Asymmetric reproductive isolation between terminal forms of the salamander ring species Ensatina eschscholtzii revealed by fine-scale genetic analysis of a hybrid zone
© Devitt et al; licensee BioMed Central Ltd. 2011
Received: 24 March 2011
Accepted: 22 August 2011
Published: 22 August 2011
Ring species, exemplified by salamanders of the Ensatina eschscholtzii complex, represent a special window into the speciation process because they allow the history of species formation to be traced back in time through the geographically differentiated forms connecting the two terminal forms of the ring. Of particular interest is the nature and extent of reproductive isolation between the geographically terminal forms, in this case E. e. eschscholtzii and E. e. klauberi. Previous studies have documented infrequent hybridization at the end of the ring. Here, we report the first fine-scale genetic analysis of a hybrid zone between the terminal forms in southern California using individual-based Bayesian analyses of multilocus genetic data to estimate levels and direction of hybridization and maximum-likelihood analysis of linkage disequilibrium and cline shape to make inferences about migration and selection in the hybrid zone.
The center of the hybrid zone has a high proportion of hybrids, about half of which were classified as F1s. Clines are narrow with respect to dispersal, and there are significant deviations from Hardy-Weinberg equilibrium as well as nonrandom associations (linkage disequilibria) between alleles characteristic of each parental type. There is cytonuclear discordance, both in terms of introgression and the geographic position of mitochondrial versus nuclear clines. Genetic disequilibrium is concentrated on the eschscholtzii side of the zone. Nearly all hybrids possess klauberi mtDNA, indicating that most hybrids are formed from female klauberi mating with male eschscholtzii or male hybrids (but not vice versa).
Our results are consistent with a tension zone trapped at an ecotone, with gene combinations characteristic of klauberi showing up on the eschscholtzii side of the zone due to asymmetric hybridization. We suggest that the observed asymmetry is best explained by increased discriminatory power of eschscholtzii females, or asymmetric postzygotic isolation. The relatively high frequency of hybrids, particularly F1s, contrasts with other contacts between the terminal forms, and with other contacts between other divergent Ensatina lineages, highlighting the diverse outcomes of secondary contact within a single species complex.
The conclusion that the two geographically terminal subspecies, eschscholtzii and klauberi, are reproductively isolated is central to the ring species interpretation, and also to unresolved debates about species boundaries in this complex [20–23]. Although the sympatry of the terminal forms was not yet known to Stebbins in 1949, it was later discovered that these forms are sympatric in four geographically isolated contact zones in southern California [24–27]. Brown  examined color pattern and blood serum proteins from three of these contact zones, including a region on Palomar Mountain covering approximately 10 square miles where several narrow zones of sympatry were found with little evidence of introgression . Marked differences in the habitats occupied by klauberi and eschscholtzii were noted in the Palomar region, with eschscholtzii occupying oak-chaparral associations below ~1,370 m and klauberi inhabiting pine-oak-cedar forest above this elevation . Hybridization occurred in an ecotone between these two habitat types between ~1,220-1,370 m . Wake et al.  typed samples of individuals from all four contact zones at 26 allozyme loci, finding evidence of hybridization (albeit infrequent) at all but the southernmost contact in the Cuyamaca Mountains (see also ).
Although these studies provided the first evidence for hybridization between klauberi and eschscholtzii, individuals were sampled at a relatively broad geographic scale, and analyses were limited to identification and classification of hybrid individuals. The present study extends previous work on hybridization at the end of the ring in three ways. First, the geographic scale of this study is much finer than previous work, an important consideration given that hybrid zones may vary in structure (i.e., clinal vs. mosaic) at different sampling resolutions [29–31]. In general, sampling should be undertaken at the scale at which individuals meet, mate, and produce offspring (tens of meters in Ensatina; [32, 33]). Second, although habitat isolation has been implicated as a strong barrier to gene flow between sympatric populations of Ensatina [19, 24, 28], our study is the first to examine whether habitat-genotype associations exist and to formally test the hypothesis that habitat type is structuring the hybrid zone . Finally, this study is the first to use maximum-likelihood analysis of multilocus clines and linkage disequilibrium to make inferences about selection in a hybrid zone at the end of the ring.
A solid theoretical framework exists for studying the evolution and maintenance of clines [34–37]. After any initial contact between differentiated populations, clines in allele frequencies will exist, the scale of which depends on the dispersal scale of the organism, the strength of selection and time since contact, and the shape of which will be determined by asymmetric and epistatic effects [2, 38]. If there is neutral mixing, steep gradients in allele frequencies will decay over time resulting in wide, shallow clines; conversely, if there is a strong barrier to gene flow because of selection against hybrids and/or assortative mating, steep clines may be maintained [2, 10, 39]. Selection may act against hybrids independent of geographic location, or along environmental gradients where local adaptation favors one parental form in one habitat, and the other in another [34–36]. In the latter scenario, migrants dispersing into the neighboring habitat keep the taxa mixed and prevent further local adaptation. All of these models assume that dispersal is random with respect to phenotype and the environment. However, it is possible that an individual's decision about whether and where to disperse depends on its fitness in a given environment, with different genotypes moving into preferred habitat patches where they are locally adapted [40, 41]. It seems reasonable that such fitness-dependent dispersal may result in fine-scale genotype-habitat associations ; when such associations exist, simple hybrid zone models may not effectively describe spatial variation in allele frequencies [43, 44], but can be extended to take such associations into account . In addition, even when selection against hybrids/hybridization is not tied to the environment, tension zones resulting from endogenous selection will tend to seek out geographic barriers to gene flow and/or habitat suitability troughs in the environment, where they will become trapped . The interaction of endogenous and exogenous factors where tension zones come to rest may further steepen clines. For these reasons, incorporating information on local environmental variation is important in understanding the balance between local adaptation and dispersal and its effect on hybrid zone structure.
Here, we present a detailed genetic analysis of a hybrid zone on Palomar Mountain between the geographically terminal forms of the Ensatina complex. Our goal is to analyze the barrier to gene flow between these sympatric forms to provide insight into the nature and extent of reproductive isolation between the terminal forms of a ring species. We use two complementary analytical approaches. First, we use individual-based Bayesian methods for identifying and classifying hybrids using multilocus genetic data to estimate levels and direction of hybridization [46, 47]. We then use maximum-likelihood analysis of linkage disequilibrium and cline shape to make inferences about dispersal and selection in the hybrid zone [48, 49]. We test for genotype-habitat associations, and whether including this variation improves the fit of clines [cf. 45]. If a strong barrier to gene flow exists between the hybridizing taxa, we would expect to see: 1) concordant clines among independent loci that are narrow with respect to dispersal; 2) deviations from Hardy-Weinberg equilibrium at diagnostic loci due to nonrandom mating and/or selection against hybrids; and 3) nonrandom associations (linkage disequilibria) between alleles characteristic of each parental type, due predominantly to dispersal of parental individuals into the hybrid zone [10, 48–50]. By examining patterns of cytonuclear disequilibria, we provide insight into possible mechanisms of selection and patterns of mating in the hybrid zone  that may be important for maintaining species boundaries. Finally, we discuss the evolutionary implications of introgression and reproductive isolation at the end of the ring for divergence within the Ensatina complex as a whole.
PCR annealing temperatures, sequence length, and primer sequences
# variable sites (bp)
Primer sequence (5'-3')
Identification and classification of hybrids
Structure [47, 60, 61] and NewHybrids [46, 62] were used to identify and classify hybrids using the multilocus sequence data. In the context of a two-population hybrid zone, Structure jointly assigns individuals probabilistically to the two parental populations [47, 60, 61], while NewHybrids computes the posterior probability that an individual belongs to distinct genotype frequency classes (e.g., parentals, F1s, F2s, and backcrosses) arising from early generation matings between two species [46, 62]. We coded each unique haplotype as a different allele (Additional file 5). In Structure, we used the admixture model and assumed two populations with independent allele frequencies (λ = 1). We ran 100,000 sweeps of five chains after a burn-in of 50,000 sweeps and checked for convergence by comparing the estimated membership coefficient (Q) for each individual across the 5 runs. For NewHybrids, we used the default genotype categories for first- and second-generations of crossing and ran 100,000 sweeps of five chains started from overdispersed starting values after a burn-in period of 50,000 sweeps following the software author's recommendation. Uniform priors were used for the mixing proportions and allele frequencies. To check for convergence, we visually inspected P(z) values from the different runs which were then averaged across the 5 runs. A threshold hybrid index (Q-value) was calculated to classify individuals as "hybrids" or "pure parentals" in Structure. For three diagnostic loci (six alleles), there are seven categories (i.e., an individual may contain 0, 1, 2, 3, 4, 5, or 6 alleles from taxon 1) and thus "hybrids" were defined as any individual with a Q-value between 0.1 [i.e., (0+1/7)/2] and 0.9 [i.e., (1+6/7)/2], while any individual with a Q-value < 0.1 or > 0.9 was considered to be a pure parental. The same threshold was used to distinguish hybrids from pure parentals for the NewHybrids analysis, with posterior probability values summed across hybrid classes for an individual [46, 62]. Information about the taxon of origin of individuals was not used for either analysis.
Phased haplotypes were used to identify allelic states associated with each source taxon (i.e., "eschscholtzii" and "klauberi" alleles) for cline analysis. All loci were diagnostic for one parental form or the other, allowing nearly all haplotypes to be unambiguously assigned. Two haplotypes found in four individuals for one of the loci (SLC8A3) could not be unambiguously assigned and were excluded from the cline analysis. We assumed that allele frequencies at individual loci did not change significantly over the three-year sampling period. Individuals were not pooled into discrete samples for cline fitting.
where c is the cline center, w is the cline width and (x - c) is the distance from the cline center . We fit sigmoid clines and did not explore more complex stepped cline models because doing so only seems justified when there is sufficient sampling in the tails of the cline . Parameter estimates are given as maximum-likelihood estimates, along with two-unit support limits analogous to 95% confidence intervals .
where the locus expectation can deviate from x = y as a function of the expected heterozygosity, He = 2HI(1 - HI). The α parameter (directionality) shifts introgression toward one or the other genome (in this case, toward eschscholtzii) and the β parameter (abruptness) allows the introgressive change at a locus to be more or less abrupt than the equal-introgression expectation .
Barton's concordance method  is an individual-based method independent of geographic information. We also fitted geographic clines to each locus separately and assessed cline coincidence using the likelihood method described by Phillips et al. . The likelihood surface of each locus was explored stepwise along axes for both center position (c) and width (w) while allowing other parameters to vary at each point . Likelihood profiles  were constructed for both c and w and summed over all loci, resulting in a log-likelihood profile for the ML shared center or width . The shared ML estimate was compared to the sum of noncoincident profile ML estimates using a likelihood ratio test . Twice the difference in log likelihood (G = 2ΔLL) between the two models under comparison is significant at level α if G = 2ΔLL > χ2df, α with the degrees of freedom equal to the difference in the number of parameters between the two models [48, 49].
Testing for genotype-habitat associations
We tested whether hybrids and pure parentals show differences in elevation and vegetation type to see if a simple clinal hybrid zone model was appropriate for describing spatial variation in allele frequency. If significant genotype-habitat associations exist, a simple clinal model may be a poor description of the observations  relative to a clinal model that takes such associations into account . We used elevation estimates taken at the point of capture for each individual and vegetation data from a floristic study of Palomar Mountain State Park . Individuals were classified as coming from one of two vegetation series, Mixed Montane Woodland (MMW) or Montane Woodland with Pseudotsuga macrocarpa (MWP) (Figure twenty-eight in ). These series intergrade  and do not have sharply defined borders in and around the hybrid zone, but there is a discernible transition from MMW in the northwestern, lower-elevation portion of the transect to MWP in the southeastern, higher-elevation portion of the transect (Figure 2). Statistical analyses were performed using SPSS Statistics 17.0 (IBM).
To examine whether genotype-habitat associations contributed to the observed spatial structure of the hybrid zone, we compared a model incorporating variation in habitat and elevation (the "habitat-and-cline" model) to one assuming no genotype-habitat associations (the "cline only" model" model) using likelihood-ratio tests [cf. 45], accepting the more complex (i.e., more parameter rich) model only when justified by a significant increase in the likelihood of the observations.
Estimating linkage disequilibria
Average pairwise linkage disequilibrium (D) was estimated in sets of sampled genotypes using the likelihood approach implemented in Analyse 1.3 . Sets of genotypes were formed in a window sliding across the most likely orientation of the hybrid zone using a window size of 200 meters moved in 100 meter increments along the cline using a routine written in Mathematica  and implemented in Analyse 2.0beta. D is a measure of the statistical association of allelic states across loci, which ranges from -0.25 through zero to 0.25. When considering a single diploid locus (rather than two haploid loci), the analog of D is heterozygote deficit . We calculated: 1) within locus disequilibrium (the ML estimate of heterozygote deficit over the three diploid nuclear loci); 2) between nuclear loci disequilibrium (the ML estimate of pairwise D over the three possible pairs of the nuclear loci); and 3) cytonuclear disequilibrium (the ML estimate of pairwise D over the three possible pairs of nuclear/mt markers).
Hybrid zone genotypes
Parental forms are associated with different vegetation series in the contact zone (Chi-square test, p < 0.001). Eschscholtzii are found almost exclusively in the MMW series along with > 80% of the hybrids, while klauberi individuals are found at roughly equal frequencies in the two vegetation series. Eschscholtzii occupies a broader, but lower elevational range (1200-1601 m; mean 1360 m) than klauberi (1297-1694 m; mean 1470 m) and hybrids (1342-1601 m; mean 1445 m) (One-way ANOVA, Tukey's HSD, p < 0.001). Modification of the underlying clines according to vegetation type [cf. 45] gave no significant improvement in cline fit, and modification according to elevation provided only a slight improvement (results not shown).
Cline shape and concordance
- 0.112 (- 0.138, - 0.087)
0.764 (0.704, 0.830)
- 0.202 (- 0.274, - 0.136)
0.718 (0.580, 0.898)
All nuclear loci
- 0.097 (- 0.125, - 0.070)
0.765 (0.700, 0.838)
- 0.091 (- 0.140, - 0.045)
0.770 (0.661, 0.901)
- 0.113 (- 0.164, - 0.065)
0.799 (0.687, 0.934)
- 0.087 (- 0.134, - 0.042)
0.725 (0.620, 0.851)
Earlier work based on allozymes has shown that hybridization is infrequent, or even absent in at least one area of sympatry at the end of the Ensatina ring [27, 28]. Our fine-scale analysis revealed a much higher frequency of hybridization compared to previous estimates for Palomar Mountain as well as other contacts between eschscholtzii and klauberi based on allozymes and morphology [27, 28]. Pure parentals and F1s dominated the sample. The hybrid zone is narrow (cline width estimates of 718-799 m) with respect to per generation dispersal estimates (maximum ~120 m; ). Although significant genotype-habitat associations exist, modification of the underlying clines according to vegetation type did not improve the fit of clines, and modification by elevation provided only a minor improvement. The predominance of genetic disequilibrium (including heterozygote deficit and cytonuclear disequilibrium) on the eschscholtzii side of the hybrid zone, coupled with the fact that nearly all hybrids have klauberi mtDNA suggests that either: 1) the hybrid zone may be moving (toward the range of eschscholtzii) and/or 2) the zone is static, but gene flow is asymmetric (from klauberi to eschscholtzii). Without long-term data on the spatiotemporal dynamics of the zone, these hypotheses are difficult to disentangle . Barton and Hewitt  argued that most hybrid zones are clines maintained by a balance between dispersal and selection against hybrids ("tension zones" ), and can move from place to place because they are not maintained by local environmental conditions. Tension zone movement may be caused by differences in fitness, density, dispersal, or hybridization asymmetry between parental forms [2, 10, 73, 74]. For this reason, although they are not maintained by the local environment, tension zones will move toward and become associated with barriers to gene exchange, including environmental factors that reduce density or dispersal . Our observations are consistent with a tension zone trapped at an ecotone, with gene combinations characteristic of klauberi showing up on the eschscholtzii side of the zone due to asymmetric hybridization, giving a strong signal of genetic disequilibria. Given that nearly all of the hybrids possess klauberi mtDNA, either: 1) hybridization between eschscholtzii and klauberi is (mostly) unidirectional, with F1 hybrids formed from female klauberi mating with male eschscholtzii (but not vice versa), implying asymmetric prezygotic isolation, or 2) hybridization is reciprocal, but offspring resulting from female eschscholtzii mating with male klauberi (or male hybrids) are inviable (implying asymmetric postzygotic isolation ). Hybrids often possess the mitochondrial DNA of only one of two parental species in nature [76–78], a pattern predicted in species with female-choice when females of a rare species are unable to find conspecific males because they are scarce, and eventually accept matings with heterospecific males of a more common species . However, the two taxa appear to be equally abundant in zones of overlap at Palomar Mountain, suggesting that factors other than rarity of conspecific klauberi males are responsible for the disproportionate percentage of hybrids with klauberi mtDNA. The presence of four hybrid individuals with eschscholtzii mtDNA suggests matings between female eschscholtzii and male klauberi (or male hybrids) are possible, but appear to be rare, or that the offspring usually do not survive. Like many animals, female Ensatina are the choosier sex because they invest relatively more than males in reproduction, and are courted by promiscuous males, both of their own and closely related species [1, 80]. If there are costs associated with heterospecific matings (e.g., because hybrids are less fit, or, because these matings produce fewer offspring), there may be strong selection acting on female mating preferences toward increased ability to discriminate between conspecific and heterospecific males [81–83]. This in turn could drive indirect selection on males to track female mating preferences .
It is instructive to compare the nature of hybridization between the terminal forms to that between another pair of morphologically and genetically distinct coastal and inland lineages of Ensatina -- xanthoptica and platensis, respectively -- in the foothills of the central Sierra Nevada [19, 24, 28]. These morphological analogs of eschscholtzii and klauberi are thought to have come into contact at some point during the Pleistocene when climate in California was cooler and wetter, allowing xanthoptica to cross the now arid Central Valley and invade the Sierras from the San Francisco Bay area . Hybrids are abundant in the Sierran contact zones, but few, if any, F1s have been detected , in contrast to our results. Alexandrino et al.  suggested that reduced opportunities for heterospecific encounters due to habitat preference and/or stronger selection against F1s compared to later generation hybrids could explain this pattern. They estimated cline widths comparable to, or wider than those observed here (730-2000 m), and inferred strong selection against hybridization (46-75%). For comparison, selection against hybridization has been estimated at 32% for distinct lineages of lizards of the Sceloporus grammicus complex [85, 86] and 17-22% for the toads Bombina bombina and B. variegata [48, 49]. Although it is tempting to try and estimate selection against hybridization in the contact zone studied here, because the hybrid zone is dynamic and/or asymmetric, we cannot sensibly do so under a standard tension zone model, which assumes a symmetric, static hybrid zone. Nonetheless, it is clear that overall selection against hybrids/hybridization is strong in both cases, yet the two contact zones differ markedly in the frequency of different hybrid genotype frequency classes.
Geographic variation in hybridization frequency among the four contact zones at the end of the ring [27, 28] suggests that reproductive isolation may not be uniform across contact zones, perhaps due to differences in local ecological conditions that limit the opportunity for hybridization [see 87], and/or different outcomes of reinforcement [88, 89] driven by selection against hybrids owing to the spatial structure of the hybrid zone [43, 90, 91]. This variation provides a rare opportunity to investigate variation in the strength of reproductive isolating barriers within a single species pair in nature, while controlling for among-taxa variation in age, degree of differentiation, strength of postzygotic isolation, etc. [see 1, 87, 92].
The narrow width, shape, and overall concordance of clines for presumably unlinked loci suggest a strong barrier to gene flow between eschscholtzii and klauberi on Palomar Mountain. Introgression of neutral alleles will be delayed, but favorable alleles could quickly cross if prezygotic isolation is weak or absent [93, 94]. The particular markers themselves probably do not have a direct effect on fitness, but rather they are in linkage disequilibrium with other loci that are under selection. If the amount of linkage disequilibrium that connects selection against hybrids (or hybridization) with an evolving prezygotic isolating mechanism is sufficiently high, then premating isolation may evolve through reinforcement . Future work incorporating information about patterns of mating and gamete utilization will be critical for understanding the role of selection in generating and maintaining species boundaries at the end of the ring [83, 96, 97].
We thank Jimmy A. McGuire, David B. Wake, and George K. Roderick for comments on an earlier draft of this manuscript. Eric Anderson kindly provided an OSX version of NewHybrids and guidance with analyses. We thank R. K. Lauri for providing an electronic copy of his unpublished thesis and vegetation map of Palomar Mountain State Park and Michelle Koo for assistance digitizing the map. We are grateful to Susan Cameron for help with vegetation analysis. We thank the ESRI Conservation Program for providing ArcGIS software for our use. For help in the field, we thank Mike Anguiano, Rayna Bell, Chuck Brown, Susan Cameron, Jessica Castillo, Becky Chong, Erin Conlisk, Brandon Endo, Matt Fujita, Emilio Gabbai-Saldate, Zach Hanna, Kory Heiken, Jasmine Junge, Megan Lahti, Ben Lowe, Matt McElroy, Greg Pauly, Ricardo Pereira, Tod Reeder, Mark Roll, Sean Rovito, Kevin Rowe, Frank Santana, Sean Schoville, Sonal Singhal, Tate Tunstall, and John Wiens. We especially thank Kim and Donna Rosier, Bill Stephenson, and the rest of the staff of the Palomar Mountain Christian Conference Center for their hospitality and access to their property. Permission to work in Palomar Mountain State Park was granted by the State of California Department of Parks and Recreation; we thank Mark Jorgensen, Nedra Martinez, and Jeff Lee for access to the park. This work was conducted under a scientific collecting permit issued by the California Department of Fish and Game (SC-007654). We thank Kevin Fleming and Art Fong (CDF&G) for assistance with permitting. Funding for this work was provided by Sigma Xi, the National Science Foundation (Doctoral Dissertation Improvement Grant DEB-0909821 to TJD and NSF DEB-0641078 to CM), the University of California Department of Integrative Biology, and the Museum of Vertebrate Zoology Martens and Louise Kellogg funds.
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