BARRIERS TO SYMPATRY BETWEEN AVIAN SIBLING SPECIES (PARIDAE: BAEOLOPHUS) IN LOCAL SECONDARY CONTACT1

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q 2004 The Society for the Study of Evolution. All rights reserved.

Evolution, 58(7), 2004, pp. 1573–1587

BARRIERS TO SYMPATRY BETWEEN AVIAN SIBLING SPECIES(PARIDAE: BAEOLOPHUS) IN LOCAL SECONDARY CONTACT1

CARLA CICEROMuseum of Vertebrate Zoology, 3101 Valley Life Sciences Building, University of California, Berkeley, California 94720-3160

E-mail: [email protected]

Abstract. Range limits and secondary contact zones often occur at ecotones between major associations of habitatand climate. Therefore, understanding processes that limit sympatry between species in such areas provides an importantframework for testing biogeographic and evolutionary hypotheses. Theoretical and empirical work has shown that theevolution of species borders is influenced by a complexity of factors, including gene flow from central to peripheralpopulations and the ability of species to adapt locally to environmental conditions. However, few studies have usedbioclimatic models, combined with molecular and morphological data, to predict geographic range limits in the contextof gene flow across a secondary contact zone. In this study, I applied these methods to test specific hypotheses aboutbarriers to sympatry between closely related species where they approach and contact each other. Specifically, Iexamined the importance of historical isolation, local adaptation, and symmetry of gene flow in limiting sympatryand range expansion of ecologically distinct species across environmental gradients. Molecular (mitochondrial DNA,allozymes), morphological, and bioclimatic data were obtained for two avian sibling species (Baeolophus inornatusand B. ridgwayi) that exist in recent, narrow secondary contact in northern California. These species are broadlyallopatric and occupy rangewide associations of oak and pinyon-juniper woodlands, respectively, although B. inornatusalso inhabits mixed or juniper woodlands locally. Patterns of molecular variation generally were congruent withmorphological and bioclimatic data, and support prior evidence for a history of isolation, adaptation, and divergencein distinctive, species-specific vegetation-climate associations. However, molecular and morphological clines fall eastof the limit of oaks, and individuals of B. inornatus in this juniper-associated contact zone experience bioclimatesthat are more similar to B. ridgwayi than to B. inornatus in oak habitat. Thus, B. inornatus is able to adapt and expandlocally into the range of its close relative, but not vice versa. These data support the hypothesis that gene flow isasymmetrical where peripheral populations meet at range boundaries. Physiological differences between species mayplay an important role in influencing these patterns. Empirical studies that highlight the importance of local adaptationand patterns of gene flow in which closely related species contact across ecotones are central to understanding limitson geographic ranges, sympatry, and introgression—a cornerstone of biogeographic and speciation theory.

Key words. Bioclimatic modeling, gene flow, historical isolation, local adaptation, range limits, secondary contact,sympatry.

Received May 5, 2003. Accepted March 29, 2004.

A fundamental question in evolutionary biology concernsbarriers that isolate species and limit their distributions. Be-cause hypotheses of biogeographic and evolutionary historyare crucial to understanding patterns and processes of speci-ation, much attention has been devoted to testing alternativepredictions about species’ ranges using both theoretical andempirical data (e.g., Kirkpatrick and Barton 1997; Case andTaper 2000; Johnson and Cicero 2002). These studies haveshown that the evolution of species’ borders is influenced bya complex interplay of evolutionary, ecological, and physio-logical processes. For example, gene flow from central to pe-ripheral populations may inhibit local adaptation to environ-mental conditions, thereby preventing expansion of species’ranges (Mayr 1963; Hoffman and Blows 1994; Garcıa-Ramosand Kirkpatrick 1997; Kirkpatrick and Barton 1997). Wheretwo species are locally sympatric, this process may be me-diated by interspecific competition (Case and Taper 2000).Although the geographic ranges of sister taxa do not alwaysreflect ecological differences on the time scale of speciation(Peterson et al. 1999), segregation into distinctive vegetation-climate zones clearly plays a role in dictating patterns of dis-tribution and speciation in some closely related groups (e.g.,sibling species of Empidonax flycatchers; Johnson and Cicero

1 This paper is dedicated to my mentor, colleague, and closestfriend Ned K. Johnson, who has been a great source of encour-agement, support, and inspiration.

2002). In addition, differences in temperature regulation canhave a direct role in separating sister species into allopatricranges (Hayworth and Weathers 1984). Finally, the relativeimportance of evolutionary versus ecological factors at theborder of species’ ranges will depend on the strength of en-vironmental gradients (Case and Taper 2000).

Studies of closely related taxa, especially where they occurin secondary contact along ecotones, provide a valuable frame-work for examining distributional limits and evolutionary bar-riers to gene exchange (e.g., Harrison and Arnold 1982; Lattaand Mitton 1999; Dessauer et al. 2000). The common asso-ciation between secondary contact zones and environmentalgradients can be explained by a balance between dispersal andselection (Barton and Hewitt 1985), the latter acting either ongenotype-specific responses to the environment (Endler 1977)or on fitness of intermediate genotypes (Barton and Hewitt1981, 1985). Alternatively, ecotones may be the most likelysites of sympatry between differently adapted parental taxa(Arnold 1997; Case and Taper 2000).

Although studies of secondary contact zones abound in theliterature (e.g., Sattler and Braun 2000; Rohwer et al. 2001;Matocq 2002; Garcıa-Parıs et al. 2003), relatively little at-tention has been directed toward the border between essen-tially allopatric sibling species (morphologically similar bi-ological species; Mayr 1942). Such cases, especially in birds,are ideal for testing alternative hypotheses about evolutionaryprocesses and limits on species’ distributions for several rea-

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sons. To begin with, sibling species are at relatively earlystages of divergence and thus offer special insight into pro-cesses such as gene flow, ecological diversification, and evo-lution of geographic ranges (Richman 1996; Price et al. 1997;Johnson and Cicero 2002). Furthermore, avian sibling speciesoften occupy broad ecogeographic regions that are allopatricwith those of their closest relatives, in contrast with manyavian congeners that display striking phenotypic differencesbut share broad-scale habitat formations. Importantly, allop-atry in these species-specific vegetation-climate zones ismaintained long after speciation, and habitat segregation con-tinues to play an important role where sibling species arelocally sympatric (Johnson 1963, 1980; Johnson and Cicero2002). Finally, sibling species often occur in reduced den-sities where their distributions approach and/or intermingle;therefore, populations at range boundaries probably are in-fluenced strongly by gene flow and environmental gradients.

In assessing the role of gene flow and environment onpatterns of species’ distribution and variation, it is essentialto separate the effects of history and current processes (Ma-tocq et al. 2000; Mahoney 2004). If geographic range limitsare attracted to ecotones (Case and Taper 2000), then twospecies with a history of isolation and adaptation in differentecological regions would be expected to approach sympatrywhere their preferred habitats and climates meet. Similarly,if gene flow inhibits adaptation of peripheral populations tolocal ecological conditions, especially where population den-sities decline (Garcıa-Ramos and Kirkpatrick 1997), then per-sistent directional selection will limit range expansion andcontact across these environmental gradients. These pro-cesses may or may not act symmetrically (Servedio and Kirk-patrick 1997), such that differences in the abilities of eachspecies to adapt locally should influence the extent and di-rection of introgression across range boundaries.

To test the role of evolutionary and ecological processesin influencing distributional limits and patterns of variation,I used molecular, morphological, and bioclimatic data to ex-amine barriers to sympatry between two avian sibling species(Baeolophus inornatus [oak titmouse] and B. ridgwayi [ju-niper titmouse]; Cicero 1996; American Ornithologists’Union 1997) in local secondary contact. These taxa are partof a diverse assemblage of birds with representatives thatmeet along a physiographic break in the western Great Basin(Johnson 1978; Johnson and Johnson 1985; Johnson and Mar-ten 1988). The two species have essentially nonoverlappinggeographic and habitat distributions (Cicero 2000), with B.inornatus inhabiting oak woodlands along the Pacific slopeand B. ridgwayi inhabiting pinyon-juniper woodlands of theIntermountain Region. However, recent analysis of cyto-chrome b sequences (Cicero 1996) revealed that secondarycontact may occur on the Modoc Plateau in extreme northernCalifornia, a physiographic province that links the Great Ba-sin with the Cascade-Sierra Nevada mountain chain. Impor-tantly, this study showed eastward expansion of B. inornatusinto pure juniper habitat more typical of B. ridgwayi, in anarea previously unknown to harbor titmice of either form(Grinnell and Miller 1944). Baeolophus inornatus also isknown to occupy pinyon-juniper woodland very locally insouthern California (Cicero 1996), and thus appears to havebroader ecological tolerance than B. ridgwayi.

Specific goals of this study were to: (1) understand thedynamics of population range expansion in B. inornatus andB. ridgwayi that may have led to secondary contact; (2) usemolecular and morphological data to characterize clines andpatterns of gene flow in relation to gradients in vegetationand climate, and to confirm that the species are in secondarycontact; and (3) use bioclimatic models to identify conditionsthat may limit sympatry and range expansion of peripheralpopulations across environmental gradients. Although fewstudies have applied bioclimatic models to predict geographicrange limits in the context of gene flow across a secondarycontact zone, such data can offer insight into ecological nich-es and the ability of different taxa to adapt locally to envi-ronmental conditions at range margins. Because there are nophysical barriers to introgression in the contact zone, I ex-pected that adaptation to distinctive vegetation-climate zonesduring historical separation would limit sympatry betweenthese species where they approach and potentially contact.Furthermore, because habitat specificity breaks down at theperiphery of the range of B. inornatus (Cicero 1996), I pre-dicted that gene flow (if any) would be asymmetrical towardthe range of B. ridgwayi.

MATERIALS AND METHODS

Sampling of Specimens

Previous study of geographic variation in B. inornatus andB. ridgwayi (Cicero 1996) entailed collection of 525 speci-mens from 32 sample areas throughout the western UnitedStates. In that analysis, all individuals were assayed allo-zymically and a subset of 52 specimens (27 sites) were ex-amined using mitochondrial DNA (mtDNA) sequencing tech-niques (cytochrome b). The sampling and results of that studyformed the framework for the current analysis, in which 211specimens from 14 sample sites were examined to addressthe questions of range limits and secondary contact on theModoc Plateau. These included: 131 specimens from six pre-viously studied sites in northern California and southernOregon, which were assigned as references either to B. in-ornatus or B. ridgwayi based on known, fixed mtDNA dif-ferences (because of their phenotypic similarity, morphologycould not be used to distinguish between species); 66 spec-imens (seven sample sites) collected from the Modoc Plateauregion specifically for this study; and 14 specimens from adisjunct peripheral population south of the Modoc Plateauthat was not studied previously and thus had unknown tax-onomic affinity. The density of individuals at various sitesdictated sample sizes; densities generally were low on theModoc Plateau, and some sites required multiple trips tocollect birds for analysis. The assessment of population rangeexpansion in the two species was based on 57 samples from30 sites (52 from the original study plus an additional five).Both species are nonmigratory (Cicero 1996), and thus spec-imens collected during either summer or fall were includedin the study. Specimens and tissues are deposited in the Mu-seum of Vertebrate Zoology (MVZ), University of California,Berkeley (http://www.mip.berkeley.edu/mvz); see Appendixfor MVZ numbers.

1575BARRIERS TO SYMPATRY IN A CONTACT ZONE

Sequencing, PCR-RFLP, and Analysis ofMitochondrial DNA

Because this study focused on the putative contact zone, the300-bp fragment of cytochrome b sequenced manually in themacrogeographic study (Cicero 1996: see table 4 for primers)was used to examine broad-scale genetic patterns of populationdivergence and range expansion in the two species. Geographicstructuring was assessed for all sequences (B. inornatus andB. ridgwayi combined, n 5 57) by neighbor joining usingPAUP*, beta test version 4.0b8a for Power Macintosh (Swof-ford 2001; Tamura-Nei and Kimura two-parameter distances).Trees were rooted with two sequences of Baeolophus bicolor(Cicero 1996). Subsequently, sequences were pooled accord-ing to unique haplotype and analyzed separately for each spe-cies using standard population genetic measures (Arlequin ver-sion 2.0, Schneider et al. 2000). Nucleotide diversity (Nei1987) and its variance were computed to estimate the meannumber of nucleotide differences among haplotypes. Tajima’sD (Tajima 1989a,b) was obtained to test for deviation fromselective neutrality or constant population size within eachgroup; assuming neutrality, this statistic can be used to inferdemographic history, whereby a value of zero is expected forstable populations and a negative value may indicate recentchanges in effective population size. Analysis of molecularvariance (AMOVA; Excoffier et al. 1992) was performed toexamine hierarchical patterns in the distribution of geneticdiversity. This method, which provides an analysis of genefrequencies while taking into account the number of mutationsbetween molecular haplotypes, was used to obtain standardvariance components and F-statistics at three hierarchical lev-els of subdivision; that is, between species, among populationswithin species, and within populations. Mismatch distribu-tions, which compare the frequency of observed pairwise dif-ferences among haplotypes, were obtained to examine whetherthe two species show patterns typical of an expanding popu-lation (Slatkin and Hudson 1991; Rogers and Harpending1992). Finally, pairwise M was calculated between species toestimate gene flow (Nm) using the relationship M 5 (1 2 FST)/2FST (Slatkin 1993).

Sequencing of a 900-bp fragment of cytochrome b waslimited initially to two individuals from each of the six pre-viously studied reference sites, both to verify levels of se-quence divergence—which were essentially identical to the300-bp fragment—and to identify diagnostic restriction en-zymes for a more comprehensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) anal-ysis of contact zone samples (see below). On the basis ofresults of the latter method, 18 additional individuals weresequenced for the same 900-bp fragment for sequence veri-fication. Double-stranded products were extracted and am-plified under the same general conditions as the smaller frag-ment (Cicero 1996), but sequences were obtained with anABI Prism 377 (Applied Biosystems Inc., Foster City, CA)automated sequencer rather than manually (for protocols, seeCicero and Johnson 2001). Additional primers were used toamplify and sequence this longer fragment (L15236, H15706;Cicero and Johnson 2001, table 2). Two negative controlswere included in all extraction and amplification experiments,fragments were sequenced in both directions, and there was

no evidence of nuclear copies (for criteria to guard againstcoamplified peaks, see Sorenson and Quinn 1998; Johnsonand Cicero 2002). Sequences are deposited in GenBank (ac-cession numbers AY607659–AY60788).

Given the large number of specimens (n 5 211), PCR-RFLP was employed as a rapid and cost-effective method oftyping individuals with respect to mtDNA haplotype. Thisapproach has been used in phylogeographic studies (Rueggand Smith 2002; Stone et al. 2002) as well as detailed anal-yses of contact zones (Ruedi et al. 1997; Rohwer et al. 2001).Sequences were surveyed for diagnostic restriction enzymesusing the Restrict algorithm of MacMolly (ver. 3.0; Scho-neberg and Priedemuth 1989). Of the 164 possible enzymes,four (Sty I, Dpn II, Hinf I, and Alu I) were found to be mostinformative and cost efficient. Individuals were assayed witheach enzyme separately by performing restriction digests onthe same PCR-amplified target of 900 bp. Total reaction vol-umes of 10 ml included the enzyme (0.5–1.0 ml), an enzyme-specific buffer (1 ml), 4 ml of double-stranded PCR product,and a balance of double-distilled water; Sty I also requiredthe addition of bovine serum (1 ml). Reaction conditions,including incubation (overnight in a water bath at 378C) andheat inactivation (658C for 20 min, if needed), followed therecommendations of the commercial enzyme vendor (NewEngland BioLabs, Beverly, MA). Digested products were vi-sualized on 2.5% agarose gels stained with ethidium bromide,then scored according to haplotype pattern.

Analysis of Polymorphic Allozyme Loci

Broad-scale analysis of allozyme variation in the two spe-cies involved a survey of 41 loci, of which 16 were poly-morphic (n 5 525, 32 samples, overall FST 5 0.106; Cicero1996). Although none of the loci had fixed allelic differences,three showed relatively strong geographic structuring acrossthe Modoc Plateau: aminopeptidase leucyl-glycyl-glycine(LGG; Enzyme Commission [EC] no. 3.4.11.4; FST 5 0.160across all samples), glucose-6-phosphate isomerase (GPI; EC5.3.1.9; FST 5 0.081), and purine-nucleoside phosphorylase(NP; EC 2.4.2.1; FST 5 0.059). Consequently, individualshaplotyped for mtDNA (sequencing or PCR-RFLP) were alsoassayed for these loci to assess concordance in patterns ofallele frequency change. Protocols and conditions for allo-zyme electrophoresis were identical to those used in the ma-crogeographic study (Cicero 1996). F-statistics were com-puted using BIOSYS-1 (Swofford and Selander 1981), andgene flow (Nm) was estimated with Wright’s (1951) formulaNm 5 1/4(1/FST 2 1).

Morphometrics and Discriminant Function Analysis

Eight linear external measurements (wing length, tail length,tarsus length, length of middle toe, hallux length, bill length,bill width, bill depth) were recorded from the same specimensused in the molecular analyses to assess concordance betweenmorphology and molecules near the putative contact zone.Measurements were taken according to standard protocols(Cicero 1996) on adult birds of known sex; juvenile or im-mature birds as determined by incomplete skull ossificationwere excluded. Body mass (gm) also was recorded from spec-imen data and transformed to cube root for analysis. Mor-

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phometric data were grouped by samples and compared usingstepwise discriminant function analysis (StatSoft 2001). Males(n 5 97) and females (n 5 55) were analyzed separately be-cause of sexual dimorphism in size (Cicero 1996).

Bioclimatic Data

Recent advances in geographic information systems (GIS)and bioclimatic modeling have enabled detailed examinationand/or prediction of broad-scale species’ distributions in re-lation to environmental factors (Chen and Peterson 2000;Peterson 2001). Such models have been applied in a varietyof organisms to address questions of ecology, biogeography,geographic variation, systematics, evolution, conservation bi-ology, climate change, and ecological niche conservatism(e.g., plants: Sykes et al. 1996; Gignac 2000; insects: Ritchieet al. 2001; snakes: Nix 1986; birds: Peterson et al. 2001;Feria and Peterson 2002; mammals: Fischer et al. 2001; Limet al. 2002; sister taxa of birds, mammals, and butterflies:Peterson et al. 1999). Few models, however, have focusedon areas of contact between close relatives (e.g., Ritchie etal. 2001). In such cases, bioclimatic modeling combined withmolecular and/or morphological data can provide a powerfultool for examining barriers to sympatry between species atrange boundaries.

Prior canonical correlation analysis across the ranges of B.inornatus and B. ridgwayi (Cicero 1996) revealed a significantassociation of phenotype and genotype with 26 environmen-tal variables (elevation, latitude, and longitude of specimenlocalities, plus 23 temperature and precipitation measurestaken from weather stations during breeding and winteringseasons). These data suggested that the two species occupydifferent climatic regimes and that physiological differencesmay be important in limiting their respective distributions.To examine climatic variation across the contact zone, I useda more refined bioclimatic modeling approach in which 15bioclimatic indices (temperature, 8C; precipitation, mm) wereextracted with the BIOCLIM algorithm (Nix 1986) across a1-km-resolution grid of points; the program DIVA-GIS (Hij-mans et al. 2004) was used for this analysis. BIOCLIM var-iables included annual mean temperature, maximum tem-perature of the hottest month (BC2), minimum temperatureof the coldest month (BC3), annual temperature range (BC2– BC3), mean temperature of the wettest three-month quarter,mean temperature of the driest three-month quarter, minimumdaily temperature range, mean daily temperature range, max-imum daily temperature range, annual mean precipitation,mean precipitation of the wettest month (BC11), mean pre-cipitation of the driest month (BC12), annual precipitationrange (BC11 – BC12), mean precipitation of the wettestthree-month quarter, and mean precipitation of the driestthree-month quarter.

BIOCLIM data were extracted using georeferenced local-ities (latitude, longitude) of all B. inornatus and B. ridgwayispecimens in the MVZ collections. Three models were runto map predicted ranges based on the bioclimatic data: (1)all specimens of pure B. inornatus, that is, all specimensexcept for those from the putative contact zone (n 5 887specimens, 262 unique localities, and 256 points, where apoint represents the center of a 1-km grid cell; thus, localities

in the same grid cell were counted as one point); (2) allspecimens of pure B. ridgwayi (n 5 577 specimens, 116 lo-calities and points); and (3) specimens of B. inornatus/B.ridgwayi from localities on the Modoc Plateau where theywere not known to occur prior to this study (Grinnell andMiller 1944), and where they putatively contact (n 5 56specimens, 17 localities, 12 points). Statistical analyses (t-tests; StatSoft 2001) were run to compare mean values forthe 15 variables between pure B. inornatus and B. ridgwayi.Maps of the predicted distributions of pure B. inornatus andB. ridgwayi enabled analysis of the extent to which the twospecies share bioclimatic envelopes (5.0–95.0% and 2.5–97.5% percentiles of points). Likewise, mapping of points onthe Modoc Plateau permitted localization of the contact zonerelative to predicted species-specific distributions. BIOCLIMvariables plotted by site allowed examination of climatic gra-dients in relation to habitat and species’ range boundaries.

RESULTS

Macrogeographic Mitochondrial DNA Variation andPopulation Range Expansion

A broad-scale analysis of 300-bp cytochrome b sequencesin B. inornatus (n 5 31) and B. ridgwayi (n 5 26) revealedsix haplotypes and two major clades that are strongly dif-ferentiated (mean 5 4%, Fig. 1). Within B. inornatus, threesubclades are recognized: subclade A on the western slopeof the Sierra Nevada, subclade B in southwestern California,and a third subclade that includes all other samples in Cal-ifornia and southern Oregon. According to the AMOVA (Ta-ble 1), over 90% of the variation is apportioned between thetwo species and approximately 10% is due to inter- or intra-population differences in haplotype within species. Nucleo-tide diversity within both species also is very low (Table 2),although diversity is an order of magnitude higher in B. in-ornatus than B. ridgwayi. Because neutrality (Tajima’s D,Table 2) cannot be rejected for either B. inornatus or B. ridg-wayi, the negative values may be used to infer recent changesin effective population size. Likewise, mismatch distributionsshow a unimodal pattern that is not significantly differentfrom that expected under a sudden expansion model (Fig. 2).Estimated gene flow (M) between species is low (0.024);values greater than one are expected to prevent populationsfrom differentiating due to drift alone (Slatkin 1993; Millsand Allendorf 1996).

A more detailed analysis of 900-bp mtDNA sequencesfrom the six reference sites in northern California and south-ern Oregon revealed four haplotypes within B. inornatus thatdiffer by one to three mutations (Fig. 3). The most distinctivehaplotype (Oroville sample) corresponds with subclade A onthe western slope of the Sierra Nevada (Fig. 1). Individualsfrom two additional populations (Shasta Valley, Fall RiverMills) shared haplotypes with their geographic neighbors.Because Fall River Mills represents a disjunct and previouslyunsampled population, the data corroborate that those birdsare allied with B. inornatus rather than B. ridgwayi. On thebasis of the haplotype network generated by these sequences,patterns of range expansion toward the putative contact zonecan be hypothesized (Fig. 3). Although previous study (Cic-ero 1996) provided evidence for northward dispersal from a


FIG. 1. Neighbor-joining tree of cytochrome b sequences (top) andmap of broad haplotype distribution (bottom) for Baeolophus in-ornatus and B. ridgwayi. Dark and light shading represents rangesof the two species, respectively. Haplotypes that define two sub-clades within B. inornatus are designated as A and B; asterisksadjacent to letters on the map indicate sites that are polymorphicfor two B. inornatus haplotypes (A or B and the more widespreadhaplotype). Sequences of B. ridgwayi are identical except for aunique haplotype in northwestern Utah (one bp difference, not il-lustrated on map), and an ancestral polymorphism for the inornatusB haplotype in the Providence Mountains of southeastern California(indicated by the letter in parentheses). The neighbor-joining treeis based on Kimura two-parameter distance; an identical tree wasobtained with Tamura-Nei distance. Numbers above the branchesindicate branch lengths.

southwestern origin of diversification in these species, thesource of B. inornatus on the Modoc Plateau was unknown.Current data resolve that question. A single-step mutation inmtDNA haplotype from the Coast Range (Clear Lake sample)to the northern edge of the Sacramento Valley, with a sub-sequent leap and mutation across the Mount Shasta region—which is inhospitable to titmice—into Shasta Valley and the

Rogue River Valley (Medford sample), suggests that popu-lations from the Coast Range are the probable ancestors. Thiscontrasts with the unlikely hypothesis that birds from thewestern slope of the Sierra Nevada (Oroville sample), whichare more distinct in mtDNA as well as morphology (Cicero1996), served as the source for those on the Modoc Plateau.Occupancy of mixed oak-juniper habitats in Shasta Valleyenabled eastward dispersal toward the range of B. ridgwayi,which likewise spread northward along the western edge ofthe Great Basin from a southwestern ancestor (Cicero 1996).Dispersal of individuals across the Modoc Plateau from bothwestern and eastern sources thus provided opportunity forsecondary contact in this region.

Mitochondrial DNA Haplotype Variation across the Zone ofSecondary Contact

Analysis of 900-bp cytochrome b fragments using PCR-RFLP and verified by sequencing detected three groups ofhaplotypes (Fig. 4). All samples of B. inornatus north of MountShasta (sites 1 through 8) shared a single PCR-RFLP haplo-type, which was distinct from the haplotype that characterizedB. inornatus south of Mount Shasta (sites 14 and 15, plusClear Lake and Oroville). However, two of 61 individuals fromthe southern group (3.3%; one each from North SacramentoValley and Clear Lake) were diagnosed with the more northernhaplotype. A third fixed haplotype diagnosed individuals ofB. ridgwayi (sites 11 through 13 and site 16).

The PCR-RFLP data clearly identified two sample sites aspolymorphic for B. inornatus/B. ridgwayi haplotypes (sites 9and 10, Fig. 4), with a sharp shift in mtDNA haplotype fre-quency between them. These sites are approximately 11–18km apart, and efforts to find birds at intermediate localitieswere unsuccessful; if one or both species occur, they pre-sumably are at very low density. Of the 14 individuals sam-pled from the western site (Lava Beds National Monument),11 (79%) had the mtDNA haplotype characteristic of B. in-ornatus. Likewise, 10 of 13 individuals from the eastern site(77%, Tule Lake Highway) had the haplotype characteristicof B. ridgwayi. Evidence for limited mtDNA introgressionacross these sites also comes from data on mated pairs. Al-though such pairs are difficult to collect on the Modoc Pla-teau, especially given the low population densities, I obtainedeight pairs during the course of several trips to the two sitesin the contact zone (59.3% of individuals analyzed). Of thefour pairs from each site, only two (one per site) were iden-tified as heterospecific matings based on mtDNA haplotypes;in both cases, the male had a B. inornatus haplotype and thefemale a B. ridgwayi haplotype. This percentage is equivalentto the overall ratio of mtDNA haplotypes at the two sites,and thus might reflect random rather than selective matings.Nonetheless, the sharp change from one mtDNA lineage toanother over a narrow distance suggests that strong selectionis limiting mtDNA introgression in this region.

Concordance between Mitochondrial DNA, Allozymes,and Morphology

Allozyme loci, especially LGG and GPI, showed strongfrequency differences across the mtDNA contact zone (Fig.5). These results are congruent with patterns observed across

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TABLE 1. Hierarchical analysis of molecular variance for cytochrome b haplotypes of Baeolophus inornatus and B. ridgwayi (n 5 57,six haplotypes, two groups). F-statistics are analogous to Wright’s (1951) F-statistics and indicate the correlation of random haplotypesat each hierarchical level. Significance of the variance components and F-statistics was tested using 1023 nonparametric permutations(haplotypes permuted among populations among and within groups, and populations permuted among groups). P-values were obtainedby comparing observed values with those generated by the random permutations.

Source of variation df

Observed partition

Variance % Variation P F

Among groups (FCT)Among populations within groups (FSC)Within populations (FST)

12827

4.980.130.37

90.832.426.76

,0.00010.002

,0.0001

0.9080.2630.932

TABLE 2. Nucleotide diversity indices and Tajima’s D statistics for haplotypes of Baeolophus inornatus and B. ridgwayi. Values arebased on 300-bp sequences of the cytochrome b gene. Neutrality in samples of either species could not be rejected on the basis ofTajima’s D.

TaxonSample

sizeNo. of

haplotypesNucleotidediversity Tajima’s D

B. inornatusB. ridgwayi

3126

42

0.00292 6 0.002340.00026 6 0.00057

20.8274 (P . 0.05)21.1556 (P . 0.05)

a broader geographic scale (Cicero 1996). Samples of B. in-ornatus were essentially fixed for one allele at the GPI locus,with a second allele appearing just west of the contact zone(Mt. Dome sample, site 8 in Fig. 4) and increasing in fre-quency eastward across the zone to pure B. ridgwayi. Thispolymorphism was absent at Double Head Mountain (site11), a result that likely reflects small sample size (n 5 4).Similarly, B. ridgwayi showed a sharp increase in frequencyof the less common alleles at the LGG locus, with a cleartransition across the mtDNA contact zone. Although the pat-tern presented by NP was less obvious, samples of B. ridgwayialso showed reduced polymorphism at this locus relative toB. inornatus. Changes in frequency of both allozyme andmtDNA loci were not coincident with the eastern limit of theoak zone occupied by titmice in northern California (Fig. 5).On the basis of these three allozyme loci, gene flow estimatesranged from 1.09 to 2.47 migrants per generation, which iscomparable to the value estimated for the entire range of bothspecies based on 32 samples and 16 polymorphic loci (Nm5 2.11; Cicero 1996).

Discriminant function analysis of morphometric datashowed strong congruence with the molecular data in sepa-rating specimens of B. inornatus from the larger B. ridgwayi(Fig. 6). Specimens of B. inornatus north and south of MountShasta, although distinguishable by mtDNA haplotype, wereequivalent in overall size patterns. Importantly, birds fromthe mtDNA contact zone (Lava Beds National Monument,Tule Lake Highway) were intermediate in size, revealing astep-cline in morphology that corresponds geographicallywith shifts in frequency of both mtDNA and allozyme mark-ers. However, mtDNA type did not appear associated withsize among individuals taken from this region; for example,three relatively small birds had B. ridgwayi haplotypes, andthree relatively large birds had B. inornatus haplotypes.

Bioclimatic Models and the Environmental Gradient

Mean bioclimatic indices generated for pure B. inornatusand B. ridgwayi showed significant differences in 14 of 15

variables (t-tests, P , 0.0001), thus providing strong evidencethat the two species occupy different bioclimatic regimes. Theonly variable that was nonsignificant was maximum temper-ature of the hottest month (P 5 0.33). These results suggestthat both species tolerate warm summer temperatures, but B.ridgwayi occupies regions of more extreme winter tempera-tures and temperature seasonality (daily and annual). In con-trast, seasonality in precipitation is greater for B. inornatusthan B. ridgwayi. As expected, B. ridgwayi is subject to lowerwinter precipitation but higher summer rainfall (e.g., summermonsoons in the southwestern United States).

The predicted distributions for the two species showed dif-ferent patterns (Fig. 7) and, importantly, barely overlapped. Ingeneral, the distribution of B. inornatus closely matched theoccurrence of datapoints from southwestern Oregon to northernBaja California. However, a few points representing B. inornatusoccurred within the predicted distribution of B. ridgwayi. In-terestingly, these were in areas where overlap was most ex-pected: (1) on the Modoc Plateau near sites of secondary contact(three points; see below for more detail); and (2) on the south-eastern slope of the Sierra Nevada north of Walker Pass andthe Kern River area (two points), where coastal and interiorbiotas meet and B. inornatus occupies mixed habitats (Cicero1996, 2000). The model for B. ridgwayi likewise showed arelatively good fit to the points at the western edge of the range,where point density is higher. However, in other areas the pre-dicted distribution had a greater geographic range than reflectedby the datapoints, extending from eastern Oregon southeastwardto eastern Wyoming, Colorado, the panhandle of Oklahoma,and western Texas. While the species does occur in centralColorado, western Oklahoma, and the Guadalupe Mountains ofextreme northwestern Texas, it is not known from many areaswhere it was predicted by the model (northeastern Colorado,southeastern Wyoming, southwestern Idaho, and eastern Oregonnorth of Steens Mountain; see Cicero 1996). In some areas, themodel did not predict B. ridgwayi (even suboptimally) whereit is known to occur; that is, northeastern Utah, northwesternColorado, and extreme southwestern Wyoming. Inclusion of


FIG. 2. Mismatch distributions for Baeolophus inornatus and B.ridgwayi based on 300 bp sequences of cytochrome b. The observedand expected distributions are not significantly different (P . 0.05)and are consistent with rapid range expansions.

additional datapoints from non-MVZ specimens, modeling ofmore variables, or application of other modeling algorithms withdifferent rules (e.g., genetic algorithm for rule-set prediction[GARP], Anderson et al. 2002), may yield different patterns. Itis noteworthy that there were no points representing B. ridgwayiwithin the predicted range of B. inornatus.

The B. inornatus model accurately predicted a distribu-tional gap across the Mount Shasta region, supporting evi-dence from mtDNA that populations in extreme northern Cal-ifornia and southwestern Oregon are isolated from those fur-ther south. On the Modoc Plateau, the predicted distributionsof B. inornatus and B. ridgwayi were essentially parapatric(Fig. 7). Although points from the contact zone included B.inornatus in juniper habitat (as identified by mtDNA, thisstudy), their predicted occurrence was completely allopatricfrom the prediction for B. inornatus modeled separately. Allof these contact zone points fell within the extreme westernedge of the prediction for B. ridgwayi.

Plots of climatic gradients across sites on the Modoc Pla-teau shed further light on patterns in this region, particularlyin relation to changes in vegetation (Fig. 8). All variablesshowed clear gradients except for precipitation of the driestmonth. In general, temperature and precipitation declineseastward across the region, whereas daily and annual tem-perature range increases; these patterns coincide with an in-crease in elevational gradient eastward across the Modoc Pla-

teau. For most variables, the sharpest gradient occurs at thetransition from oak to juniper-dominated woodlands. Thus,populations of pure B. inornatus in juniper (sites 6 through8, Fig. 4) experience bioclimates that are more similar to B.ridgwayi than to B. inornatus in oak habitat. Bioclimateswhere B. inornatus and B. ridgwayi contact (sites 9 and 10)fall intermediately along the gradients.

DISCUSSION

This study provides empirical data that intersect two majorfields of inquiry in evolutionary biology: the evolution ofgeographic ranges, where much attention has focused onrange limits (Grinnell 1917; Johnson 1978; Case and Taper2000), range sizes (Rapoport 1982; Price et al. 1997; Webband Gaston 2000), range changes (Johnson 1994), and extentof overlap between close relatives (Johnson and Cicero2002); and the role of secondary contact zones, which typ-ically occur at the periphery of geographic ranges, in elu-cidating evolutionary processes (Harrison 1990, 1993). Al-though unraveling the complex interplay of evolutionary andecological processes at species’ borders is a challenging task(Garcıa-Ramos and Kirkpatrick 1997; Case and Taper 2000),it is central to understanding factors that promote and/or re-inforce reproductive isolation between species. Likewise, ex-amining the symmetry of such processes is a crucial com-ponent of this task (Servedio and Kirkpatrick 1997). Thesedata, together with evolutionary and ecological niche theory(e.g., Garcıa-Ramos and Kirkpatrick 1997; Peterson et al.1999), offer a valuable framework for elucidating patternsand processes of speciation and biogeography, and for ad-dressing pertinent conservation issues such as the effect ofenvironmental change on range boundaries (e.g., Johnson1994).

Historical Isolation, Post-Pleistocene Expansion, andAdaptation to Distinctive Habitats and Climates

Broad-scale analysis and calibration of molecular diver-gences between B. inornatus and B. ridgwayi, combined withdata on the evolutionary history of plant communities in thewestern United States, suggest a scenario of historical iso-lation, divergence, and postglacial range expansion that ac-counts for current patterns of distribution, phenotypic traits,and genetic variation (Cicero 1996). Mitochondrial DNA dis-tances place the split of these sibling species in the earlyPleistocene (1.9 million years ago), whereas estimates basedon allozyme distances indicate much more recent (Late Pleis-tocene, t 5 64,400–196,200 years ago) divergence times(Cicero 1996). Regardless of this discrepancy, the evolutionand biogeography of B. inornatus and B. ridgwayi closelyparallel the Pleistocene/Holocene history of oak and pinyon-juniper woodlands that characterize the habitats and rangesof these species, respectively (Cicero 1996 and referencestherein; Betancourt 1987; Betancourt et al. 1990; Byrne etal. 1991; Thompson and Anderson 2000). Although glacialplant communities were compressed and mixed relative tomodern vegetation types (Graham et al. 1996), California andGreat Basin woodlands have had independent evolutionaryhistories since the late Pliocene or early Pleistocene (Axelrod1958, 1973). Thus, populations of B. inornatus and B. ridg-

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FIG. 3. Haplotype network (900-bp sequences of cytochrome b)and hypothesis of dispersal toward the putative contact zone (shownby asterisk) for samples of Baeolophus inornatus and B. ridgwayiin northern California and southern Oregon. Ranges are shaded asin Figure 1. Reference samples for PCR-RFLP include: B. inornatus,Medford, Northern Sacramento Valley, Clear Lake, Oroville; B.ridgwayi, Warner, and Northeastern California. Shasta Valley andFall River Mills were included to show probable patterns of dis-persal toward the eastern limits of the range of B. inornatus. North-ward versus southward movement is hypothesized because of asouthwestern origin of diversification in these species (Cicero1996), and because the paleobotanical history of oak and juniperwoodlands (see Discussion) makes this the only plausible scenario.Furthermore, allozyme evidence suggests a recent founding eventin the Rogue River Valley of southern Oregon (Medford sample,Hobs 5 0.006; Cicero 1996).

wayi have had a long opportunity for isolation, adaptation,and genetic drift in distinctive vegetation-climate zones.

Through glacial and interglacial periods, B. inornatus andB. ridgwayi probably experienced repeated range shifts as-sociated with climate changes that enabled southern ancestralpopulations to move northward via dispersal by pioneersalong the Pacific slope and Intermountain Region, respec-tively (Cicero 1996). The fact that molecular divergence isgreatest between northernmost populations supports the no-

tion that these have had the longest history of separation,both ecologically and physically (i.e., by the Cascade-SierraNevada barrier). Southwestern populations, on the otherhand, have been in relatively recent contact. MitochondrialDNA data from B. ridgwayi in the eastern Mojave Desertrevealed one of 15 individuals with a B. inornatus haplotype,and allozyme distances were found to be relatively minorwhen compared to Pacific slope samples (Cicero 1996, C.Cicero, unpubl. data). Interestingly, hierarchical analysis ofallozyme data for these populations yielded an estimated di-vergence time of only t 5 9850 years ago, which coincideswith the regional expansion of southwestern deserts duringthe Pleistocene/Holocene transition (Axelrod 1983).

The role of ecological adaptation in driving speciation andgeographic ranges has been well documented in other groupsof closely related species (e.g., Richman and Price 1992;Price et al. 1997; Johnson and Cicero 2002). Likewise, nu-merous studies have emphasized post-Pleistocene environ-mental changes in driving observed or potential secondarycontact of close relatives through expansion of populationranges (e.g., Cicero and Johnson 1998; Demboski and Cook2001; Stone et al. 2002). Along the same lines, the mtDNAdata for B. inornatus and B. ridgwayi strongly support a sce-nario of rapid range expansions, which followed the Holocenespread of favorable climates and habitats for the two siblingspecies (Cicero 1996). Although this scenario is repeatedamong a diversity of organisms, the application of bioclimaticmodels in a phylogeographic framework (e.g., Zink 1996;Avise 2000) enable closer examination of geographic rangesin the context of evolutionary and ecological diversification.The bioclimatic and modeling data for Baeolophus reflectcurrent rather than historical conditions, but unequivocallysupport the hypothesis that pure populations of B. inornatusand B. ridgwayi are tied to species-specific, broadly non-overlapping climatic regimes associated with their preferredhabitat types. Furthermore, adaptation to these vegetation-climate zones has occurred essentially in allopatry, with pos-sibly some gene exchange to the south as recently as the LatePleistocene. This historical perspective suggests that isola-tion and associated evolutionary processes (i.e., selection,drift) dictate the modern distributions of B. inornatus and B.ridgwayi where they approach on the Modoc Plateau. How-ever, differences in the ability of the two species to adaptlocally to environmental conditions (habitat, climate) appearparamount in setting boundaries to sympatry and in estab-lishing geographic range limits in this region. Empirical (thisstudy) and theoretical (Garcıa-Ramos and Kirkpatrick 1997;Case and Taper 2000) data on the importance of regional andlocal adaptation in determining species borders are crucialfor understanding how habitat tracking and colonization abil-ity influence biogeographic patterns at all scales (e.g., rangeexpansions in western North American birds, Johnson 1994;continental ranges of Phylloscopus, Price et al. 1997; geo-graphic overlap of clades of Empidonax, Johnson and Cicero2002).

Secondary Contact, Asymmetry of Gene Flow, and LocalAdaptation as a Barrier to Sympatry

Studies of sympatry and introgression between close rel-atives require an understanding of both historical and ongoing


FIG. 4. Sample sites and mtDNA haplotypes of Baeolophus inornatus and B. ridgwayi in northern California and southern Oregon.Haplotype frequencies are based on PCR-RFLP analysis of a 900-bp fragment of cytochrome b using four restriction enzymes (seeMaterials and Methods). Samples are plotted on a vegetation map to illustrate associations with the preferred habitat type of the twospecies (oak and juniper-dominated woodland, respectively). Note the patchiness of juniper in the interior. Sampling is more concentratedwest of the contact zone because of efforts to pinpoint the eastern limit of B. inornatus in atypical juniper habitat, and because of thenotably low density of B. ridgwayi in this region (Cicero 1996). Site names and sample sizes are as follows: 1, Medford (n 5 15); 2,Copco (n 5 10); 3, Little Shasta River (n 5 3); 4, Juniper Flat (n 5 11); 5, Secret Spring Mountain (n 5 11); 6, Dorris (n 5 5); 7,Cedar Mountain (n 5 8); 8, Mount Dome (n 5 12); 9, Lava Beds National Monument (n 5 14); 10, Tule Lake Highway (n 5 13); 11,Double Head Mountain (n 5 4); 12, Big Sage Reservoir (n 5 10); 13, Warner (n 5 16); 14, North Sacramento Valley (n 5 16); 15,Fall River Mills (n 5 14); and 16, Northeastern California (n 5 18). Clear Lake (n 5 17) and Oroville (n 5 14) also were analyzed byPCR-RFLP but are not illustrated on this map; see Figure 3 for site locations.

processes. For example, it is crucial in such analyses to dis-tinguish between secondary contact and in situ differentiationwhen explaining patterns of variation where species overlap.Although these two processes may be difficult to distinguishusing clinal analysis of single-locus traits (Endler 1977; Har-rison 1993; Durrett et al. 2000), evidence for secondary con-tact is provided by concordant clines of different traits, es-pecially some that are known to be neutral (Barton and Hewitt1981; Dessauer et al. 2000). Similarly, tracking the timingof secondary contact relative to the history of isolation is

critical for explaining current patterns and processes. Mo-lecular and morphological data often are used both to examinepatterns in secondary contact zones and to infer historicalevents (e.g., Matocq 2002; Garcıa-Parıs et al. 2003; Mahoney2004). However, an independent assessment of such eventsallows for much stronger inference and hypothesis testing.

Baeolophus inornatus and B. ridgwayi exhibit concordantpatterns of variation in morphology, mtDNA, and allozymes(Cicero 1996; this study), thus providing convincing evidencethat the two species are in secondary contact on the Modoc

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FIG. 5. Shifts in frequency of mtDNA haplotypes (black dots) andalleles at three allozyme loci (NP, LGG, GPI, white symbols) acrossthe contact zone (samples 1 through 13, Fig. 4). Letters in paren-theses after the locus name refer to allele designations; allelic fre-quency data are available from the author. The position of the con-tact zone is determined by the sharp change in cytochrome b se-quences. The eastern limit of oaks, the preferred habitat of Baeo-lophus inornatus, is illustrated for reference; all other sites are injuniper woodland.

FIG. 6. Discriminant function analysis of size data for specimensfrom 16 sample areas in northern California and southern Oregon(see Fig. 4). Some samples were combined for analysis because ofgeographic proximity and to boost sample sizes. Because onlyadults were measured, the total number of specimens is less thanfor the mtDNA and allozyme analyses. Samples north and south ofMount Shasta were analyzed as separate transects, and individualsare coded according to mtDNA haplotype (Fig. 4). Sample areasare numbered as in Figure 4.

Plateau in northern California. Furthermore, these data gen-erally are congruent with information derived from biocli-matic modeling. Two independent lines of evidence suggestthat contact between the two species occurred within ap-proximately the last 5000 years: (1) the paleobotanical recordshows rapid northward expansion of pinyon-juniper wood-lands in the Great Basin after the Pleistocene (Betancourt1987); furthermore, western juniper (Juniperus occidentalis)either first appears in the fossil record in the northwesternGreat Basin (including the Modoc Plateau), or shows an in-crease in abundance there, at 5300 years ago (Lava BedsNational Monument, Siskiyou County, California, the site ofsecondary contact; Mehringer and Wigand 1987) or 4800–4000 years ago (Diamond Pond, Diamond Craters, HarneyCounty, Oregon; Wigand 1987); and (2) oak pollen in Cal-ifornia shows an elevational increase relative to modern lev-els between the early and mid-Holocene (10,000–5,000 yearsago; Byrne et al. 1991). The latter is especially significantbecause an upward shift in oaks would have provided a cor-ridor for northward dispersal of B. inornatus across the MountShasta region and into extreme northern California. Similarly,the expansion of juniper around Lava Beds National Mon-ument into areas dominated by sagebrush-grass steppe wouldhave created an east-west vegetative link between peripheralpopulations of B. inornatus and B. ridgwayi. Although theplant fossil data suggest that this connection may date to the

mid-Holocene, additional evidence from modern stands ofwestern juniper indicate that it may, in fact, be much morerecent. In particular, western juniper communities on the Mo-doc Plateau and elsewhere in the northwestern Great Basinhave shown an unprecedented increase in both density anddistribution of trees since European settlement, a trend at-tributed to changes in grazing and fire regimes as well asclimate (Miller and Rose 1995; Chambers et al. 1999; Schae-fer 1999). These changes transformed western juniper wood-lands from open, savannahlike stands to those with highercanopy cover favorable for titmice.

Data from this study indicate a very low level of intro-gression between B. inornatus and B. ridgwayi, especially in


FIG. 7. Predicted distributions of Baeolophus inornatus, B. ridgwayi, and ‘‘contact zone’’ based on 15 BIOCLIM variables, extractedand modeled separately (see Materials and Methods for further explanation). Points labeled ‘‘contact zone’’ represent localities near andincluding the two sites of secondary contact (Fig. 4), in an area previously unknown to harbor either species (Grinnell and Miller 1944);thus, the westernmost points in red represent B. inornatus in juniper habitat. The box outlines the detailed study area illustrated in Figure4, and the heavy arrow points to the exact location of contact between the two species. The contact zone model showed an extremelytight prediction that is hidden by the points. This model also predicted a tiny area (not illustrated because of scale) in the Klamath regionof south-central Oregon. Although either species may exist there, records are scarce (only two specimens from 6 mi E Lorella, KlamathCounty, Oregon; University of Puget Sound museum nos. 14288 and 14289) and I searched for them during two trips without success.Also not illustrated is a single point at the southern tip of the Baja California peninsula, where B. inornatus occurs in an isolatedpopulation; the model for B. inornatus did not provide any prediction around this point. A thin arrow shows where two points representingB. inornatus occurred within the predicted range of B. ridgwayi on the southeastern slope of the Sierra Nevada (see Results). The predicteddistributions of B. inornatus and B. ridgwayi overlapped by ,0.2%.

mtDNA. Whether this interaction constitutes a hybrid zonesensu Arnold (1997, p. 4), that is, whether crosses betweenthe two species ‘‘form viable and at least partially fertileoffspring,’’ remains to be seen. Nonetheless, the small geo-graphic scale of contact combined with low population den-sities suggest that opportunities for hybridization are severelylimited. Furthermore, although dispersal distances of juve-niles in this region are unknown, key behavioral character-

istics (i.e., nonmigratory tendency, long-term pair bonds, andhigh philopatry to year-round territories; Cicero 1996, 2000)likewise restrict the potential for introgression. In contrastto other well-studied cases of established secondary contactor hybridization in birds (e.g., Paridae: Baeolophus bicolorand B. atricristatus, Dixon 1955; Poecile atricapilla and P.carolinensis, Robbins et al. 1986; Bronson et al. 2003), thetenuous conditions on the Modoc Plateau may not result in

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FIG. 8. Bioclimatic gradients for six of 15 variables across sitesin northern California and southern Oregon; site numbers corre-spond to Figure 4. The remaining nine variables were correlatedand showed similar patterns. Baeolophus inornatus and B. ridgwayiare illustrated by white and black symbols, respectively. The twocontact zone sites (9 and 10) are illustrated in gray. The transitionbetween oak and juniper habitat is shown for comparison.

long-term stable contact between B. inornatus and B. ridg-wayi. However, further study is needed to address this ques-tion (e.g., Arntzen and Wallis 1991; Rohwer et al. 2001).

Because sibling species of birds lack distinctive plumagecolors and patterns that serve as premating reproductive iso-lating mechanisms in many avian groups (Johnson and Cicero2002), other factors must compensate. For example, ecolog-ical and behavioral diversification has played a key role inspeciation and patterns of sympatry in Empidonax flycatchers(Johnson and Cicero 2002) and Phylloscopus warblers (Rich-man and Price 1992; Richman 1996). Although B. inornatusand B. ridgwayi are distinguished by species-specific vocal-izations (Cicero 1996, 2000), song characteristics in the Mo-doc Plateau region may be more complex (C. Cicero, unpubl.data). Thus, the extent to which vocalizations serve as cuesto isolate these two taxa where they contact is unknown and

currently under investigation. On the other hand, differencesin the ability of these species to adapt locally to habitat and/or bioclimates clearly play a central role in dictating patternsof sympatry and introgression in this region. Baeolophus in-ornatus is closely associated with warm, dry oak woodlandsthrough most of its range, but locally occupies juniper orpinyon-juniper woodland in a few areas beside the ModocPlateau (Scodie Mountains, Kern County, California; LittleSan Bernardino Mountains, Riverside and San Bernardinocounties, California; Cicero 1996). Thus, B. inornatus is pre-disposed to inhabiting woodlands more typical of B. ridgwayi.In contrast, B. ridgwayi shows no comparable movement intothe oak habitat of B. inornatus, and clearly prefers juniper tooaks where they intermingle in the southwestern UnitedStates (Marshall 1957; Gaddis 1987; Cicero 1996). This ex-ceptional behavior of B. inornatus undoubtedly facilitatedeastward expansion from oak-dominated habitat in ShastaValley and enabled secondary contact in patchy junipers onthe Modoc Plateau. The fact that concordant morphologicaland molecular clines fall east of the limit of oaks in the region,and that sites of secondary contact occur at the margin of thepredicted range of B. ridgwayi based on bioclimatic models,indicates that B. inornatus has adapted locally to the preferredhabitat and climate of B. ridgwayi. Therefore, gene flow isasymmetrical toward B. ridgwayi. Furthermore, this patternprobably is intensified by the relatively lower population den-sity of B. ridgwayi compared to B. inornatus, both in thecontact zone and throughout their ranges (Cicero 1996; alsosee Case and Taper 2000). Nonetheless, historical isolationand divergent selection in distinctive, rangewide associationsof climate and vegetation likely restricts further expansionand overlap of the two species. Although additional work isneeded to test the relative importance of endogenous (genetic-based) and exogenous (environmental-based) selection on B.inornatus and B. ridgwayi in this region (Bronson et al. 2003),findings from this study provide important insight into thecomplex interplay of history, adaptation, and gene flow ininfluencing distributions and local sympatry of close relativesin recent secondary contact.

The Importance of Physiology: Implications forFuture Research

The role of climate in influencing the occurrence of birdand other species has been known for at least a century (e.g.,Grinnell 1904, 1914, 1917). Within the past few decades,however, the physiological basis for these patterns has gainedprominence in the literature. Researchers have approachedthis topic from a variety of perspectives, including ecology(Richter et al. 1997; Weathers and Greene 1998), behavior(Cooper 1999; Wolf 2000), geographic variation in seasonalacclimatization (O’Connor 1996), and biogeography (Hay-worth and Weathers 1984; Root 1988a; Cooper 1997). Ingeneral, these studies have shown that energy constraints andphysiological adaptation are crucial to species’ distributionsat different scales, ranging from local habitat selection(Weathers and Greene 1998) to continentwide range bound-aries (Root 1988a). At least for birds, northern range limitsmay be especially affected by environmental and physiolog-ical factors (Root 1988a,b; but see Repasky 1991).


Studies that compare physiological constraints in closelyrelated and/or sympatric species are especially valuable forunderstanding biogeographic patterns. For example, Hay-worth and Weathers (1984) showed that temperature regu-lation and climatic adaptation in black-billed (Pica hudsonia)and yellow-billed (P. nuttalli) magpies, two allopatric sisterspecies (American Ornithologists’ Union 2000), act directlyto restrict their distributions to distinctive climatic regimes.Similarly, studies on sympatric but nonsister species of parids(Baeolophus and Poecile) have shown that physiological dif-ferences are important in determining habitat selection, be-havior, and northern range limits (Weathers and Greene 1998;Cooper 1999, 2000). Baeolophus inornatus and B. ridgwayiare ideal for such a comparative analysis because they (1)are proven sister taxa, (2) have broadly allopatric distribu-tions that are coincident with distinctive bioclimates, and (3)occur very locally in sympatry at the northern or northwesternmargins of their ranges, where they contact across a strongclimatic gradient. Although physiological data are availablefor B. ridgwayi at two sites (northern Utah, Cooper 1997;southeastern Arizona, Weathers and Greene 1998), data fromnear the contact zone—and data for B. inornatus from any-where in its range—are lacking. Physiological data taken forB. inornatus and B. ridgwayi along west-to-east transects innorthern California (per sites analyzed in the current study)would provide crucial insight into the role of energetic con-straints on their evolution and distribution. Future work willentail a collaborative approach to this question.

ACKNOWLEDGMENTS

The California Department of Fish and Game, Oregon Di-vision of Wildlife, U. S. Fish and Wildlife Service, and Uni-versity of California Berkeley Animal Care and Use Com-mittee issued all of the permits and protocols for this study.N. K. Johnson collected some specimens, and T. Schlenkehelped with DNA extractions and PCR-RFLP experiments.J. Wiezcorek and R. Hijmans provided georeferenced local-ities from the MVZ specimen database. R. Hijmans and C.Graham generated the climate layers that enabled GIS anal-ysis, and R. Hijmans extracted the BIOCLIM variables, pro-duced the models of predicted species’ distributions, and cre-ated the resulting figure. K. Klitz prepared the final figures.C. Benkman, N. K. Johnson, M. Mahoney, C. Moritz, A. T.Peterson, and one anonymous reviewer provided valuablecomments that greatly improved this manuscript. I am grate-ful to all of these individuals and agencies for their assistance.

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Corresponding Editor: C. Benkman

APPENDIX

Museum of Vertebrate Zoology (University of California, Berke-ley; MVZ) catalog numbers of specimens (n 5 211) examined forthis study; numbers in parentheses (sites 1 through 16) refer toFigure 4: Medford (1): 173082–173096; Copco (2): 171853–171862; Little Shasta River (3): 172855–172857; Juniper Flat (4):172858–172865, 173120–173122; Secret Mountain (5): 171863,171865–171874; Dorris (6): 173097–173101; Cedar Mountain (7):172922–172923, 173114–173119; Mount Dome (8): 173102–173113; Lava Beds National Monument (9): 173140, 173485–173487, 173488, 173948–173950, 174016–174018, 177138–177140; Tule Lake Highway (10): 173142, 173143, 173145,172924–172925, 173141, 173144, 173146–173151; Double HeadMountain (11): 177258–177261; Big Sage Reservoir (12): 179183–179192; Warner (13): 171908–171917, 171920, 171922–171923,171925–171926, 171928; Northern Sacramento Valley (14):173123–173131, 173133–173139; Fall River Mills (15): 173489–173490, 174019–174024, 177141–177146; Northeastern California(16): 171639–171641, 171940–171945, 171948, 171929–171931,171933–171936, 171939; Clear Lake: 173152–173168; Oroville:171893–171896, 171876–171881, 171883, 171886–171887,171890. Detailed data for each specimen are available by searchingthe MVZ database (http://elib.cs.berkeley.edu/mvz).

BARRIERS TO SYMPATRY BETWEEN AVIAN SIBLING SPECIES (PARIDAE: BAEOLOPHUS) IN LOCAL SECONDARY CONTACT1

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