Nuclear Loci and Coalescent Methods Support Ancient ...lithornis.nmsu.edu/~phoude/Peters_et_al_2007_Nuclear_loci_and... · original article doi: 10. 1111/j. 1558-5646.2007.00149.x

Nuclear Loci and Coalescent Methods Support Ancient Hybridization as Cause of MitochondrialParaphyly between Gadwall and Falcated Duck (Anas SPP.)Author(s): Jeffrey L. Peters, Yuri Zhuravlev, Igor Fefelov, Anne Logie and Kevin E. OmlandSource: Evolution, Vol. 61, No. 8 (Aug., 2007), pp. 1992-2006Published by: Society for the Study of EvolutionStable URL: http://www.jstor.org/stable/4627014 .

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ORIGINAL ARTICLE

doi: 10. 1111/j. 1558-5646.2007.00149.x

NUCLEAR LOCI AND COALESCENT METHODS SUPPORT ANCIENT HYBRIDIZATION AS CAUSE OF MITOCHONDRIAL PARAPHYLY BETWEEN GADWALL AND FALCATED DUCK (ANAS SPP.) Jeffrey L. Peters,1,2,3 Yuri Zhuravlev,4 Igor Fefelov,5 Anne Logie,1'6 and Kevin E. Omland1'7 'Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21250 2E-mail: [email protected]

4lnstitute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences, Vladivostok 690022, Russia E-mail: [email protected]

5Research Institute of Biology, Irkutsk State University, Irkutsk 664003, Russia E-mail: [email protected] 6E-mail: alogie [email protected] 7E-mail: [email protected]

Received July 24, 2006

Accepted March 21, 2007

Many species have mitochondrial DNA lineages that are phylogenetically intermixed with other species, but studies have rarely tested the cause of such paraphyly. In this study, we tested two hypotheses that could explain mitochondrial paraphyly of Holarctic

gadwalls (Anas strepera) with respect to Asian falcated ducks (A. falcata). First, hybridization could have resulted in falcated duck mitochondrial DNA (mtDNA) introgressing into the gadwall gene pool. Second, gadwalls and falcated ducks could have diverged so

recently that mtDNA lineages have not sorted to reciprocal monophyly. We used coalescent analyses of three independent loci to

distinguish between these two hypotheses. Two lines of evidence support introgression. First, analyses of the three loci combined show that some introgression is necessary to explain current genetic diversity in gadwalls. Second, we generated alternative

predictions regarding time since divergence estimated from mtDNA: falcated ducks and gadwalls would have diverged between 65,000 and 700,000 years before present (ybp) under the introgression hypothesis and between 11,000 and 76,000 ybp under the incomplete lineage sorting hypothesis. The two independent nuclear introns indicated that these species diverged between 210,000 and 5,200,000 ybp, which did not overlap the predicted time for incomplete lineage sorting. These analyses also suggested that ancient introgression (-14,000 ybp) has resulted in the widespread distribution and high frequency of falcated-like mtDNA (5.5% of haplotypes) in North America. This is the first study to use a rigorous quantitative framework to reject incomplete lineage sorting as the cause of mitochondrial paraphyly.

KEY WORDS: Anatidae, ancient introgression, coalescence, Holarctic, incomplete lineage sorting, mitochondrial paraphyly, nuclear introns.

In a survey of published phylogenetic and phylogeographic studies, Funk and Omland (2003) documented that 23% of surveyed

animal species had mitochondrial DNA (mtDNA) that was not

monophyletic. Yet, few studies have attempted to differentiate between possible causes of such gene-tree paraphyly and polyphyly (paraphyly hereafter). Two major causes of paraphyly are

introgression and incomplete lineage sorting (Avise 2000; Funk 3Current address: Institute of Arctic Biology, University of Alaska Fairbanks, 909 Koyukuk Dr., Fairbanks, Alaska 99775.

? 2007 The Author(s). Journal compilation ? 2007 The Society for the Study of Evolution. 1 992 Evolution 61-8: 1992-2006



TESTING CAUSES OF PARAPHYLY

and Omland 2003). Introgression can occur when interspecific hybridization results in DNA crossing species boundaries; the re-

cipient species will have DNA that is paraphyletic with respect to the other. Alternatively, incomplete lineage sorting can result in

paraphyly if species divergence was too recent for ancestral polymorphisms to have sorted to reciprocal monophyly. Mitochondrial

paraphyly is particularly important because mtDNA should sort more rapidly than nuclear DNA owing to its smaller effective population size (Avise et al. 1987; Moritz et al. 1987; Moore 1995). Distinguishing between introgression and incomplete lineage sort-

ing is difficult because both can produce similar topologies in gene trees (Wakeley 1996; Holder et al. 2001), and most studies that have addressed the cause of paraphyly gave ad hoc explanations without statistically rejecting alternative hypotheses. Three main

approaches have been used to date: examining the topology of the mtDNA tree, comparing mtDNA with nuclear DNA, and comparing the geographic distributions of different clades.

The topology of the mtDNA tree has been used as a heuris- tic approach to determining the cause of paraphyly. For exam-

ple, a shallow genealogy that lacks distinct clades suggests that

species have diverged too recently for mtDNA to have sorted to

monophyly (e.g., Klicka et al. 1999; Baker et al. 2003). This in-

terpretation is especially supported when shared haplotypes are ancestral whereas species-specific haplotypes are derived (Baker et al. 2003; Donnelly et al. 2004; Omland et al. 2006). Other studies have used the more rigorous method of nested clade analysis (Templeton 1998) to objectively evaluate these genealogical signatures (e.g., Liebers et al. 2004; Morando et al. 2004), but this method has been criticized because it does not provide a statistical framework for rejecting alternative scenarios (Knowles and Maddison 2002). Although the topology of a single gene tree can

provide some evidence regarding incomplete lineage sorting or

introgression, the randomness of mutation and genetic drift pre- vents any strong conclusions if analyses do not account for this stochastic variance (Knowles and Maddison 2002; Rosenberg and

Nordborg 2002; Knowles 2004). A second commonly used approach has been to compare

mtDNA to nuclear DNA (nDNA). The absence of shared nuclear

polymorphisms (i.e., differences were fixed between species) has

been interpreted as evidence of a deep species divergence and

introgression of mtDNA (e.g., Tegelstrom 1987; Ballard 2000; Weckstein et al. 2001; Redenbach and Taylor 2002). In contrast, extensive sharing of nDNA polymorphisms has been interpreted as evidence for a recent divergence and the retention of ancestral polymorphisms (e.g., Wilson et al. 2000; Sefc et al. 2005). However, these comparisons implicitly assume that mtDNA has

higher potential for moving across species boundaries and that nDNA will better reflect phylogenetic history (see Brumfield et al. 2001; Funk and Omland 2003). If hybridization is common, then nuclear polymorphisms can be shared, even when species di-

verged a long time ago. Alternatively, species might exhibit fixed differences at some loci due to chance, even when species diver-

gence was recent (Palumbi et al. 2001; Hudson and Turelli 2003). Although comparing mtDNA to nDNA is a promising method of

determining the cause of mitochondrial paraphyly, more rigorous methods are needed to test alternative hypotheses (see Buckley et al. 2006).

Finally, a third approach has been to compare sympatric and

allopatric populations. Under the introgression hypothesis, lin-

eage sharing might be more common where the two species are

sympatric, because there are more opportunities for hybridization (e.g., Hare and Avise 1998; Masta et al. 2002; Donnelly et al.

2004; Kulikova et al. 2004, 2005; Morando et al. 2004; Sefc et al.

2005). In contrast, incomplete lineage sorting does not necessar-

ily make any predictions regarding the geographic distribution of

lineages. This method has merit if hybridization is ongoing and

dispersal is low, but it may not be applicable to all study systems. If species have high dispersal capabilities that facilitate gene flow between distant populations, then introgressed DNA might not be restricted to sympatry. A range expansion or positive selection can also cause introgressed DNA to spread beyond areas of sympatry. As such, finding shared lineages between allopatric populations does not necessarily reject hybridization as the cause of paraphyly.

Rigorous tests of explicit hypotheses are needed to distin-

guish between causes of paraphyly, and these tests need to account for the stochastic variance of genetic processes (Knowles and Maddison 2002; Rosenberg and Nordborg 2002; Knowles

2004). The isolation-with-migration (IM) coalescent method ac- counts for genetic stochasticity by sampling from the range of genealogies that are consistent with the data (Nielsen and

Wakeley 2001; Hey and Nielsen 2004). Isolation-with-migration estimates six parameters, including effective population sizes of the ancestral population and each daughter population, im-

migration rates into each daughter population, and time since

divergence (Hey and Nielsen 2004). Using IM, the posterior distributions of migration rates can be examined to determine whether gene flow is necessary to explain phylogenetic patterns. Posterior distributions that include the lowest value of migration examined suggest that the data are consistent with com-

plete isolation (e.g., Hey 2005; Peters et al. 2005a; Omland et al. 2006). In this case, paraphyly is consistent with incomplete lineage sorting. In contrast, posterior distributions that do not include the lowest value examined (e.g., Won and Hey 2005) suggest that gene flow is necessary to explain phylogenetic patterns. Thus, IM provides a quantitative framework for evaluating the likelihood of gene flow in species exhibiting paraphyletic DNA.

STUDY TAXA-GADWALL AND FALCATED DUCK

In this study, we tested hypotheses regarding mitochondrial paraphyly of gadwalls (Anas strepera) with respect to falcated ducks

EVOLUTION AUGUST2007 1 9 9 3



JEFFREY L. PETERS ET AL.

A mtDNA 1 1 B nDNAo 2-14

6 (CHD, LDH of ....e 1 5 6 1,0 ,2] ,2c; 1 351,2 ,4tfantit, 1 54 12Atlantic , , 2,

Vir,'a n)Ocea n Ocean

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cto!Oe

,, cean.. 4O acean

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40 65,18

ga1 d falcated 8 gadwall dc , 3 ~~duck 51

Sbreeding - 1 2 .

.;k...... %.," .jll, ?wintering lJ

[_- .'...i 3:r. . ..;,. sam pling .... . .- ,'i

Figure 1. Breeding and wintering ranges of gadwall and falcated ducks. Sampling location and sample sizes for (A) mtDNA and (B) introns (CHD, LDH) are shown in ellipses. (An additional mtDNA haplotype and CHD allele were sampled from a captive falcated duck.)

(A. falcata). The gadwall has a Holarctic distribution that extends across Europe, Asia, and North America, whereas the falcated duck is restricted to East Asia (Fig. 1). Within North America, 5.5% of 348 gadwalls had mtDNA (clade B) that was more similar to falcated ducks than to the majority of gadwalls (clade A), rendering gadwall paraphyletic with respect to falcated duck

(Peters and Omland 2007); none of the clade B haplotypes were shared between species. Clade B was geographically widespread throughout North America, but was not found in a small sample of gadwalls from Eurasia (Peters et al. 2005b). The relatively high frequency and widespread distribution of clade B in North Amer- ica seem consistent with incomplete lineage sorting, especially given the Asian distribution of falcated ducks. Furthermore, the mtDNA tree was consistent with a monophyletic falcated duck nested within gadwall (Peters and Omland 2007); widespread species having paraphyletic mtDNA relative to restricted-range species is consistent with expectations under models of recent

speciation via peripheral isolation (Harrison 1991; Omland 1997). However, gadwall and falcated ducks are known to hybridize in the wild (Johnsgard 1960), and introgression of mtDNA from falcated ducks into gadwalls could explain the pattern of paraphyly. Because these two species are each other's closest extant relatives

(Johnson and Sorenson 1999; Peters et al. 2005b), both incomplete lineage sorting and hybridization are viable hypotheses.

To test the cause of mitochondrial paraphyly between gadwalls and falcated ducks, we applied the approaches reviewed

above, but we made explicit predictions and tested those predictions using coalescent models. Following species divergence, DNA progresses through various stages of polyphyly and para-

phyly before ultimately sorting to reciprocal monophyly (Neigel and Avise 1986; Omland et al. 2006). Thus if hybridization with falcated ducks resulted in the introgression of clade B haplotypes into gadwalls (i.e., mtDNA has otherwise sorted to reciprocal monophyly), then theory predicts a deeper species divergence (tINT; Fig. 2A) than predicted if clade B is an ancestral polymorphism that has incompletely sorted (tiLs; Fig. 2B). The probability of monophyly, however, is also a function of effective population sizes (smaller populations will sort to monophyly more rapidly) and stochastic processes. Therefore, we first estimated the range of possible divergence times consistent with mtDNA sorting to

A Introgression B Incomplete lineage sorting

Present Clade A Clade B Clade A Clade B G G F G G F

TMRCA - -

Figure 2. Alternative hypotheses to explain mitochondrial paraphyly of gadwalls (G) with respect to falcated ducks (F). Thick lines show gene trees within species trees. (A) Clade B haplotypes introgressed into the gadwall gene pool after species divergence (tINT). (B) Clade B is found in both species as a result of incomplete lineage sorting following a more recent divergence (tlLS). TMRCA is the time to most recent common ancestor of all mtDNA haplotypes.

1 994 EVOLUTION AUGUST 2007




reciprocal monophyly (tINT) and those consistent with incomplete sorting of ancestral polymorphisms (tiLS). Specifically, we used IM to jointly estimate effective population sizes and time since di-

vergence, while accounting for stochastic variance, to determine whether estimates of time since divergence differed between these two models. We then tested these predicted divergence times using two independent nuclear loci. Second, combining all loci to better account for stochastic among-locus variation, we estimated rates of gene flow to determine whether some introgression was nec-

essary to explain the observed phylogenetic patterns. Finally, we

compared falcated ducks to allopatric and to sympatric gadwalls to test the prediction that introgression rates would be higher in

sympatry.

Materials and Methods LABORATORY METHODS We sampled 42 gadwalls and 47 falcated ducks from areas of sympatry in East Asia (Fig. lA) for comparisons with the 348 gadwalls sampled from North America by Peters and Omland (2007; GenBank accession numbers are given in online Supplementary Material). DNA was extracted from muscle or feathers using the

DNeasy tissue kit (Qiagen, Valencia, CA). For each individual, we sequenced 658-659 bp of the mtDNA control region using primers L78 and H774 (Sorenson and Fleischer 1996; Sorenson et al. 1999) following standard protocols (see McCracken et al.

2001). (Internal primers were used for some individuals; see online Supplementary Material.) Two haplotypes from Asian gadwalls and 10 haplotypes from falcated ducks had been published previously (Peters et al. 2005b); we also included nine published sequences from allopatric Eurasian gadwalls (Donne-Gouss6 et al. 2002; Peters et al. 2005b). Introgressed haplotypes shared with Eurasian wigeons (A. penelope) and an American black duck

(Anas rubripes) were excluded from this study (Peters et al. 2005b; Peters and Omland 2007).

For a subset of individuals, we sequenced a sex-linked intron from the chromo-helicase-DNA binding gene (CHD) located on the Z-chromosome (Fig. IB). We used primers 3007 and 3112 (Ellegren and Fridolfsson 1997) following published protocols (Peters et al. 2005b) or CHDb.F (5'-AACT- GTTCTACATACAGGTTCTACTCC-3') and CHDb.R (5'- AGTTCCTCTGCACCAAACTTTA-3') with an annealing temperature of 60?C; the latter primers yielded more consistent

amplifications. The overlapping portions of the two CHD fragments included a 270-271 bp intron and 43 bp of flanking exon. CHD sequences included 50 alleles from 34 North American

gadwalls, 50 alleles from 32 East Asian gadwalls, 14 alleles from 9 European/West Asian gadwalls, and 66 alleles from 40 falcated ducks (Fig. IB; 32 gadwall and 16 falcated duck alleles were published in Peters et al. 2005b). Because females carry

a single Z-chromosome, an unambiguous allele was amplified and sequenced. The gametic phase of alleles for males that were either homozygous or polymorphic at a single position could be resolved without ambiguity. However, five male falcated ducks and three male gadwalls were polymorphic at multiple positions, and gametic phases were ambiguous. For those males, we used PHASE version 2.1 (Stephens et al. 2001; Stephens and Donnelly 2003) to infer gametic phase (unambiguous alleles were included in analyses). Inferred alleles for four falcated ducks and two

gadwalls matched known alleles, but PHASE indicated novel alleles for one individual of each species. We used allele-specific primers to preferentially amplify and sequence one allele from those two individuals (see Peters et al. 2005b), and we subtracted this allele from the polymorphic sequence to resolve the phase of the other allele. (Allele-specific primer sequences are provided in online Supplementary Material.)

Seven falcated ducks contained a polymorphic indel in CHD. Locations of indels were easy to recognize, because the 5'-ends of sequences were unambiguous, whereas 3'-ends contained nu- merous double peaks (Jennings and Edwards 2005; also see online Supplementary Material). We compared the unambiguous 5'- ends to ambiguous 3'-ends between forward and reverse strands to determine indel length (see online Supplementary Material). Furthermore, because gaps resulted in shifted peaks in chro-

matograms, we could resolve gametic phase for other polymorphisms within the sequence (see online Supplementary Material). We found one novel allele using this method; the remaining 13 alleles matched known alleles from unambiguous sequences.

We also sequenced intron 4 from the lactate dehydrogenase gene (LDH) using primers LDH4.F (5'-ATGGGAGC TTGTTCCTTCAA-3') and LDH4.R (5'-CTTCCTGCTGACG AACACCT-3') (Peters 2006). LDH sequences included 458-469

bp of intron sequence, including a variable T repeat (7-10 bp in length), and 69 bp of flanking exon. One falcated duck and four gadwalls were heterozygous for the T repeat, and resolv-

ing alleles as described above, all 10 alleles matched known alleles. One gadwall had two polymorphic sites, and we used allele specific primers to resolve alleles (online Supplementary Material). We sequenced LDH for 15 North American gadwalls (30 alleles), 12 Asian gadwalls (24 alleles), and 10 falcated ducks (20 alleles) (Fig. IB). All new sequences have been

deposited in GenBank (Accession Nos. EF537650-EF537858; EF541024).

SUMMARY STATISTICS

We tested whether the frequency of clade B haplotypes differed between North American and East Asian gadwalls using a two- tailed Fisher's exact test. For each locus, we calculated nucleotide

diversity (Tr) and haplotype diversity (h) in Arlequin version 2.001 (Schneider et al. 2000). We conducted analyses of molecular

EVOLUTION AUGUST2007 1 9 9 5




variance (AMOVA, Excoffier et al. 1992) in Arlequin to examine

population structure between falcated ducks and gadwalls and between North American and Asian gadwalls. We constructed haplotype networks for each locus using the median-joining algorithm in FLUXUS (Bandelt et al. 1999, www.fluxus-engineering.com). For all analyses, we treated each indel as a single polymorphism, regardless of its size.

COALESCENT ANALYSES- ISOLATION-WITH-MIGRATION We used a Bayesian MCMC method in the IM (Hey and Nielsen

2004) coalescent program to generate testable predictions and to test those predictions by simultaneously estimating six parameters scaled to the neutral mutation rate: u: 0cG (effective population size of gadwalls), 0 FD (effective population size of falcated ducks), 0A (effective size of the ancestral population at the time of divergence), t (time since divergence), mG (the immi-

gration rate for gadwalls; i.e., the rate at which falcated duck alleles enter the gadwall gene pool), and mFD (the immigration rate for falcated ducks). Although migration typically refers to movements of individuals between populations, the m parameter is better interpreted as the rate of DNA introgression for the pur- poses of this study. Fundamentally, there is no difference between these interpretations because both result in DNA moving between

gene pools, but for clarity, we hereafter refer to m as the rate of

introgression. Isolation-with-migration assumes no recombination within

loci and that loci are selectively neutral. There was no evidence for recombination in either CHD or LDH (four-gametes test; Hud- son and Kaplan 1985). Furthermore, an HKA test (Hudson et al.

1987) indicated that polymorphisms within and between gadwall and falcated ducks across the three loci fit expectations under neu-

trality. This result was robust to including or excluding gadwall clade B haplotypes in analyses (X 2 = 2.138, df = 4, P = 0.71 and

X 2 = 1.219, df = 4, P = 0.87, respectively). Following the approach used by Won and Hey (2005), we

first ran IM using large, flat priors for each parameter. Based on those runs, we defined narrower upper bounds that encompassed the full posterior distribution of each parameter. However, estimates of t and 0A sometimes contained distinct peaks, but the tails were flat and did not approach zero. In these cases, we defined upper bounds based on other information obtained from preliminary runs (see below). We used a uniform (flat) prior distribution between zero and the defined upper bound, which we assumed to be uninformative. Using those priors, we repeated runs with a burnin of at least 200,000 steps. To check that the program was converg- ing, we assessed autocorrelations and effective sample size (ESS) for each parameter throughout the run (Hey and Nielsen 2004). We ran the program until the lowest ESS was at least 150. We also repeated each run using a different random number seed to check

Table 1. Summary of data partitions used for generating and testing predictions for introgression and incomplete lineage sorting in IM.

Models of Falcated Gadwall divergence duck

Generating predictions Deep divergence mtDNA All populations- (introgression) mtDNA (clade

A only) Shallow mtDNA All populations- divergence mtDNA (clades (incomplete A & B) lineage sorting)

Testing predictions Isolation-with- CHD & LDH All populations- migration- CHD & nDNA LDH Isolation-with- mtDNA, CHD, & East Asia-mtDNA migration LDH (clades A & B), (sympatry) CHD, & LDH Isolation-with- mtDNA, CHD, & North America- migration LDH mtDNA (clades (allopatry) A & B), CHD,

& LDH

that multiple runs gave similar results. Details of each analysis are

provided below and are summarized in Table 1.

Generating predictions with mtDNA We first generated predictions regarding time since divergence based on two alternative hypotheses: mtDNA introgression and

incomplete lineage sorting. Under the introgression hypothesis, clade B became incorporated into the gadwall's gene pool after they diverged from falcated ducks, but mtDNA has otherwise sorted to reciprocal monophyly. We used IM to estimate a range of

possible divergence times (tiNT; Fig. 2A) consistent with this hypothesis by comparing gadwall clade A and falcated duck mtDNA

(we excluded clade B gadwalls) using a model of isolation (only four parameters were estimated: 0FD, 0G, 0A, and t). Doing so caused the Markov chain to sample from parameter space consistent with mtDNA having sorted to reciprocal monophyly (the- oretically, deep divergence times and small effective population sizes). Because the posterior distribution of 0A was flat, we defined an upper bound for the prior by assuming that the ancestral

population size was not greater than the combined size of the two

daughter populations (OFD + OG); we summed the upper 95%

highest posterior distribution (HPD) for OFD

and 0G to set OA (upper bound = 110).

We estimated a range of possible divergence times under the incomplete lineage sorting hypothesis (tILS; Fig. 2B) by

1 9 9 6 EVOLUTION AUGUST 2007




analyzing all gadwall (clades A and B) and falcated duck mtDNA with a model of strict isolation. Excluding the possibility of gene flow caused the Markov chain to sample from parameter space consistent with species still sharing ancestral polymorphisms (the- oretically, shallow divergence times and large effective population sizes). Using this model, the full posterior distribution for each parameter was contained within the priors.

Testing predictions with nuclear introns To test predictions generated from mtDNA, we obtained independent estimates of time since divergence using the nuclear introns CHD and LDH (tnDNA). We excluded flanking exons from analyses because coding regions are more likely to be under direct selection. We treated introns as HapSTRs, which are loci that have

single nucleotide polymorphisms (SNPs) linked to short-tandem

repeats (STR) that vary in length (Hey et al. 2004). We defined inheritance scalars in the input files to reflect modes of inheritance for CHD (0.75, sex-linked on the Z-chromosome) and LDH (1.0, biparentally inherited). By doing so, parameters estimated from CHD were readjusted by the program to be on the same scale as autosomal loci (Hey and Nielsen 2004). We analyzed nDNA

using a model of strict isolation and a model of isolation with

migration to evaluate the sensitivity of results to assumptions re-

garding gene flow. Using a model of isolation with migration, the

posterior distributions of 0A and t contained flat tails. We defined an upper bound for 0A by summing the upper 95% HPD of OFD and 0G as described above (upper bound = 2.5). To set an upper prior for t, we assumed that time since divergence could not be older than TMRCA (time to most recent common ancestor of all

sequences). Averaging the posterior distributions of TMRCA for the two loci, we used the upper 95% HPD of TMRCA as the upper bound for t (upper bound = 1.25). Using a model of isolation, the full posterior distributions of all parameters were encompassed by the priors.

We compared the posterior distributions of tnDNA to tINT and

tILS to determine whether we could reject one of the divergence times estimated from mtDNA. Because mtDNA and nDNA evolve at different rates, we converted estimates of t to biologically in- formative values, using the equation t = Tu, where T is time in

units of years and u is the neutral mutation rate for the locus. Because our mutation rates were calibrated as the number of sub-

stitutions per year (see below), no corrections were needed for

generation time. For the intron dataset, u was the geometric mean of the mutation rates for the two loci. HapSTR loci require two mutation rate calibrations: one for the STR portion and one for the sequence portion. Because we did not have calibrations for the STR portion, we used mutation rate scalars estimated in IM

(Hey and Nielsen 2004; Won et al. 2005). For conversions, we

multiplied t by the geometric mean of the mutation rate scalars for the sequence portions of loci, and then divided this value

by the geometric mean of calibrated mutation rates (Won et al.

2005).

Testing predictions using all loci Isolation-with-migration can better account for the stochasticity of mutation and lineage sorting when all loci are examined simul-

taneously. Therefore, we further tested predicted divergence times

using a model of isolation with migration that included mtDNA

(both clade A and clade B haplotypes), CHD, and LDH (Table 1). We also made two predictions regarding the migration parameter, m, under a hypothesis of mtDNA introgression. First, the 95% HPD of m would exclude zero and hence reject the possibility of

"no introgression." Second, we predicted that introgression rates would be higher where gadwalls and falcated ducks are sympatric. We treated allopatric North American gadwalls and sympatric East Asian gadwalls in separate analyses. We defined inheritance scalars as 0.25 for mtDNA, 0.75 for CHD, and 1.0 for LDH to reflect their modes of inheritance (Hey and Nielsen 2004). In both

analyses, we used the sum of the upper 95% HPDs of OFD and G

to set upper bounds for 0A (upper bound = 4) and the upper 95% HPD of TMRCA (averaged across loci) for t (upper bound = 1.9) (see above).

Isolation-with-migration can also record the time of any intro-

gression events detected within genealogies sampled throughout the run ("time of migration," Won and Hey 2005). We examined time of migration to determine whether any introgression was more consistent with recent or past hybridization.

MUTATION RATE CALIBRATIONS For mtDNA, we used the control region mutation rate calibrated

by Peters et al. (2005a) of 4.8 x 10-8 substitutions/site/year (s/s/y; range = 3.1 x 10-8 -6.9 x 10-8 s/s/y). This calibration was based on relative rates of control region to mtDNA coding regions in

multiple duck taxa. The rate for coding regions was calibrated by Paxinos et al. (2002) using fossil evidence that the goose genera Anser and Branta diverged during the Pliocene (four to five million

years ago [mya]). We calibrated mutation rates for CHD and LDH introns based

on the goose-duck split (subfamilies Anserinae and Anatinae, re-

spectively); hence our intron calibrations are independent of the mtDNA calibration. Fossil records suggest that these subfamilies

likely diverged during the Oligocene (23-38 mya; Howard 1964). Fossils assigned to each subfamily have been recovered from the

Oligocene and afterwards (Howard 1964; Livezey 1996, 1997b; Louchart et al. 2005), whereas earlier fossils have been assigned to lineages sister to Anatidae (Livezey 1997a; Ericson 1997; Dyke 2001; Clarke et al. 2005). We used d = 2u7r for calibrations, where d is the average percent sequence divergence between ducks and

geese (Subfamilies Anserinae and Anatinae), u is the mutation

EVOLUTION AUGUST 2007 1 9 9 7




rate, and r is the time of divergence. We estimated d between Anserinae and Anatinae for 20 pairwise comparisons for CHD

(n = 2 Anserinae, n = 10 Anatinae; J. L. Peters et al. 2005b; Humphries and Peters unpubl. data, this study) and 40 pairwise comparisons for LDH (n = 4 Anserinae, n = 10 Anatinae; Kraft et al. 1993, St. John et al. 2005; A. Logie and J. L. Peters, unpubl. data, this study; see online Supplementary Material). We corrected d using the best-fit mutation model that was selected

using Akaike Information Criterion (AIC) in ModelTest version 3.7 (Posada and Crandall 1998). For CHD, the TVM model was

selected, which includes a general time reversible model of substitution (A-C, A-G, A-T, C-G, C-T, and G-T = 0.6, 2.4, 0.5, 2.8, 2.4, and 1.0, respectively). For LDH, the TrN model was selected, which includes different rates for transversions (1.0) and for A-G and C-T transitions (3.9 and 6.1, respectively). When multiple sequences were available for a single species, we calculated the

average value of d. As a best approximation to mutation rates, we defined T as the midpoint of the Oligocene (30.5 mya) and used the average d among all pairwise comparisons between subfamilies. To set a lower bound, we used the lowest value of d and the

beginning of the Oligocene (38 mya); to set an upper bound, we used the maximum value of d and the end of the Oligocene (23 mya). Thus, our calibrations incorporate uncertainty in the exact

divergence date between subfamilies and stochastic variance in mutations.

To test the reliability of rate calibrations, we compared time since divergence estimated from introns and mtDNA coding re-

gions. In those comparisons, we grouped gadwalls and falcated

ducks, because divergence between those species is the parameter of interest in this study. For both CHD and LDH, we estimated time since divergence for 36 nonindependent pairwise comparisons within Anatinae (Kraft et al. 1993; Peters et al. 2005b, unpubl. data, this study). For species that had multiple available sequences, we calculated average sequence divergences. For mtDNA, we estimated time since divergence using published cytochrome b and ND2 sequences (Johnson and Sorenson 1999; see online Supple- mentary Material) and a divergence rate of 2.1 % per million years (Paxinos et al. 2002). We used PAUP* (Swofford 1999) to correct

pairwise divergences for mtDNA using the TIM + I + F model chosen using AIC in ModelTest version 3.7 (Posada and Crandall 1998). This model includes a general time reversible substitution model (A-C, A-G, A-T, C-G, C-T, and G-T = 3.0, 106.1, 2.0, 0.2, 30.3, and 1.0, respectively), invariant sites (I = 0.71), and a

gamma distribution (F = 5.1). We plotted time since divergence estimated from introns against time since divergence estimated from mtDNA to determine whether there was general agreement between markers. We also performed a Mantel test with 100,000 permutations in the program ZT (Bonnet and de Peer 2002) to determine whether there was a significant positive correlation between those estimates.

A mtDNA B nDNA - CHD

Clade 8

12 substitutions & 1 indel

C nDNA- LDH

Clade A/ * 1bp*

,.- , l 7bp

70 I -

falcated duck - nucleotide substitution gadwall - North America - - insertion/deletion

* gadwall - East Asia hypothetical unsampled * gadwall - Europe/West Asia haplotype/allele

Figure 3. Haplotype networks for (A) mtDNA, (B) CHD, and (C) LDH. Circle area is proportional to the number of shared sequences. Indel sizes are shown next to the corresponding branch; an asterisk (*) indicates STR portions that were included in coalescent analyses. Note that one clade B haplotype and one CHD allele (sampled from different individuals) from Asian gadwalls were shared with falcated ducks, whereas none of the clade B haplotypes sampled from North America were shared with falcated ducks.

Results PATTERNS OF GENETIC DIVERSITY Within North America, 19 of 348 gadwalls (5.5%) had clade B mtDNA haplotypes that grouped with falcated ducks to the exclu- sion of gadwall clade A haplotypes (Fig. 3A; Peters and Omland

2007). All 47 falcated ducks had clade B haplotypes (h = 0.68, ar =

0.0020), none of which were shared with North American gadwalls (Fig. 3A). Clade B haplotypes were strongly structured between falcated ducks and North American gadwalls (PST = 0.47; P < 0.00001). One of the 42 gadwalls from East Asia also had a clade B haplotype (2.4% of haplotypes; the frequency of clade B did not differ significantly between Asia and North America; P =

0.71). The Asian clade B haplotype differed from North American

clade B by 1-6 bp and was identical to the most common falcated duck haplotype (Fig. 3A). The remaining 41 East Asian gadwalls

1998 EVOLUTION AUGUST 2007




had clade A haplotypes (h = 0.89, rr = 0.0028). (Within clade A, mtDNA was significantly structured between hemispheres

[srT = 0.12; P < 0.00001].) For CHD, we found 19 different alleles among the 66 fal-

cated duck (h = 0.74, - = 0.0043) and 114 gadwall (h = 0.43,

T = 0.0018) sequences. Ten alleles were unique to falcated ducks and eight alleles were unique to gadwalls. Only one allele was shared between species-one allele from an East Asian gadwall was identical to 12 falcated duck alleles (Fig. 3B). CHD alleles were strongly structured between falcated ducks and gadwalls (Fig. 3B; (ST =

-0.71; P < 0.00001). For LDH, we found eight dif-

ferent alleles among the 20 falcated duck (h = 0.19, w = 0.00043) and 54 gadwall (h = 0.49, a = 0.0012) sequences. Two fixed indels between the three falcated duck and the five gadwall alleles resulted in LDH being strongly structured (Fig. 3C; srT = 0.81; P < 0.00001). (Both CHD and LDH were also structured between North American and Asian gadwalls [CHD: OSTr = 0.088; P = 0.004; LDH: (sr = 0.23; P < 0.00001].)

INTRON MUTATION RATE CALIBRATIONS On average, CHD alleles from ten species of Anatinae were 8.9%

divergent from Anserinae alleles (range = 7.5% to 10.2%). Using the midpoint of the Oligocene, 30.5 mya, we estimated a mutation rate for CHD of 1.5 x 10-9 s/s/y. Average divergence for LDH alleles was 12.1% (range = 10.9% to 14.1%) between subfamilies suggesting a mutation rate of 2.0 x 10-9 s/s/y. Incorporating possible sources of error, we defined a range of plausible mutation rates for CHD (1.0 x 10-9-2.2 x 10-9 s/s/y) and LDH (1.4 x

10-9 -3.1 x 10-9 s/s/y). Despite using a different calibration point (goose-duck split) than that used for mtDNA (Anser-Branta split),

S 16- CHD -- c o 14- LDH ---

1- 2 -? S.- 12

a ) i(A 0-

U c 0 6 Ce o 4 - g ' O

0 2 4 6 8 10 12 14 16 time since divergence (millions of years) -

mtDNA

Figure 4. Correlation between time since divergence estimated from mtDNA coding regions and two nuclear introns. The solid line is the expected line for a perfect correlation. Dashed lines show the correlation between mtDNA and introns with the origin forced through zero. There is a general agreement between divergence times estimated from different markers.

there was general agreement between divergence times estimated from nuclear introns and mtDNA (Fig. 4). We found significant positive correlations between mtDNA and CHD (r = 0.80, P =

0.00008), between mtDNA and LDH (r = 0.92, P = 0.0001), and between CHD and LDH (r = 0.62, P = 0.0006).

COALESCENT ANALYSES-ISOLATION-WITH-MIGRATION Time since divergence Estimates of time since divergence differed and 95% HPDs did not overlap between the two mtDNA models (Fig. 5A; see Tables 2 and 3 for the full sets of parameters estimated from the various IM models). Given mutation rate estimates, gadwalls and falcated ducks most likely diverged about 290,000 ybp (tiNT = 9.1) under a model that assumed mtDNA has sorted to reciprocal monophyly

TILS (lineage sorting) 0.005 A mtDNA 0.004 - TINT (introgression)

0.003- 0.002 ' i 0.001

C, 0

0- 1 10 100 1000 10000

C I TnDNA(I) a 0.007 BnDNA DBnDNA

0.006 0.005

cn 0.004 T S 0.003 TnDNA(IM) 0.002 0.001

1 10 110 1000 10000

0005 T 0.005 C mtDNA + nDNA Tallopatry 0.004

, sympatry

0.003 - 0.002 - 0.001

0? w1

1 10 100 1000 10000 Time since divergence

(T; thousands of years; log scale)

Figure 5. Time since divergence (T; t = Tu): posterior distributions

(thick, solid lines). Lower and upper 95% HPDs adjusted using fast and slow mutation rate calibrations, respectively, are shown by the thinner lines. (A) T predicted from mtDNA under hypotheses of incomplete lineage sorting (TILS) and mtDNA introgression (TINT). (B) T estimated from nDNA under models of strict isolation (TnDNA(i)) and isolation with migration (TnDNA(IM)). (C) T estimated from the three loci combined, comparing falcated ducks to Asian (Tsympatry) and North American (Tallopatry) gadwalls using a model of isolation- with-migration. The vertical dashed line indicates the threshold for rejecting a hypothesis of incomplete lineage sorting (upper 95%

HPDadj for TB). (See online Supplementary Material for posterior distributions of other parameters.)

EVOLUTION AUGUST 2007 1 999



4 m 3111 ~1 3a m ?e r ?b c3 12~ 5a v,

m c3

r(

0CD 0- 0 0 o" 0 H0 o0 ?o o st -oI Or~o y

N ot l?

3 O("D (D (D r5 CD 3 C <0- p C o CD' a bC C-D C-D nDCc v DCDt, =t O 5 C V s : D r r a 0 z CD P-CD V C) f o- -cm " ? ~ a a p CD NE f of D ; O s rD t O O CPr lO , 3 p~ rv Yl cr l 3 C

CD P) 00 ? CD P.C

rD P o0, 55 > I CD z C, . :z (D CD CD CD~nw C ~O D

0D WCD 00

~< 0~ 0CD P- CDSo ~ ~ <

CD Q 0--0 CD CDE CD n CD CD a~Dc ~ r;c

0- 0 a~ J CD C0

p n ? 3 CD CD ?z c-, ,3cDCQ~~ SCD p) rD CD C). CD r),r r Ct QV CD P.(0 (D CD CD 3 o a o s9 C "CI E 3

l I I -

CD E3

0 5 -1 C. a 0~~0 ~ '0 C0L~ 0'

kC tj ?Z q 0 =1 *" Cd C C?, r. Vv

0 CL 0-0 f Y 000 a 0 00 E3 CD CD CD CD V, (D~?CD CD Q p CDDo 0 C CD , t. "r b 5aCC CD E3 ctP P CD l ?zr Ef` cr cr n t fi bCTI mc CJ)CD cr cr.c e~P)O3 ~ OO CDO CD ( t CC 0

CL 0-. f-+ ?z r- CDd ~ a aCD CD p CDv Cf CD rD CD; I s o C cr ~=t Y "vi cr CDE3 CD , p~

rD CD CD CD C?Q v Cr Os pr oz, CD ?7n1 Eo3 p p a v ua g ?7 o 1 - CD E3 Cd ,3 O CD nJ

Table 2. Parameters scaled to the neutral mutation rate u estimated from the various IM models (95% HPD).

Models of divergence 0FD 0G 0A t mED MG Mutation scalar1 1

Deep divergence (introgression 22 43 0.06 9.1 - - - 3.2x 10 of clade B)-mtDNA (11-40) (31-61) (-0-101) (2.6-14) (2.0x 105-4.5x 10-)

Shallow divergence (incomplete 25 59 17 0.88 - - - 3.2x 10- lineage sorting)-mtDNA (11-53) (40-89) (7.2-43) (0.51-1.6) (2.0x 10-5-4.5x 10-)

Isolation with 0.49 0.28 0.001 0.27 0.004 0.39 1.84 6.1 x 10- migration-nDNA (0.11-1.7) (0.08-0.78) (--0-2.2) (0.11-1.2) (--0-1.8) (--0-3.5) (4.2x 10-7-9.2x 10-7

Isolation-nDNA 1.2 0.39 0.16 0.19 - - 1.62 6.1 x 107 (0.35-4.4) (0.16-0.99) (0.004-1.1) (0.09-0.40) (4.2 x 10-7-9.2x 10-7

Isolation with migration 0.95 0.63 0.15 0.44 0.0035 0.47 2.10 2.3x 10-6

(sympatry)-all loci (0.31-2.1) (0.20-1.5) (--0-3.5) (0.17-1.7) ('--0-0.84) (0.046-2.7) (1.5x 10-6-3.4x 10-6 Isolation with migration 1.1 0.89 0.34 0.42 0.0035 0.30 2.59 2.3 x 10-6

(allopatry)-all loci (0.47-2.2) (0.39-1.7) ('-0-3.1) (0.17-1.0) (-0-0.64) (0.011-1.4) (1.5x 10-6-3.4x 10-6

1 Geometric mean for the sequence portions of loci in models that included multiple loci; u is the mutation rate per locus, not per site.

NJ 0 0

0

z



z

C12

0

Table 3. Parameters estimated from the various IM models converted using calibrated mutation rates1.

Models of divergence Ne-effective number of individuals T-ybp M-migrants per generation O=4Neu2 t=Tu M=Om/2

Falcated duck Gadwall Ancestral Falcated duck Gadwall

Deep divergence (introgression 58,000 110,000 160 290,000 of clade B)-mtDNA (20,000-160,000) (57,000-250,000) (~0-410,000) (65,000-700,000)

Shallow divergence (incomplete 65,000 160,000 46,000 28,000 lineage sorting)-mtDNA (20,000-220,000) (74,000-360,000) (13,000-180,000) (11,000-76,000)

Isolation with 120,000 70,000 330 820,000 0.001 0.05 migration-nDNA (18,000-640,000) (13,000-290,000) (--0-810,000) (220,000-5,300,000) (~0-1.6) (~0-1.4)

Isolation-nDNA 270,000 86,000 35,000 510,000 (51,000-1,400,000) (24,000-320,000) (590-360,000) (150,000-1,600,000)

Isolation with migration 73,000 49,000 12,000 410,000 0.002 0.15 (sympatry)-all loci (16,000-240,000) (10,000-170,000) (~0-400,000) (110,000-2,300,000) (-~0-0.88) (0.005-2.0)

Isolation with migration 100,000 85,000 32,000 480,000 0.002 0.21 (allopatry)-all loci (30,000-310,000) (25,000-240,000) (-0-440,000) (130,000-1,800,000) (~0-0.70) (0.009-2.3)

1 For Ne and T, uncertainty in mutation rates was incorporated into conversions by using the upper bound of mutation rates (fast rate) to convert lower 95% HPDs and using the lower bound (slow rate) to convert upper 95% HPDs (see Table 2); 95% HPDs for M were adjusted using the 95% HPD of 0. 2Estimates of Ne assume an average generation time of three years (Peters 2006). This same formula was used for converting estimates from mtDNA because defining an inheritance scalar of 0.25 resulted in 0 being adjusted to the same scale as autosomal loci.

0

z

oJ 0




0.010 A mG 0.008

< 0.006 . 0.004 -o 0.002 .-

0.000 0 1 2 3 4 5 6

o 0.040 B mFD

-;-- allopatry c 0.030 - sympatry 0.020

0.010

0.000 0 1 2 3 4 5 6

introgression rates (m)

Figure 6. Introgression rates scaled to the neutral mutation rate, u, estimated using all three loci. (A) The introgression rate of falcated duck DNA into gadwalls (mG), and (B) the introgression rate of gadwall DNA into falcated ducks (mFD).

Discussion MITOCHONDRIAL PARAPHYLY-INTROG RESSION OR INCOMPLETE LINEAGE SORTING? Mitochondrial paraphyly is common among animal taxa, but few studies have attempted to quantitatively differentiate between

possible causes of paraphyly, especially by accounting for the stochastic variance of lineage sorting (Funk and Omland 2003). In this study, we accounted for this stochasticity, as well as uncertainties in mutation rates, and rejected incomplete lineage sorting as the cause of mitochondrial paraphyly of gadwalls with respect to falcated ducks. Two lines of evidence support introgression. First, analyzing three independent loci with an isolation-with-migration

0.18 0.1 -- allopatry: mtDNA

0.16 - sympatry: mtDNA C 0.14 0.14 sympatry: CHD -0 " 0.12 o

o0.10o o 0.08 CL 76 0.06

.-0.04 2 0.02

0.00 0.001 0.01 0.1 1

time of migration (log scale)

Figure 7. Posterior distributions of time of migration (i.e., introgression; scaled to the neutral mutation rate) for those loci that showed evidence of introgression. Both mtDNA and CHD are consistent with recent and/or ongoing hybridization in sympatry, whereas mtDNA is more indicative of ancient introgression from falcated ducks to allopatric gadwalls in North America.

coalescent model indicated that some gene flow is necessary to

explain patterns of genetic diversity in both North American and Eurasian gadwalls. Specifically, the 95% HPD of introgression rates did not overlap zero in either comparison. Importantly, this result is not sensitive to mutation rates, and simultaneously ana-

lyzing three loci at least partially accounted for stochastic among- locus variance.

Second, lineage sorting is nearly complete for the two nuclear introns examined, despite mitochondrial paraphyly. LDH contained two fixed differences between species, and CHD contained one nearly fixed difference (only one allele was shared; Fig 3). Qualitatively, these among-locus comparisons suggest in-

trogression of mtDNA as the cause of paraphyly, because lineage sorting should proceed more rapidly in mtDNA. Formally testing this hypothesis using coalescent methods resulted in quantitatively rejecting incomplete lineage sorting for mtDNA. Specifically, the

strong between-species differences in nDNA suggested that gadwalls and falcated ducks diverged between 220,000 and 5,300,000 ybp (Middle Pleistocene or earlier). However, for incomplete lin-

eage sorting to explain the patterns in mtDNA, then these species likely diverged between 11,000 and 76,000 ybp (Late Pleistocene), a date that does not overlap that estimated from nDNA. In contrast, a model that assumed introgression of clade B into the gadwall gene pool suggested that these species diverged between 65,000 and 700,000 ybp (Middle-Late Pleistocene), a date that is consistent with that estimated from nDNA. In other words, even after

accounting for the stochastic variance of genetic processes (and uncertainties in mutation rates), nDNA is too deeply structured for the pattern observed in mtDNA to be explained by incomplete lineage sorting.

Although comparing estimates of time since divergence de-

pends on accurate mutation rates, three observations suggest that our conclusions were not sensitive to possible calibration errors.

First, we adjusted our converted parameters by incorporating a

range of plausible mutation rates that incorporate both uncer-

tainty in outgroup divergence times and stochastic variance in mutations. Second, we found a strong agreement in divergence times estimated from mtDNA and from introns among waterfowl

species, despite using different fossil data for rate calibrations.

Third, rejecting a hypothesis of no gene flow (see above) was not dependent on mutation rates. Therefore, rejecting incomplete lineage sorting seems to be well supported despite depending on mutation rates for some analyses.

DNA INTROGRESSION There was no evidence that introgressed DNA was more abun- dant in Asia where gadwalls and falcated ducks are sympatric. Peters and Omland (2007) estimated that between 77,000 and 190,000 North American gadwalls had clade B haplotypes and those haplotypes were widely distributed (i.e., Alaska, California,





Utah, Alberta, North Dakota, Arkansas, Quebec, Delaware, and

Maryland). A combination of factors likely contributed to the rela-

tively high frequency and widespread distribution of clade B. First, estimates of time of migration suggested that clade B in North America resulted from ancient introgression ('-14,000 ybp). Al-

though closely related, none of the clade B haplotypes were shared between falcated ducks and North American gadwalls, and these data are inconsistent with recent, ongoing hybridization. Second, mtDNA supported a rapid historical population expansion in North America (Peters and Omland 2007), and observational data have revealed substantial population and range expansions during the

past century (LeSchack et al. 1997). Therefore, a likely explana- tion for the strong incorporation of clade B in North America is that introgression occurred when the gadwall population size was much smaller than it is today and periods of expansion facilitated the spread of clade B.

In sympatry, hybridization seems to have been recent and/or ongoing, yet we found no evidence of introgression from gadwalls to falcated ducks. All analyses that included the migration parameter suggested asymmetrical gene flow (Table 2); DNA seemingly has introgressed from falcated ducks to gadwalls more often than the reverse (although 95% HPDs for m were broadly overlapping). One hypothesis is that this asymmetry resulted from differences in species' range sizes, coupled with male-biased sex ratios

(Kulikova et al. 2004). Because of their restricted distribution, va-

grant female falcated ducks sometimes might randomly disperse to areas where male falcated ducks are absent. In contrast, gadwalls are widely distributed, and by random chance, female gadwalls will be more likely to disperse to areas where conspecific males are present. Waterfowl sex ratios tend to be male-biased

(Baldassarre and Bolen 1994), so unpaired males may mate with females of either species rather than forego breeding. Under this

scenario, most hybridization would involve female falcated ducks and male gadwalls, and also as a consequence of differences in

population size and geographic distributions, hybrid offspring would be more likely to mate with gadwalls. If this scenario is

correct, then introgressed DNA should also be present in other Eurasian populations of gadwalls, especially those that are para- patric with falcated ducks.

ISOLATION-WITH-MIGRATION ASSUMPTIONS

Isolation-with-migration makes a number of implicit assumptions, including selective neutrality, no recombination, that the two populations being compared are each panmictic and randomly sampled, that those populations are not exchanging genes with other

populations or species, and that population sizes and migration rates have been constant over time (Hey and Nielsen 2004; Won et al. 2005). These assumptions will be violated to some extent in most (if not all) systems, but the sensitivity of IM results to these violations needs additional study (see Hey 2005). In this study,

Asian and North American gadwalls were significantly structured at all three loci suggesting at least three distinct populations of interest: falcated duck, Asian gadwall, and North American gadwall. There also exists significant mtDNA structure within North America, suggesting the possibility of additional populations (Pe- ters and Omland 2007). However, IM can only accommodate two

populations, leaving two options for handling structure: (1) lumping multiple populations as a single population, which violates the assumption of panmixia, or (2) treating each population in

separate analyses, which would likely violate the assumption of no gene flow with other populations. We obtained similar results

regarding time since divergence when lumping North American and Asian gadwalls in the same analyses (i.e., nDNA) and when

treating them in separate analyses (i.e., analyzing all three loci to-

gether). Therefore, the main conclusions of this study do not seem

overly sensitive to violations of assumptions regarding population structure.

The assumption of constant population sizes was also likely violated in this study. All three populations likely experienced recent population growth (Table 2; Peters and Omland 2007). Be- cause lineage sorting is a function of effective population size, as-

suming constant population sizes could potentially bias parameter estimates when species experienced more complex demographies. Isolation-with-migration can incorporate population growth into models of divergence, but this model assumes a constant rate of

exponential growth (Hey 2005), which may also be unrealistic for

many species. However, allowing for growth in one of our models (Asian gadwalls vs. falcated ducks) suggested that gadwalls and faclated ducks most likely diverged about 380,000 ybp (95% HPDadj = 110,000 to 1,100,000 ybp; data not shown), which is similar to time since divergence estimated when assuming constant population sizes. Therefore, time since divergence, in this

case, is robust to changing the assumption of past population de-

mography. Isolation-with-migration assumes that introgression rates

have been constant since species divergence, but hybridization between gadwalls and falcated ducks may be a recent phe- nomenon that occurred after secondary contact. Indeed, estimates of "time of migration" suggested that introgression has occurred

recently relative to species divergence, probably after the last

glacial maximum. If migration has not been constant, but instead these species were strictly isolated during most of their history and only recently came into secondary contact, then current in-

trogression rates might be much higher than estimated in this

study.

USING nDNA TO TEST HYPOTHESES OF MITOCHONDRIAL PARAPHYLY

Comparing nDNA to mtDNA offered strong resolution for dis-

tinguishing between incomplete lineage sorting and introgression

EVOLUTION AUGUST2007 2003




in this study. Furthermore, two nuclear introns were sufficient to

reject a hypothesis of incomplete lineage sorting, despite being a smaller sample size than other similar studies (e.g., Won and

Hey 2005; Won et al. 2005). The signal in those introns was in- creased by using information provided by the STR portions of

sequences (Won et al. 2005). For example, LDH contained two fixed indels (one of which was part of an STR) between species but no fixed differences in nucleotide substitutions (Fig. 3C). The

deep species divergence time relative to the recent introgression of mtDNA also likely contributed to the strong resolution provided by only two introns. Because the stochastic variance of genetic processes results in large confidence intervals, especially when few loci are studied, systems exhibiting smaller differences in diver-

gence times or more complex patterns of paraphyly will require more loci to reject alternative hypotheses (Edwards and Beerli

2000). Our approach of using multiple loci and coalescent methods

can be applied to any paraphyletic system. However, it is important to appreciate that introgression and incomplete lineage sort-

ing are not necessarily mutually exclusive hypotheses, and both

might have contributed to paraphyly within some systems (e.g., Kulikova et al. 2005; Sefc et al. 2005; Won et al. 2005). Under such a scenario, it might be difficult to generate and test predictions that unequivocally demonstrate that both have contributed to paraphyly.

In conclusion, despite some potentially limiting assumptions of IM, our approach provides a quantitative framework to better understand causes of gene tree paraphyly between closely related species. Furthermore, this is the first study to incorporate the stochastic variance of genetic processes and reject incomplete lineage sorting as the cause of paraphyly. Finally, this study illus- trates a compelling case of ancient hybridization that has lead to DNA crossing species boundaries and becoming strongly incor-

porated into the gene pool of the recipient.

ACKNOWLEDGMENTS We thank J. Bell and B. Alemi of the UMBC Department of Mathe- matics and Statistics and the UMBC Bioinformatics Research Center for providing computer resources. S. Freeland and N. Keulman, provided additional computer resources and consultation. The Academy of Natural Sciences, the University of Washington Burke Museum, and the Delaware Museum of Natural History contributed tissues from museum specimens. M. Maystrenko, P. Yung, and N. Yakovchits assisted with field work in Russia, and E. Humphries helped with laboratory work. T. Roberts, R. Fleischer, J. Leips, P. Sokolove, S. Freeland, B. Kondo, K. McCracken, and five anonymous reviewers provided valuable feedback during various stages of this study. We also thank the US Fish and Wildlife Service for issuing import permits. This research was funded by Delta Waterfowl Foundation; KEO was supported by National Science Foundation DEB- 0347083. Alaska EPSCoR (EPS-0346770) provided salary for JLP during the final stages of revisions.

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Associate Editor: K. Crandall

Supplementary Material The following supplementary material is available for this article:

Table S1. GenBank accession numbers for falcated ducks and gadwalls. Table S2. Additional numbers used for amplifying and/or sequencing smaller fragments of the mtDNA control region and allele

specific primers used for introns with multiple polymorphic positions. Table S3. GenBank accession numbers of sequences used for intron clock calibrations and for comparing divergence times estimated from introns and mtDNA. Table S4. List of vouchered specimens with GenBank accession numbers.

Figure St. Resolving simple indels.

Figure S2. Resolving indels with nucleotide polymorphisms. Figure S3. Marginal posterior densities of theta (effective population size scaled to the neutral mutation rate) estimated from the various models used.

Figure S4. Marginal posterior densities of introgression rates (scaled to the neutral mutation rate) estimated from nuclear introns

(CHD and LDH) using a model of Isolation with Migration.

This material is available as part of the online article from:

http://www.blackwell-synergy.com/doi/abs/10. 1111/j.1558-5646.2007.00149.x (This link will take you to the article abstract).

Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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