BACKGROUND
the study of speciation has been influenced during the last decade by increasing awareness that different parts of the genome tell different evolutionary stories  <cit> .
previously, speciation studies relied heavily on mitochondrial dna , given the numerous benefits offered by these data for inferring evolutionary history at diverse timescales   <cit> .
yet, along with these benefits come drawbacks  <cit> , among them that mtdna is a single recombinational unit that can present a biased snapshot of evolutionary history when mtdna data are not balanced by independent evidence from nuclear markers.
one case in which mtdna data can be particularly misleading occurs during gene flow between populations in secondary contact.
haldane was the first to note that when two differentiated lineages come back into reproductive contact, the heterogametic sex  often shows reduced fertility  <cit> .
the mechanistic reasons behind haldane’s rule, as it came to be known, are under intense study  <cit> .
while we may not understand why haldane’s rule occurs, the consequences for female-heterogametic species like birds and butterflies are clear: if female hybrids show reduced fertility, then mtdna, which is inherited as a single recombinational unit from the female parent, is less likely to be passed on to the next generation.
as a result, mtdna might show little or no evidence of hybridization, despite the fact that hybridization occurs and paternal, nuclear alleles move between lineages.
the potential mismatch between gene flow estimated from mtdna and gene flow estimated using nuclear markers is not just theoretical; it has been demonstrated empirically by studies of female-heterogametic organisms  <cit> .
this phenomenon is of interest to biologists, among other reasons, because of the continuing influence of the biological species concept  and its emphasis on cessation of gene flow as a necessary criterion for species delimitation  <cit> .
while the bsc may not be applied as commonly in practice as is claimed  <cit> , it is nevertheless evident that many attempts to split species taxonomically fail due to the existence of gene flow and the perceived importance of the bsc.
as empirical evidence continues to mount demonstrating mito-nuclear discordance  <cit> , the use of nuclear markers has become essential to species delimitation and the study of speciation.
western scrub-jays  are an excellent model for testing the mismatch between mtdna and nuclear markers across a hybrid zone predicted by haldane’s rule because previous work documented a zone of secondary contact and hybridization between two divergent lineages.
western scrub-jays are widely distributed in north america from british columbia, canada to oaxaca, mexico and east to the great plains .
prior research delimited several lineages based on morphology, allozymes, and mtdna variation  <cit> .
two of these lineages – one found primarily in oak woodlands along the pacific slope and the other found in pinyon-juniper or pine-oak habitats in the interior us and mexico – meet and hybridize in a few mountain ranges in western nevada east of lake tahoe , but are otherwise separated from one another by the high-elevation conifer forests of the sierra nevada.
these two lineages  are thought to have diverged from each other anywhere from  <dig> to  <dig> million years ago  based on divergence times calibrated with a fossil and estimated molecular substitution rates  <cit> .
coastal individuals are easily distinguished from those in the interior by their brighter blue plumage, bolder white supercilium, and more pronounced blue collar.
coastal individuals also have stout, hooked bills, which are better for husking acorns than the smaller, more pointed bills of interior individuals, which are better suited for extracting pine seeds from cones  <cit> .
the hybrid zone was originally described by pitelka  <cit>  who traversed the zone and analyzed numerous museum specimens.
he found intermingling of coastal and interior phenotypes in western nevada, as well as individuals that appeared to be f <dig> hybrids.
the hybrid zone has never been studied in detail using genetic techniques, although previous work documented a relatively rapid turnover of mtdna types and mismatches of taxonomy and genotype of birds from this zone  <cit> .
nevertheless, a prior proposal to elevate the coastal and interior lineages to species status  <cit>  failed because of doubts concerning the extent of gene flow in the contact zone.
the purpose of this study was to test for a prediction of haldane’s rule that there will be a mismatch between mtdna and nuclear gene flow across the contact zone.
specifically, we predict that if haldane’s rule is operating in this system, we will see a narrower  cline in the transition of mtdna haplotypes compared to nuclear markers.
before addressing this hypothesis, however, we needed to determine the exact nature of the contact zone in western nevada and whether there were any undescribed contact zones nearby that might confound our results.
we therefore first assessed broad-scale patterns of divergence and gene flow among western scrub-jays across their entire geographic range, using deep sampling of populations and a suite of nuclear markers.
previous molecular studies sampled only one or a few individuals of different populations, focusing largely on allozymes  <cit>  or mtdna  <cit> .
our study design includes nearly  <dig> individuals sampled from throughout the range of the species, thus providing a comprehensive portrait of genetic divergence and allowing us to locate potentially undescribed contact zones.
finally, we correlated proportion of hybrid ancestry with phenotypic traits for a subset of individuals in the contact zone, as a way to control for the influence of environment and therefore test for a genetic basis of phenotypic differences between the coastal and interior lineages.
if phenotypic traits are genetically based, we expect to see a correlation between neutral genetic assignment to coastal or interior lineages and the phenotypic differences known to characterize those lineages.
RESULTS
geographic structure of coastal and interior mtdna types
to assess gene flow throughout the range of western scrub-jays, we conducted simple mtdna typing of all  <dig> individuals using restriction digest, which showed that most populations are composed entirely of coastal or interior mtdna types, with very few populations showing mixing of mtdna types.
the four populations showing mixing were  the previously described contact zone in western nevada, which featured several populations having mixed types 100 km east and southeast of reno and carson city ;  a single interior type  found at the base of the western sierra 100 km east of stockton, ca ;  an even mix of coastal and interior types in the eastern sierra nevada to the west of the owens valley near independence, ca ; and  one remarkable interior type  in the santa ana mountains in orange county, los angeles .
broad-scale geographic structure of microsatellite variation
to complement the mtdna typing results with information from nuclear markers, we genotyped all  <dig> individuals at  <dig> microsatellite loci.
no pairs of loci were physically linked based on linkage tests.
of the  <dig> loci, one locus  showed significant deviations from hwe across many populations following bonferroni correction.
we removed this locus from the data set.
four other loci showed deviations from hwe across some populations.
we ran downstream analyses with and without these loci to investigate whether they were driving any of the patterns of genetic structure we observed.
results among analyses including and excluding loci were qualitatively similar, so we retained the larger group of  <dig> loci.
no loci showed consistent signs of other microsatellite artifacts like large-allele drop-out or null alleles.
initial structure runs from k = 1– <dig> found increasing likelihood  values until k =  <dig>  followed by a plateau in lnl until k =  <dig>  followed by a decrease from k = 14– <dig>  structure runs on successively smaller clusters revealed five distinct geographic clusters of nuclear dna variation  that were largely uniform in their population assignment.
these genetic clusters were  a pacific slope group;  an interior us group;  a group from the edwards plateau in texas;  an interior group from mexico north of the transvolcanic belt ; and  an interior group from mexico south of the transvolcanic belt .
phylogeny from mtdna
to determine if the novel genetic clusters found with microsatellites  could also be found in a mtdna phylogeny, we sequenced a subset of individuals at the cytochrome b gene , including sampling from throughout the genus.
we chose samples to both capture the major genetic lineages within western scrub-jays and outgroups, and also to sample deeply within certain microsatellite groups of interest .
the larger phylogeny, including all aphelocoma samples, is available through dryad  <cit> .
in figure  <dig>  we show a trimmed version of this phylogeny including only scrub-jays, which confirms that the current concept of the western scrub-jay is paraphyletic because it includes the island scrub-jay .
in addition to the island scrub-jay, phylogenetic analyses placed three other highly supported and divergent clades within western scrub-jays:  a pacific slope clade that was sister to the island scrub-jay ;  a clade from mexico south of the transvolcanic belt ;  a clade of all other interior individuals .
within this last clade, we recovered a weakly supported  group from the edwards plateau in texas, with the exception of one individual that grouped elsewhere in the interior clade .
there was no support in mtdna for a split between interior us and northern mexico , although some geographic localities grouped together with high pp.
geographic cline analysis
both the mtdna and microsatellite data showed a step cline pattern of variation across the hybrid zone, and the null model of no clinal variation had much higher aicc scores in each case .
model  <dig> with no scaling was accepted as the most likely model for both datasets.
estimates of the center of the cline  were similar and had overlapping confidence intervals.
the center of the mtdna cline was ~300 km , and the center of the nuclear dna cline was ~315 km .
the width of the mtdna cline was ~131 km , and the width of the nuclear dna cline was ~331 km .
the estimated width of the mtdna cline fell outside the  <dig> log likelihood confidence intervals for the nuclear dna, and vice versa, therefore the mtdna cline was significantly narrower than the microsatellite cline .
disparity in dispersal distances between males and females did not account for the difference in cline width between mtdna and microsatellites because our time-of-contact estimates under a model of neutral diffusion, which included estimates of male and female dispersal distance , did not overlap .
in other words, even taking sex-biased dispersal into account, mtdna showed less diffusion.
correlation between phenotype and genotype
we observed strong correlations between hybrid values  from structure and many phenotypic traits for the full data set of  <dig> individuals that included many pure coastal and interior individuals outside the hybrid zone, especially for plumage traits and principal components  axes describing bill shape .
however, by including individuals from allopatric ranges, this test potentially confounds genetic and environmental causes of trait variation.
within the hybrid transect and inside the core hybrid zone, where individuals experience the same environment, plumage traits, but not morphological traits, were correlated with q-scores .
1pc <dig> describes general size.
2pc <dig> describes a trade-off between body and bill size.
3pc <dig> describes a trade-off between bill length and bill width.
ns = not significant, *p <  <dig> , **p <  <dig> , ***p <  <dig> .
discussion
haldane’s rule and the mismatch in cline width between mtdna and microsatellites
our results support a key prediction from haldane’s rule that mtdna will show a steeper cline across a hybrid zone than nuclear markers.
although haldane’s rule may explain the clinal pattern we observed, this pattern could also result from divergent selection on mtdna types  <cit>  or sex-biased dispersal, wherein females  tend to disperse less than males.
although we have not looked at selection, what little is known about natal dispersal in western scrub-jays suggests that, to the contrary, females disperse about three times farther than males on average  <cit> .
the results from our time-since-contact analyses, which incorporated estimates of dispersal distances, also support predictions from haldane’s rule, with less diffusion of mtdna compared to nuclear markers.
our results showing steeper clines in mtdna compared to nuclear markers add to a gathering corpus of research showing discordant rates of dna transmission across hybrid zones among different marker types  <cit> , a pattern that has often been attributed to haldane’s rule  <cit> .
whatever the root cause, these results caution that conclusions about individual-level gene flow made on the basis of mtdna alone may be underestimated.
in our study, gene flow is generally restricted in both mtdna and nuclear markers, due to the low dispersal distances of western scrub-jays.
in other species, such as long-distance dispersers with no sex-biased dispersal, mismatches could be much more pronounced.
recent work on the genetic basis for haldane’s rule suggests it results from incompatibilities among genes that are exposed to selection on hybrid female z chromosomes, causing sterility  <cit> , whereas only those incompatibilities that are dominant are visible to selection in hybrid zz males.
if haldane’s rule is indeed the cause of the clinal mismatch we observed, then our results suggest that the coastal and interior lineages of western scrub-jay have begun the process of reproductive isolation.
ultimately, haldane’s rule as the cause of the mismatch in marker introgression rates could be determined through field studies or captive mating experiments that document the reproductive viability of female offspring resulting from hybrid crosses  <cit> .
clarifying the hybrid zone between coastal and interior western scrub-jays
our results also help clarify the nature of the hybrid zone between coastal and interior lineages of western scrub-jays.
the fact that our hybrid transect populations could be fit to a cline suggests that pitelka  <cit>  was correct that the major axis of gene flow is from the northwest to southeast, as coastal populations continue around the north side of the sierra nevada and meet with interior populations in western nevada.
however, the coastal influence of the woodford site  warrants further study.
in addition to gene flow from the north, some gene flow may link the southern end of the sierra nevada to the woodford site, with coastal individuals inhabiting canyons of the southeastern sierra that were not sampled for this study.
our results also support pitelka’s  <cit>  conclusions that the hybrid zone is narrow and the result of secondary contact.
even the oldest time estimates from our time-since-contact analysis across the hybrid zone  are younger than divergence estimates of the original split between coastal and interior lineages , supporting the hypothesis that the hybrid zone results from secondary contact and not in situ divergence.
in terms of broader biogeographic evidence, several other species have secondary contact zones where coastal and interior lineages meet in this general region of the great basin  <cit> .
our results also confirm that the contact zone in western nevada is the only place where coastal and interior lineages come into contact to any great degree.
outside of western nevada, two sites  appear to contain examples of rare, long-distance dispersers from pure interior populations because the nuclear dna profile of the two individuals matches their mtdna type .
in contrast, at a third site in the eastern sierra nevada , the three individuals with interior mtdna had strongly coastal nuclear dna profiles , suggesting that they were advanced backcrosses from older hybridization events .
thus, although there appears to be one major contact zone, the exceptions show that the reality of dispersal and gene flow among jays in and around the sierras – and across the mojave desert farther south – is more complex.
fine-scale sampling, combined with habitat-based methods of identifying dispersal corridors  <cit> , could provide a more complete picture of gene flow across and around the sierra nevada for this and other species.
previous studies documented phenotypic differences between coastal and interior western scrub-jays  <cit> , but ours is the first to provide evidence for a genetic basis for some traits.
there was a correlation between nuclear genetic variation and phenotypic traits, such as bill shape and plumage traits, when considering all individuals, even those from pure populations .
however, including individuals from different geographic locations potentially confounds environmental and genetic sources of phenotypic variation.
still, many of these relationships remained, for the plumage traits at least, within the contact zone where there are hybrid individuals and all variety of backcrosses.
the correlation between proportion of interior vs. coastal genetic ancestry and phenotypic traits for individuals that were fledged in the hybrid zone and therefore experienced the same environment since fledging provides evidence for a genetic basis of plumage traits, at the very least.
why we did not observe similar relationships for bill traits is not clear, but could be due to low statistical power, stabilizing selection, or greater plasticity in bill traits.
further studies with more individuals and better genomic coverage will help discriminate between these alternate hypotheses, as will studies that attempt to identify the genetic loci or environmental pressures responsible for the phenotypic differences.
genetic differentiation across north america’s scrub woodland
previous genetic studies of species inhabiting north america’s pine-oak and scrub woodland have uncovered unexpectedly deep phylogenetic structure within species  <cit> .
results of the current study, which are the most comprehensive in terms of geographic, individual, and genetic sampling of western scrub-jays, indicate at least five genetic clusters with strong spatial structuring .
the pacific coast and southern mexico groups have been recovered previously in studies of phenotype, allozymes, and mtdna  <cit> .
the edwards plateau group and the split between interior us and northern mexico individuals are novel, although the inferred population clusters align with current subspecies designations  <cit> .
this study is the first to show clear evidence of nuclear and mtdna differentiation of western scrub-jays on the edwards plateau in texas .
all individuals in the edwards plateau population except one possess a unique mtdna haplotype differing from other interior us haplotypes by two substitutions.
nuclear and mtdna differentiation on the edwards plateau is interesting given the history of isolation of this region and its unique flora and fauna.
the oak savanna and ashe juniper woodland of the edwards plateau are separated from other scrub woodland to the west by an extension of the chihuahuan desert in the pecos valley.
fossil and pollen data suggest, however, that pygmy woodland was continuous across this region, and connected the edwards plateau to western forests throughout the last glacial maximum until about  <dig>  years ago  <cit> .
at least one other distinct bird species with a similar geographic range occurs on the edwards plateau, the golden-cheeked warbler , and other organisms in this region have distinct populations or subspecies  <cit> .
our results lend additional support to the distinctiveness of the biota in this region, where native habitat is under persistent conservation threat from urbanization.
the nuclear genetic break between the interior us and northern mexico populations, occurring roughly where the sierra madre occidental and oriental turn into a network of sky islands near the us border, could be explained either by a break in suitable habitat or a sampling artifact.
addressing the latter point, observations posted on ebird  confirm observations of western scrub-jays in southeastern arizona and northern sonora , but these records could represent wandering individuals.
likewise few observations exist in the vicinity of the genetic break in southwestern texas, and virtually no observations exist from northern coahuila, where congeneric mexican jays  occur at lower elevations, seemingly occupying the western scrub-jay niche  <cit> .
thus, while available evidence suggests that the genetic break between interior us and mexico populations relates to a real distributional break near the us border, the exact causes of this break remain unclear.
future niche modeling could be used to determine if there are cryptic breaks in suitable habitat.
lack of genetic differentiation along the pacific coast
in contrast to the highly structured genetic portrait of the interior us and mexico, western scrub-jays along the pacific slope show a remarkable lack of genetic structure despite  <dig>  km of nearly unbroken sampling from northern oregon to the southern tip of baja california.
lack of genetic structure over large distances suggests two possible scenarios:  high levels of dispersal and gene flow link coastal populations; or  recently expanded coastal populations have had insufficient time to accumulate genetic differences.
available evidence suggests that western scrub-jays generally have low dispersal  <cit> , notwithstanding a few rare, long-distance dispersal events discussed above.
furthermore, the original split between coastal and interior lineages occurred between 1– <dig> million years ago  <cit> , providing ample time for genetic structure to emerge in coastal populations, absent other demographic events.
thus, the hypothesis that seems most likely is a recent expansion following a population bottleneck.
however, pleistocene niche models of western scrub-jay niches suggest ample habitat along the pacific slope during the last glacial maximum  <cit> .
further study is needed to determine the cause of the apparent genetic homogeneity of pacific slope populations.
taxonomic recommendations
we recommend splitting the current concept of the western scrub-jay into three species, as has already been suggested previously  <cit> .
the pacific slope lineage  is phenotypically distinct  <cit> , possesses a bill morphology adapted to local resources  <cit> , is monophyletic in mtdna, and is sister to another recognized species, the island scrub-jay  <cit> .
this study shows that the pacific slope lineage is also well-differentiated in nuclear markers and has only a narrow zone of contact with interior populations.
the genetic clines across the hybrid zone are generally steep, and the mismatch between mtdna and nuclear clines suggests a measure of reproductive isolation.
these results would appear to meet criteria for species distinction under even the most stringent species concept.
southern mexico populations  also meet criteria for species recognition.
they are phenotypically distinct  <cit> , with behavioral evidence that some southern populations are cooperative breeders  <cit> , whereas other western scrub-jays breed in territorial pairs.
this study demonstrates that these southern populations are reciprocally monophyletic to other interior populations in mtdna and well-differentiated in nuclear markers.
finally, we recommend that the remaining interior populations  be elevated to species level, given their diagnostic phenotype  <cit> , adaptive bill morphology  <cit> , high support for monophyly in mtdna in previous work  <cit>  and this study , and differentiation in nuclear markers .
within this group, we do not recommend elevating the genetically distinctive edwards plateau population to species level at this time because our study sampled only one population.
more information is needed on potential gene flow with other interior us populations, as well as a modern multivariate analysis of their phenotypic differentiation and studies of their ecological and behavioral divergence.
likewise, we do not currently recommend splitting interior us from northern mexico populations, unless, at minimum, further sampling near the us-mexico confirms that the break seen in microsatellite variation is not the result of a sampling artifact.
CONCLUSIONS
we report a mismatch in cline widths between mtdna and nuclear markers across a hybrid zone in western scrub-jays.
this result is consistent with predictions based on haldane’s rule, where there is less mtdna introgression between lineages in female-heterogametic species.
this study adds to a growing list of studies that support predictions of haldane’s rule using cline analysis of multiple loci of differing inheritance modes.
structure results of microsatellite variation confirm that there is only one major area of contact between coastal and interior lineages.
furthermore, structure identified at least five clusters of individuals with strong spatial structuring: pacific slope, interior us, edwards plateau, northern mexico, and southern mexico.
based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: a. californica ; a. woodhouseii ; a. sumichrasti .
the distinctive edwards plateau population in texas, which was also monophyletic in mtdna except for one individual, should be studied in greater detail given conservation concerns.

 the study of speciation has been influenced during the last decade by increasing awareness that different parts of the genome tell different evolutionary stories. previously, speciation studies relied heavily on mitochondrial dna, given the numerous benefits offered by these data for inferring evolutionary history at diverse timescales. yet, along with these benefits come drawbacks, among them that mtdna is a single recombinational unit that can present a biased snapshot of evolutionary history when mtdna data are not balanced by independent evidence from nuclear markers. one case in which mtdna data can be particularly misleading occurs during gene flow between populations in secondary contact. haldane was the first to note that when two differentiated lineages come back into reproductive contact, the heterogametic sex often shows reduced fertility. the mechanistic reasons behind haldane’s rule, as it came to be known, are under intense study. while we may not understand why haldane’s rule occurs, the consequences for female-heterogametic species like birds and butterflies are clear: if female hybrids show reduced fertility, then mtdna, which is inherited as a single recombinational unit from the female parent, is less likely to be passed on to the next generation. as a result, mtdna might show little or no evidence of hybridization, despite the fact that hybridization occurs and paternal, nuclear alleles move between lineages. the potential mismatch between gene flow estimated from mtdna and gene flow estimated using nuclear markers is not just theoretical; it has been demonstrated empirically by studies of female-heterogametic organisms. this phenomenon is of interest to biologists, among other reasons, because of the continuing influence of the biological species concept and its emphasis on cessation of gene flow as a necessary criterion for species delimitation. while the bsc may not be applied as commonly in practice as is claimed, it is nevertheless evident that many attempts to split species taxonomically fail due to the existence of gene flow and the perceived importance of the bsc. as empirical evidence continues to mount demonstrating mito-nuclear discordance, the use of nuclear markers has become essential to species delimitation and the study of speciation. western scrub-jays are an excellent model for testing the mismatch between mtdna and nuclear markers across a hybrid zone predicted by haldane’s rule because previous work documented a zone of secondary contact and hybridization between two divergent lineages. western scrub-jays are widely distributed in north america from british columbia, canada to oaxaca, mexico and east to the great plains. prior research delimited several lineages based on morphology, allozymes, and mtdna variation. two of these lineages – one found primarily in oak woodlands along the pacific slope and the other found in pinyon-juniper or pine-oak habitats in the interior us and mexico – meet and hybridize in a few mountain ranges in western nevada east of lake tahoe, but are otherwise separated from one another by the high-elevation conifer forests of the sierra nevada. these two lineages are thought to have diverged from each other anywhere from to million years ago based on divergence times calibrated with a fossil and estimated molecular substitution rates. coastal individuals are easily distinguished from those in the interior by their brighter blue plumage, bolder white supercilium, and more pronounced blue collar. coastal individuals also have stout, hooked bills, which are better for husking acorns than the smaller, more pointed bills of interior individuals, which are better suited for extracting pine seeds from cones. the hybrid zone was originally described by pitelka who traversed the zone and analyzed numerous museum specimens. he found intermingling of coastal and interior phenotypes in western nevada, as well as individuals that appeared to be f hybrids. the hybrid zone has never been studied in detail using genetic techniques, although previous work documented a relatively rapid turnover of mtdna types and mismatches of taxonomy and genotype of birds from this zone. nevertheless, a prior proposal to elevate the coastal and interior lineages to species status failed because of doubts concerning the extent of gene flow in the contact zone. the purpose of this study was to test for a prediction of haldane’s rule that there will be a mismatch between mtdna and nuclear gene flow across the contact zone. specifically, we predict that if haldane’s rule is operating in this system, we will see a narrower cline in the transition of mtdna haplotypes compared to nuclear markers. before addressing this hypothesis, however, we needed to determine the exact nature of the contact zone in western nevada and whether there were any undescribed contact zones nearby that might confound our results. we therefore first assessed broad-scale patterns of divergence and gene flow among western scrub-jays across their entire geographic range, using deep sampling of populations and a suite of nuclear markers. previous molecular studies sampled only one or a few individuals of different populations, focusing largely on allozymes or mtdna. our study design includes nearly individuals sampled from throughout the range of the species, thus providing a comprehensive portrait of genetic divergence and allowing us to locate potentially undescribed contact zones. finally, we correlated proportion of hybrid ancestry with phenotypic traits for a subset of individuals in the contact zone, as a way to control for the influence of environment and therefore test for a genetic basis of phenotypic differences between the coastal and interior lineages. if phenotypic traits are genetically based, we expect to see a correlation between neutral genetic assignment to coastal or interior lineages and the phenotypic differences known to characterize those lineages.  geographic structure of coastal and interior mtdna types to assess gene flow throughout the range of western scrub-jays, we conducted simple mtdna typing of all individuals using restriction digest, which showed that most populations are composed entirely of coastal or interior mtdna types, with very few populations showing mixing of mtdna types. the four populations showing mixing were the previously described contact zone in western nevada, which featured several populations having mixed types 100 km east and southeast of reno and carson city; a single interior type found at the base of the western sierra 100 km east of stockton, ca; an even mix of coastal and interior types in the eastern sierra nevada to the west of the owens valley near independence, ca; and one remarkable interior type in the santa ana mountains in orange county, los angeles. broad-scale geographic structure of microsatellite variation to complement the mtdna typing results with information from nuclear markers, we genotyped all individuals at microsatellite loci. no pairs of loci were physically linked based on linkage tests. of the loci, one locus showed significant deviations from hwe across many populations following bonferroni correction. we removed this locus from the data set. four other loci showed deviations from hwe across some populations. we ran downstream analyses with and without these loci to investigate whether they were driving any of the patterns of genetic structure we observed. results among analyses including and excluding loci were qualitatively similar, so we retained the larger group of loci. no loci showed consistent signs of other microsatellite artifacts like large-allele drop-out or null alleles. initial structure runs from k = 1– found increasing likelihood values until k = followed by a plateau in lnl until k = followed by a decrease from k = 14– structure runs on successively smaller clusters revealed five distinct geographic clusters of nuclear dna variation that were largely uniform in their population assignment. these genetic clusters were a pacific slope group; an interior us group; a group from the edwards plateau in texas; an interior group from mexico north of the transvolcanic belt; and an interior group from mexico south of the transvolcanic belt. phylogeny from mtdna to determine if the novel genetic clusters found with microsatellites could also be found in a mtdna phylogeny, we sequenced a subset of individuals at the cytochrome b gene, including sampling from throughout the genus. we chose samples to both capture the major genetic lineages within western scrub-jays and outgroups, and also to sample deeply within certain microsatellite groups of interest. the larger phylogeny, including all aphelocoma samples, is available through dryad. in figure  we show a trimmed version of this phylogeny including only scrub-jays, which confirms that the current concept of the western scrub-jay is paraphyletic because it includes the island scrub-jay. in addition to the island scrub-jay, phylogenetic analyses placed three other highly supported and divergent clades within western scrub-jays: a pacific slope clade that was sister to the island scrub-jay; a clade from mexico south of the transvolcanic belt; a clade of all other interior individuals. within this last clade, we recovered a weakly supported group from the edwards plateau in texas, with the exception of one individual that grouped elsewhere in the interior clade. there was no support in mtdna for a split between interior us and northern mexico, although some geographic localities grouped together with high pp. geographic cline analysis both the mtdna and microsatellite data showed a step cline pattern of variation across the hybrid zone, and the null model of no clinal variation had much higher aicc scores in each case. model with no scaling was accepted as the most likely model for both datasets. estimates of the center of the cline were similar and had overlapping confidence intervals. the center of the mtdna cline was ~300 km, and the center of the nuclear dna cline was ~315 km. the width of the mtdna cline was ~131 km, and the width of the nuclear dna cline was ~331 km. the estimated width of the mtdna cline fell outside the log likelihood confidence intervals for the nuclear dna, and vice versa, therefore the mtdna cline was significantly narrower than the microsatellite cline. disparity in dispersal distances between males and females did not account for the difference in cline width between mtdna and microsatellites because our time-of-contact estimates under a model of neutral diffusion, which included estimates of male and female dispersal distance, did not overlap. in other words, even taking sex-biased dispersal into account, mtdna showed less diffusion. correlation between phenotype and genotype we observed strong correlations between hybrid values from structure and many phenotypic traits for the full data set of individuals that included many pure coastal and interior individuals outside the hybrid zone, especially for plumage traits and principal components axes describing bill shape. however, by including individuals from allopatric ranges, this test potentially confounds genetic and environmental causes of trait variation. within the hybrid transect and inside the core hybrid zone, where individuals experience the same environment, plumage traits, but not morphological traits, were correlated with q-scores. 1pc describes general size. 2pc describes a trade-off between body and bill size. 3pc describes a trade-off between bill length and bill width. ns = not significant, *p < , **p < , ***p < . discussion haldane’s rule and the mismatch in cline width between mtdna and microsatellites our results support a key prediction from haldane’s rule that mtdna will show a steeper cline across a hybrid zone than nuclear markers. although haldane’s rule may explain the clinal pattern we observed, this pattern could also result from divergent selection on mtdna types or sex-biased dispersal, wherein females tend to disperse less than males. although we have not looked at selection, what little is known about natal dispersal in western scrub-jays suggests that, to the contrary, females disperse about three times farther than males on average. the results from our time-since-contact analyses, which incorporated estimates of dispersal distances, also support predictions from haldane’s rule, with less diffusion of mtdna compared to nuclear markers. our results showing steeper clines in mtdna compared to nuclear markers add to a gathering corpus of research showing discordant rates of dna transmission across hybrid zones among different marker types, a pattern that has often been attributed to haldane’s rule. whatever the root cause, these results caution that conclusions about individual-level gene flow made on the basis of mtdna alone may be underestimated. in our study, gene flow is generally restricted in both mtdna and nuclear markers, due to the low dispersal distances of western scrub-jays. in other species, such as long-distance dispersers with no sex-biased dispersal, mismatches could be much more pronounced. recent work on the genetic basis for haldane’s rule suggests it results from incompatibilities among genes that are exposed to selection on hybrid female z chromosomes, causing sterility, whereas only those incompatibilities that are dominant are visible to selection in hybrid zz males. if haldane’s rule is indeed the cause of the clinal mismatch we observed, then our results suggest that the coastal and interior lineages of western scrub-jay have begun the process of reproductive isolation. ultimately, haldane’s rule as the cause of the mismatch in marker introgression rates could be determined through field studies or captive mating experiments that document the reproductive viability of female offspring resulting from hybrid crosses. clarifying the hybrid zone between coastal and interior western scrub-jays our results also help clarify the nature of the hybrid zone between coastal and interior lineages of western scrub-jays. the fact that our hybrid transect populations could be fit to a cline suggests that pitelka was correct that the major axis of gene flow is from the northwest to southeast, as coastal populations continue around the north side of the sierra nevada and meet with interior populations in western nevada. however, the coastal influence of the woodford site warrants further study. in addition to gene flow from the north, some gene flow may link the southern end of the sierra nevada to the woodford site, with coastal individuals inhabiting canyons of the southeastern sierra that were not sampled for this study. our results also support pitelka’s conclusions that the hybrid zone is narrow and the result of secondary contact. even the oldest time estimates from our time-since-contact analysis across the hybrid zone are younger than divergence estimates of the original split between coastal and interior lineages, supporting the hypothesis that the hybrid zone results from secondary contact and not in situ divergence. in terms of broader biogeographic evidence, several other species have secondary contact zones where coastal and interior lineages meet in this general region of the great basin. our results also confirm that the contact zone in western nevada is the only place where coastal and interior lineages come into contact to any great degree. outside of western nevada, two sites appear to contain examples of rare, long-distance dispersers from pure interior populations because the nuclear dna profile of the two individuals matches their mtdna type. in contrast, at a third site in the eastern sierra nevada, the three individuals with interior mtdna had strongly coastal nuclear dna profiles, suggesting that they were advanced backcrosses from older hybridization events. thus, although there appears to be one major contact zone, the exceptions show that the reality of dispersal and gene flow among jays in and around the sierras – and across the mojave desert farther south – is more complex. fine-scale sampling, combined with habitat-based methods of identifying dispersal corridors, could provide a more complete picture of gene flow across and around the sierra nevada for this and other species. previous studies documented phenotypic differences between coastal and interior western scrub-jays, but ours is the first to provide evidence for a genetic basis for some traits. there was a correlation between nuclear genetic variation and phenotypic traits, such as bill shape and plumage traits, when considering all individuals, even those from pure populations. however, including individuals from different geographic locations potentially confounds environmental and genetic sources of phenotypic variation. still, many of these relationships remained, for the plumage traits at least, within the contact zone where there are hybrid individuals and all variety of backcrosses. the correlation between proportion of interior vs. coastal genetic ancestry and phenotypic traits for individuals that were fledged in the hybrid zone and therefore experienced the same environment since fledging provides evidence for a genetic basis of plumage traits, at the very least. why we did not observe similar relationships for bill traits is not clear, but could be due to low statistical power, stabilizing selection, or greater plasticity in bill traits. further studies with more individuals and better genomic coverage will help discriminate between these alternate hypotheses, as will studies that attempt to identify the genetic loci or environmental pressures responsible for the phenotypic differences. genetic differentiation across north america’s scrub woodland previous genetic studies of species inhabiting north america’s pine-oak and scrub woodland have uncovered unexpectedly deep phylogenetic structure within species. results of the current study, which are the most comprehensive in terms of geographic, individual, and genetic sampling of western scrub-jays, indicate at least five genetic clusters with strong spatial structuring. the pacific coast and southern mexico groups have been recovered previously in studies of phenotype, allozymes, and mtdna. the edwards plateau group and the split between interior us and northern mexico individuals are novel, although the inferred population clusters align with current subspecies designations. this study is the first to show clear evidence of nuclear and mtdna differentiation of western scrub-jays on the edwards plateau in texas. all individuals in the edwards plateau population except one possess a unique mtdna haplotype differing from other interior us haplotypes by two substitutions. nuclear and mtdna differentiation on the edwards plateau is interesting given the history of isolation of this region and its unique flora and fauna. the oak savanna and ashe juniper woodland of the edwards plateau are separated from other scrub woodland to the west by an extension of the chihuahuan desert in the pecos valley. fossil and pollen data suggest, however, that pygmy woodland was continuous across this region, and connected the edwards plateau to western forests throughout the last glacial maximum until about  years ago. at least one other distinct bird species with a similar geographic range occurs on the edwards plateau, the golden-cheeked warbler, and other organisms in this region have distinct populations or subspecies. our results lend additional support to the distinctiveness of the biota in this region, where native habitat is under persistent conservation threat from urbanization. the nuclear genetic break between the interior us and northern mexico populations, occurring roughly where the sierra madre occidental and oriental turn into a network of sky islands near the us border, could be explained either by a break in suitable habitat or a sampling artifact. addressing the latter point, observations posted on ebird confirm observations of western scrub-jays in southeastern arizona and northern sonora, but these records could represent wandering individuals. likewise few observations exist in the vicinity of the genetic break in southwestern texas, and virtually no observations exist from northern coahuila, where congeneric mexican jays occur at lower elevations, seemingly occupying the western scrub-jay niche. thus, while available evidence suggests that the genetic break between interior us and mexico populations relates to a real distributional break near the us border, the exact causes of this break remain unclear. future niche modeling could be used to determine if there are cryptic breaks in suitable habitat. lack of genetic differentiation along the pacific coast in contrast to the highly structured genetic portrait of the interior us and mexico, western scrub-jays along the pacific slope show a remarkable lack of genetic structure despite  km of nearly unbroken sampling from northern oregon to the southern tip of baja california. lack of genetic structure over large distances suggests two possible scenarios: high levels of dispersal and gene flow link coastal populations; or recently expanded coastal populations have had insufficient time to accumulate genetic differences. available evidence suggests that western scrub-jays generally have low dispersal, notwithstanding a few rare, long-distance dispersal events discussed above. furthermore, the original split between coastal and interior lineages occurred between 1– million years ago, providing ample time for genetic structure to emerge in coastal populations, absent other demographic events. thus, the hypothesis that seems most likely is a recent expansion following a population bottleneck. however, pleistocene niche models of western scrub-jay niches suggest ample habitat along the pacific slope during the last glacial maximum. further study is needed to determine the cause of the apparent genetic homogeneity of pacific slope populations. taxonomic recommendations we recommend splitting the current concept of the western scrub-jay into three species, as has already been suggested previously. the pacific slope lineage is phenotypically distinct, possesses a bill morphology adapted to local resources, is monophyletic in mtdna, and is sister to another recognized species, the island scrub-jay. this study shows that the pacific slope lineage is also well-differentiated in nuclear markers and has only a narrow zone of contact with interior populations. the genetic clines across the hybrid zone are generally steep, and the mismatch between mtdna and nuclear clines suggests a measure of reproductive isolation. these results would appear to meet criteria for species distinction under even the most stringent species concept. southern mexico populations also meet criteria for species recognition. they are phenotypically distinct, with behavioral evidence that some southern populations are cooperative breeders, whereas other western scrub-jays breed in territorial pairs. this study demonstrates that these southern populations are reciprocally monophyletic to other interior populations in mtdna and well-differentiated in nuclear markers. finally, we recommend that the remaining interior populations be elevated to species level, given their diagnostic phenotype, adaptive bill morphology, high support for monophyly in mtdna in previous work and this study, and differentiation in nuclear markers. within this group, we do not recommend elevating the genetically distinctive edwards plateau population to species level at this time because our study sampled only one population. more information is needed on potential gene flow with other interior us populations, as well as a modern multivariate analysis of their phenotypic differentiation and studies of their ecological and behavioral divergence. likewise, we do not currently recommend splitting interior us from northern mexico populations, unless, at minimum, further sampling near the us-mexico confirms that the break seen in microsatellite variation is not the result of a sampling artifact.  we report a mismatch in cline widths between mtdna and nuclear markers across a hybrid zone in western scrub-jays. this result is consistent with predictions based on haldane’s rule, where there is less mtdna introgression between lineages in female-heterogametic species. this study adds to a growing list of studies that support predictions of haldane’s rule using cline analysis of multiple loci of differing inheritance modes. structure results of microsatellite variation confirm that there is only one major area of contact between coastal and interior lineages. furthermore, structure identified at least five clusters of individuals with strong spatial structuring: pacific slope, interior us, edwards plateau, northern mexico, and southern mexico. based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: a. californica; a. woodhouseii; a. sumichrasti. the distinctive edwards plateau population in texas, which was also monophyletic in mtdna except for one individual, should be studied in greater detail given conservation concerns. 
based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: a. californica; a. woodhouseii; a. sumichrasti. haldane’s rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtdna between species where females are heterogametic. therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. our initial phylogeographic analysis revealed that there is only one major area of contact between coastal and interior lineages and identified five genetic clusters with strong spatial structuring: pacific slope, interior us, edwards plateau, northern mexico, and southern mexico. this study adds to a growing list of studies that support predictions of haldane’s rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtdna types cannot be ruled out. we studied introgression of microsatellites and mtdna across a secondary contact zone between coastal and interior lineages of western scrub-jays to test for a signature of haldane’s rule: a narrower cline of introgression in mtdna compared to nuclear markers. consistent with predictions from haldane’s rule, mtdna showed a narrower cline than nuclear markers across a transect through the hybrid zone. that haldane’s rule appears to be operating in this system suggests a measure of reproductive isolation between the pacific slope and interior lineages. the distinctive edwards plateau population in texas, which was monophyletic in mtdna except for one individual, should be studied in greater detail given habitat threat. lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control.
 haldane’s rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtdna between species where females are heterogametic. therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. we studied introgression of microsatellites and mtdna across a secondary contact zone between coastal and interior lineages of western scrub-jays to test for a signature of haldane’s rule: a narrower cline of introgression in mtdna compared to nuclear markers.  our initial phylogeographic analysis revealed that there is only one major area of contact between coastal and interior lineages and identified five genetic clusters with strong spatial structuring: pacific slope, interior us, edwards plateau, northern mexico, and southern mexico. consistent with predictions from haldane’s rule, mtdna showed a narrower cline than nuclear markers across a transect through the hybrid zone. this result is not being driven by female-biased dispersal because neutral diffusion analysis, which included estimates of sex-specific dispersal rates, also showed less diffusion of mtdna. lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control.  this study adds to a growing list of studies that support predictions of haldane’s rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtdna types cannot be ruled out. that haldane’s rule appears to be operating in this system suggests a measure of reproductive isolation between the pacific slope and interior lineages. based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: a. californica; a. woodhouseii; a. sumichrasti. the distinctive edwards plateau population in texas, which was monophyletic in mtdna except for one individual, should be studied in greater detail given habitat threat. birdsspeciationgene flowpostzygotic reproductive isolationphylogeography 