich, St. Louis, MO), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, of the nonrandom association of alleles. Alleles were per-

3.25 mM MgCl2, 200 µM of dNTPs (Invitrogen, Carlsbad, muted among individuals within each population. Markov CA), and from 1.0 to 4.8 pmol of each forward and reverse chain methods (15 000 randomizations) were used to create

primer to produce equal fluorescent signals. The PCR cycling the contingency tables, and FIS was calculated and ranked for profile consisted of one cycle at 95n C for 2 min, 35 cycles of each. In addition, we evaluated Hardy–Weinberg equilibrium 50 sec at 95n C, 1 min at 56n or 60n C (specific to each mul- globally by permuting alleles among all populations and using

tiplex mix), and 1 min at 72n C, followed by a final extension Wright’s FIT, the total inbreeding coefficient (Wright 1951), to cycle of 30 min at 72n C. We carried out PCR by using a Dyad characterize the data. thermalcycler (Bio-Rad Laboratories, Hercules, CA). Frag- Sampling in the field was conducted without prior knowl- ment-size information was collected on an ABI Prism 3100 edge of geographic or genetic boundaries between groups of Genetic Analyzer (Applied Biosystems, Foster City, CA), and interbreeding individuals. Nest boxes at sampling sites were allele sizes were estimated with GeneMapper version 3.7 (Ap- usually situated very close to each other in clusters of as few plied Biosystems, Foster City, CA). as 10 and as many as 100. Spacing between boxes within clus- We also sequenced 966 base pairs (bp) of the mitochon- ters was between 0 (back to back on poles) and 300 m. Thus drial gene ND2. We generated PCR products and sequenced we first considered each sampling site as an a priori popula- both strands by following Lovette and Rubenstein (2007). Cy- tion. A priori populations relatively close to each other (i.e., cle sequencing was carried out with the BigDye 3.1 chemistry the 15 sites in New York/Ontario, the 3 sites in Alaska, and the (Applied Biosystems, Foster City, CA), and sequences were 3 sites in California) were tested first for genetic differentia- collected with an Applied Biosystems model 3730 automated tion. In Tennessee, 22 birds were sampled from one cluster of DNA sequencer and viewed with the software Sequencher 4.5 nest boxes (separated by ~1.7–21 km), with an additional three (Genecodes Corp., Ann Arbor, MI). birds up to 30 km away; therefore, we considered all of these birds to represent one sampling site. If there was no signifi- STATISTICAL ANALYSES cant population structure among sampling sites, we considered those birds as one geographically defined population for Where appropriate, we controlled for multiple comparisons further analyses. by calculating the P values adjusted for false discovery rate Using the microsatellite data, we determined values of (FDR) with the function compute.fdr for R 2.3.1. The library Wright’s F (Weir and Cockerham 1984) for pairs of pop- of the function is available online at http://www.stjudere- ST ulations and globally to evaluate genetic structure among search.org/depts/biostats/documents/fdr-library.R. We used a priori populations, as implemented in the software pro- the method of Benjamini and Hochberg (1995), which con- gram GENETIX (Belkhir et al. 1996), which uses permu- trols the proportion of significant results that are in fact false tation-based statistical inference (10 000 permutations) to positives (type I errors). FDR control has the advantage of be- determine the significance of the observed F values. In ad- ing less stringent than the widely used Bonferroni correction ST dition, we tested the null hypothesis of identical distribution and hence avoids the considerable loss of power associated of alleles across populations (i.e., no genetic differentiation) with this method (e.g., Moran 2003, Verhoeven et al. 2005). by a log-likelihood G-based exact test (Goudet et al. 1996) as implemented in the software package FSTAT v2.9.3.2 (Gou- MICROSATELLITES det 1995). We evaluated genotypic linkage disequilibrium by testing for We also used the Bayesian clustering method imple- nonrandom correlations between genotypes across pairs of mented by the program STRUCTURE v2.0 (Pritchard et al. loci by Fisher’s exact probability test implemented by the web 2000) to infer spatial structure from the genetic data, eliminat- version of GENEPOP v3.1d (Raymond and Rousset 1995). We ing the need to assign birds to a priori populations. The model used Markov chain methods (2000 batches, 5000 iterations clusters individuals into K groups, which minimize Hardy– per batch) to estimate the P-value of the test. Expected and Weinberg and linkage disequilibrium. Within a run, K is fixed observed heterozygosity and number of alleles per locus for to a value set by the user. Multiple runs of a range of values of

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K are performed to obtain values for ln P(D), an estimate of was done by calculating the population’s pairwise FST values the posterior probability of the data for a given K. The values from genetic distances computed as the average number of of ln P(D) are used to infer the most likely number of groups pairwise differences between haplotypes. In addition, an anal- (value of K) existing in the dataset. To find the appropriate pa- ysis of molecular variance (AMOVA) was calculated (ARLE- rameters and models to use in the study, we did pilot runs on QUIN) to partition the variation among molecular haplotypes the data set first. For the final analyses, we used the admixture within and among individuals and populations. Groupings model without prior population information with a burn-in pe- consisted of all four populations or two populations (the first riod of 500 000 Markov-chain Monte Carlo iterations followed consisting of all California birds and the second a pool of by 2 000 000 further iterations. We used the correlated allele- all other birds). P-values were determined by permutation frequency model, which allows migration and shared ancestry (10 000 permutations). among the populations. An individual value of A (the Dirich- We tested whether the observed pattern of nucleotide let parameter for the degree of admixture) was used for each polymorphism among ND2 sequences might be an indication

population, with an alphapropsd of 0.001 (alphapropsd is the of a past population expansion, by using the statistics Fs (Fu standard deviation of the distribution from which A is updated 1997) and R2 (Ramos-Onsins and Rozas 2002), which look along the runs). Runs were conducted for K between 1 and 10 for an excess of rare (i.e., young) alleles over expectation from with 10 independent repetitions at each K. We ﬁrst estimated a neutral model of evolution, as implemented in the program the most likely “true” value of K by computing the posterior DnaSP, v4.50.1 (Rozas et al. 2003). The null hypothesis for probabilities of K with the method suggested by Pritchard and both tests is a constant population size, versus the alternative Wen (2004), who noted, however, the difﬁculties in selecting of population growth. These two statistics are the most pow-

the “true” value of K, especially where K 2. For this reason, erful for detecting population expansions, Fs for larger and we also used the $K method of Evanno et al. (2005) to evalu- R2 for smaller sample sizes (Ramos-Onsins and Rozas 2002). ate the value of K. We then assigned each individual to the ge- The significance of the tests was calculated by coalescent sim- netic cluster for which its estimated coefficient of ancestry (Q) ulations conditional on the number of segregating sites with was 0.5 or higher. 10 000 replicates, also implemented by DnaSP. In addition, we used ARLEQUIN to perform Tajima’s test (Tajima 1989) MITOCHONDRIAL HAPLOTYPES D for selective neutrality. The significance of the test is calcu- For mtDNA sequence data, we computed standard indices of lated with a coalescent simulation under a model of selective haplotype (h) and nucleotide diversity P) (Nei 1987) for all neutrality and population equilibrium. birds as well as per geographic population by using DnaSP RESULTS (v4.50.1) (Rozas et al. 2003). To infer population-level divergence on the basis of an MICROSATELLITE ANALYSES independent-gene genealogy we estimated genealogical rela- Observed heterozygosities of the nine loci ranged from 0.26 tionships among the ND2 sequences in mtDNA with the pro- to 0.97, and the number of alleles was between 6 and 14 per gram TCS v. 1.21 (Clement et al. 2000). The program creates locus (Table 1). There was no evidence of linkage disequilib- a minimum-spanning network with 95% plausibility per link rium among microsatellite loci, nor was there deviation from between haplotypes. We used ARLEQUIN v3.1 (Excoffier et Hardy–Weinberg equilibrium, globally or within any of the al. 2005) to assess genetic structure among populations. This four geographically defined populations (P 0.10 for all tests

TABLE 1. Microsatellite loci used to assess genetic variability in Tree Swallows from New York, Alaska, California, and Tennessee. Ho is observed heterozygosity, HE is expected heterozygosity, k is number of alleles.

All birds New York Alaska California Tennessee

Locus k HO HE k HO HE k HO HE k HO HE k HrU7 7 0.67 0.74 7 0.59 0.73 6 0.67 0.69 6 0.64 0.73 6 IBI MP 5-29 6 0.65 0.6 5 0.57 0.69 5 0.52 0.65 6 0.64 0.64 4 TBI-104 11 0.63 0.67 10 0.86 0.81 10 0.26 0.31 7 0.84 0.84 11 TBI-81 14 0.86 0.91 14 0.97 0.89 12 0.85 0.84 12 0.84 0.91 11 Ase29 13 0.88 0.86 10 0.78 0.84 10 0.63 0.68 9 0.92 0.9 12 IBI MP 3-31 14 0.90 0.83 13 0.81 0.89 13 0.85 0.84 12 0.84 0.84 11 Pca3 11 0.61 0.67 10 0.68 0.65 10 0.37 0.35 8 0.44 0.62 9 LTR6 11 0.75 0.75 10 0.73 0.72 7 0.61 0.64 8 0.68 0.78 7 TBI-106 9 0.78 0.70 6 0.76 0.78 7 0.78 0.72 6 0.60 0.8 7 All loci 0.75 0.74 0.77 0.75 0.62 0.63 0.72 0.77

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with the exception on one locus in one population (Pca3 in TABLE 2. Pairwise FST values for four geographic populations of New York; P 0.007). the Tree Swallow based on nine microsatellite loci and the sequence of ND2. Microsatellite-based pairwise FST values among the 15 sampling sites in New York/Ontario, among the three in Cali- New York Alaska California fornia, and among the three in Alaska were universally low (–0.0132 to 0.0178) and not significant (all P 0.05). Exact Nine microsatellite loci tests for genetic differentiation among these same groups Alaska 0.00436 a were likewise not significant. On the basis of these results, we California 0.04343 b 0.03857 b ns ns b considered the birds in New York/Ontario, in California, and Tennessee 0.00043 0.00368 0.05753 in Alaska each a geographic population and grouped them ac- ND2 c cordingly for further analyses. A subset of birds from the New Alaska −0.00158 California 0.15372 a 0.10338 a York/Ontario sampling area, taken from the center of New Tennessee −0.00341 c −0.00796 c 0.12197 a York state, was selected with a random-number table to bring the sample sizes in the four populations closer to parity for aP 0.05. b further analyses. The resulting sample consisted of 159 birds P 0.001. cNot statistically significant; 10 000 permutations. from four geographic populations: New York (n 51), Alaska (n 37), California (n 46), and Tennessee (n 25) (Fig. 1).

Pairwise FST values for these four geographic populations supported this two-population subdivision, as the $K value for ranged from 0.00043 to 0.05753. The values of FST for Cali- K 2 (7.002) was greater than the $K (range: 0.67 to 1.22) for fornia compared to each of the other three populations were all other tested values of K. We assigned each individual bird all significant (P 0.001), whereas the only other significant to one of the two inferred genetic groups on the basis of coef- pairwise comparison involved the New York and Alaska pop- ficients of ancestry calculated by the STRUCTURE run for ulations (P 0.03; Table 2). Only the comparisons involving K 2 with the highest value of ln P(D). The first group included the California populations were significant in the exact tests a total of 32 birds, 29 from California, 2 from Alaska, and 1 for differentiation implemented in FSTAT (data not shown). from New York. The second group consisted of the remaining

The STRUCTURE analysis further highlighted the 127 birds. The pairwise FST between these two genetic groups differentiation of the California population and the homo- was 0.05719 and highly signiﬁcant (P 0.001). Despite the geneity of the remaining three populations. The highest pos- overall support for the presence of two genetic groups within terior probability was for subdivision into two genetic groups the continental breeding range of the Tree Swallow, most in- (K 2) (Fig. 2). The $K method (Evanno et al. 2005) also dividual birds had intermediate assignment probabilities (Fig. 3), suggesting that these two groups are not deeply differentiated. This pattern of subtle separation is particularly evident in the California population, of which the 46 genotyped individuals all fall in the middle region of the assignment distribution, with no individual strongly supported as belonging predominantly to one genetic group.

FIGURE 1. Distribution of sites where Tree Swallows were sampled (closed black circles, n number of individuals from each region) FIGURE 2. Mean values of ln P(D) (an estimate of the posterior from four regions. NY New York; AK Alaska; CA California; probability of the data for a given K) for ten STRUCTURE runs at TN Tennessee. each value of K (error bars are standard deviations).

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MITROCHONDRIAL-HAPLOTYPE ANALYSES We obtained 966 bp of the ND2 mitochondrial sequence from 155 of the 159 Tree Swallows; DNA was unavailable from four samples. Among the mtDNA sequences, overall haplotype diversity was 0.767, which varied little among populations. Nu- cleotide diversity among the four populations differed and was lowest in New York, Alaska, and Tennessee (0.0016–0.0019, mean 0.0018) and highest in California (0.0045). Overall nucleotide diversity was 0.0027 (Table 3). Collectively there were 43 haplotypes (GenBank accession numbers EU725728 through EU725770), including one that was common in all four populations and shared by a total of 74 individuals. This common haplotype is at the center of one of the two major haplotype clusters reconstructed in the spanning network (Fig. 4). All other haplotypes in the larger cluster are at low frequencies and are separated from the common central haplotype by one to three nucleotide substitutions, with no ap- parent geographic structure. The second cluster of haplotypes in the spanning network was much more geographically restricted and comprised two haplotypes found in 11 Tree Swal- lows, 10 from California and one from Alaska. This haplotype group was separated from the larger group by a minimum of nine nucleotide substitutions.

Population pairwise FST values ranged from −0.00796 to 0.15372. The values of FST for California compared to those for each of the other three populations revealed significant genetic subdivision (P 0.05), whereas all other pairwise comparisons were not significant (Table 2). Variation among populations was significant both for four populations (AM- OVA, 9.4% among populations, 90.6% within populations, P 0.0001) and when samples from New York, Tennessee, and Alaska were pooled and compared with those from Cal- ifornia (AMOVA, 16.7% among populations, 83.3% within populations, P 0.0001).

Fu’s FS test (Fu 1997) for New York (FS −22.57, P 0.0001) and Alaska (FS −9.25, P 0.003) was signiﬁcant, as was Ramos-Onsins and Rozas’ R2 test (2002), a more powerful test, for the smaller Tennessee (R2 0.067, P 0.01) population. In contrast, the null hypothesis of population stability

TABLE 3. Characteristics of mtDNA ND2 sequence in Tree Swal- lows sampled from four geographic populations from New York, Alaska, California, and Tennessee; h is haplotype diversity, P is nucleotide diversity.

Number of Geographic segregating Number of population n sites haplotypes h P

All birds 155 44 43 0.767 0.0027 FIGURE 3. Probability of membership in each of two genetic New York 51 24 22 0.795 0.0016 groups, based on the coefﬁcient of ancestry (Q) calculated by the Alaska 36 20 15 0.749 0.0019 Bayesian clustering algorithm of STRUCTURE. Gray bars repre- California 44 18 11 0.704 0.0045 sent group 1, white bars group 2. Each bar represents an individual Tennessee 24 13 10 0.815 0.0018 bird.

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second divergent cluster geographically restricted to our western sites. Neutrality tests suggest a nonrandom overabun- dance of rare or new alleles for all areas except California. Nucleotide diversity is highest in California, possibly the result of a longer period over which mutations have accumu- lated there, but certainly because the California population contains individuals from both of the differentiated haplotype

clusters. The microsatellite-based assignment tests and FST- based comparisons identify congruently subtle differentiation in the California population, suggesting that this pattern is not an artifact of a single-marker system. A number of historical scenarios could explain this pattern of general panmixia with modest differentiation in California; one possibility is FIGURE 4. Minimum-spanning network of mtDNA haplotypes that the ancestral California population experienced a period from four geographic populations of the Tree Swallow. Straight lines of isolation and differentiation before being recently recon- indicate one nucleotide substitution between extant haplotypes, small closed circles represent inferred but unsampled haplotypes, nected to the broader, expanding population. This scenario and the black rectangle represents seven inferred nucleotide substitu- is consistent with the low mtDNA haplotype variation in the tions. Circle sizes and internal divisions are proportional to number cluster restricted to the West, with the evidence for admixture of individuals (black New York state, white California, gray seen in California at both classes of markers and with the his- Alaska, cross hatch Tennessee). torical demographic signature of population expansion seen in the non-California populations. The differentiation of the could not be rejected for California, where F was not signifi- S California population could also be related to differences in cant (F 0.269, P 0.59). The test for the entire sample of S migration behavior, as Butler (1988) reported that birds from birds was highly significant for F ( 39.6829, P 0.0001). Ta- S the midwestern U.S. and Canadian prairies appear to winter jima’s D statistic, tested against a model of selective neutrality along the coast of the Gulf of Mexico and in Central America, and population equilibrium, was significant for New York whereas east-coast breeding birds winter in Florida and the (D 2.31, P 0.0007), Alaska (D 2.03, P 0.005), and Caribbean. Tennessee (D 1.72, P 0.024) but not for California (D It is notable that the population-genetic patterns revealed 0.192, P 0.63). These results are consistent with a pattern by these data are not concordant with patterns of geographic of population expansion in New York, Alaska, and Tennessee variation in life-history traits. Ardia (2005, 2006, 2007) com- but of greater population stability in California. pared life-history traits of the Tree Swallow in three of the four geographic areas we sampled (New York, Tennessee, DISCUSSION and Alaska, but not California), finding clear geographic dif- Our analyses of microsatellite and mitochondrial markers in- ferences in reproductive effort, nestlings’ growth rate, body dependently suggest that Tree Swallows have a hierarchical condition, and immune responses. Ardia attributed much of pattern of genetic structuring across the broad area encom- this geographic variation to the habitat and environmental passed in this study. Across sites that span most of their breed- factors that impose different selective pressures across these ing distribution, we found little to no evidence for genetic extremes of the Tree Swallow’s breeding range. This lack of structuring, showing that these geographically distant sites concordance among life-history traits and genetic variation is have been connected by recent or continuing gene flow. In the not surprising, as microsatellite and mitochondrial sequence West and particularly in California, both markers supported variation is generally selectively neutral, and these markers the presence of subtle but significant population differentia- are unlikely by chance to be linked closely to loci under dif- tion, suggesting that these western populations have a slightly ferential selection at these different sites. Furthermore, genetic more complicated history. The microsatellite-based pairwise variation across most of the species’ range is low, and even in

FST between Alaska and New York was signiﬁcant but very California, the degree of genetic differentiation is not high. low (0.00436). From a biological point of view, this low FST The more dramatic geographic variation in life-history traits plus the nonsigniﬁcance of the exact test of differentiation and therefore likely results either from strong local selection on

of the mtDNA-based pairwise FST between these two popula- these traits or (perhaps most likely) from phenotypic plasticity tions lead us to believe that the birds from Alaska and New (Charmantier et al. 2008, Gienapp et al. 2008, Crispo 2008). York are not genetically differentiated. In an analogous study, Blondel (2007) found wide phenotypic Mitochondrial variation at ND2 has a pattern consistent divergence in demographic and behavioral traits in the Blue with the microsatellite results, with one widespread haplo- Tit (Cyanistes caeruleus) across heterogeneous landscapes type cluster common throughout the range of the species and a in the presence of very little genetic divergence, suggesting

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phenotypic plasticity may be one of many factors leading to plasticity in response to climate change in a wild bird population. divergence of adaptive traits in the absence of genetic diver- Science 320:800–803. gence at neutral loci. CLEMENT, M., D. POSADA, AND K. A. CRANDALL. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology In summary, the low magnitude of genetic differentiation 9:1657–1660. among Tree Swallow populations that span most of this spe- CRISPO, E. 2008. Modifying effects of phenotypic plasticity on inter- cies’ extensive breeding distribution suggests that this highly actions among natural selection, adaptation and gene flow. Jour- dispersive bird has experienced substantial recent gene flow nal of Evolutionary Biology 21:1460–1469. across most of North America. A finer-scale evaluation of ge- CROSSMAN, C. C. 1996. Single locus DNA profiling in the Tree Swal- low Tachycineta bicolor: a comparison of methods. M.Sc. thesis, netic variation in the western region of the species’ breeding Queen’s University, Kingston, Ontario, Canada. range is necessary to explore the origin and geographic limits DAWSON, D. A., O. HANNOTE, C. GREG, I. R. K. STEWART, AND of the genetic structuring that caused the California popula- T. BURKE. 2000. Polymorphic microsatellites in the Blue Tit tion to be subtly but significantly differentiated at both micro- Parus caeruleus and their cross-species utility in 20 songbird satellite and mitochondrial loci. families. Molecular Ecology 9:1941–1944. DUNN, P. O., K. J. THUSIUS, K. KIMBER, AND D. W. WINKLER. 2000. Geographic and ecological variation in clutch size of Tree Swal- ACKNOWLEDGMENTS lows. Auk 117:215–221. EVA N NO, G., S. REGNAUT, AND J. GOUDET. 2005. 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