A population genomic analysis of species boundaries: neutral processes, adaptive divergence and introgression between two hybridizing plant species

Little is known about the nature of species boundaries between closely related plant species and about the extent of introgression as a consequence of hybridization upon secondary contact. To address these topics we analyzed genome-wide differentiation between two closely related Silene species, Silene latifolia and S. dioica , and assessed the strength of introgression in sympatry. More than 300 AFLP markers were genotyped in three allopatric and three sympatric populations of each species. Outlier analyses were performed separately for sympatric and allopatric populations. Both positive and negative outlier loci were found, indicating that divergent and balancing selection, respectively, have shaped patterns of divergence between the two species. Sympatric populations of the two species were found to be less differentiated genetically than allopatric populations, indicating that hybridization has led to gene introgression. We conclude that differentiation between S. latifolia and S. dioica has been shaped by a combination of introgression and selection. These results challenge the view that species differentiation is a genome-wide phenomenon, and instead support the idea that genomes can be porous and that species differentiation has a genic basis.     

Genetic structure of hybrid zones between Silene latifolia and Silene dioica (Caryophyllaceae): evidence for introgressive hybridization

Natural hybrid zones provide a valuable tool to study introgressive hybridization, because they can contain a wide variety of genotypes that result from many generations of recombination. Here we used molecular markers and morphological variation to describe the structure of two natural hybrid zones between Silene latifolia and Silene dioica in the Swiss Alps. Populations in both hybrid zones consisted of few intermediate hybrids and were dominated by backcross hybrids. The latter were also found in the parental populations at the margins of the hybrid zones. Out of 209 amplified fragment length polymorphism (AFLP) markers scored in 390 individuals, only 7 (3.3%) were species specific. These results indicate that introgression between S. dioica and S. latifolia is extensive, and that hybrid zones act as bridges to gene flow between these two species. Analysis of linkage disequilibrium identified few populations in which hybridization is ongoing, whereas in most populations linkage disequilibrium has eroded. Where hybridization is ongoing, strong changes in species-specific marker frequencies and morphological traits were observed. Plastid introgression into the hybrid zone was found to be bidirectional, but only the S. latifolia plastid haplotype was found in a nuclear S. dioica background. This unidirectional plastid introgression from S. latifolia into S. dioica is most likely due to pollen-flow from S. dioica onto S. latifolia, and results in plastid capture. Comparisons between the molecular and the morphological hybrid indices revealed that morphology in this study system is useful for identifying hybrids, but not for detailed analysis of hybrid zone structure.     

Differences in population structure of the anther smut fungus Microbotryum violaceum on two closely related host species, Silene latifolia and S. dioica

We investigated the genetic population structure of the sexually transmitted plant pathogen, the fungus Microbotryum violaceum, on the two closely related host species Silene latifolia and S. dioica using microsatellite markers. We found strong deviations from Hardy-Weinberg expectations, with significant heterozygote deficiency in almost all populations. Fungal strains from the two host species were differentiated, and these host races differed in amount of variation within populations and differentiation among populations. Anther smut from S. latifolia harboured significantly less microsatellite diversity and were more genetically differentiated from each other than those from S. dioica. Small effective population sizes, rapid population turnover, and less gene flow among populations could lead to this higher population differentiation and lower within population genetic diversity for anther smut populations on S. latifolia than on S. dioica. These results are in concordance with host ecology because S. latifolia grows in more disturbed habitats than S. dioica and may provide a shorter-lived host environment.     

Within-population structure highlighted by differential introgression across semipermeable barriers to gene flow in Anguilla marmorata

In the marine environment, differential gene exchange between partially reproductively isolated taxa can result in introgression that extends over long distances due to high larval dispersal potential. However, the degree to which this process contributes to interlocus variance of genetic differentiation within introgressed populations remains unclear. Using a genome-scan approach in the Indo-Pacific eel Anguilla marmorata, we investigated the degree of interpopulation genetic differentiation, the rate of introgression, and within-population genetic patterns at 858 AFLP markers genotyped in 1117 individuals. Three divergent populations were identified based on clustering analysis. Genetic assignments of individuals revealed the existence of different types of hybrids that tended to co-occur with parental genotypes in three population contact zones. Highly variable levels of genetic differentiation were found between populations across the AFLP markers, and reduced rates of introgression were shown at some highly differentiated loci. Gene flow across semipermeable genetic barriers was shown to generate spatial introgression patterns at some loci which define within-population structure over long distances. These results suggest that differential introgression in subdivided populations may be relevant when interpreting spatial variation patterns displayed by outlying loci in other marine fish populations.     

Intraspecific competition and mating between fungal strains of the anther smut Microbotryum violaceum from the host plants Silene latifolia and S. dioica

Abstract We studied intraspecific competition and assortative mating between strains of the anther smut fungus Microbotryum violaceum from two of its host species, Silene latifolia and S. dioica . Specifically, we investigated whether strains from allopatric host populations have higher competitive ability on their native host species and show positive assortative mating. In general, strains isolated from S. latifolia outcompeted strains isolated from S. dioica on both host species, but in female hosts, heterotypic dikaryons (i.e., dikaryons composed of a haploid strain originating from S. latifolia and a haploid strain originating from S. dioica ) were most successful in competition. Furthermore, the latency period was significantly shorter for heterokaryons that contained at least one strain originating from S. latifolia , compared to heterokaryons that only contained strains originating from S. dioica . The frequencies of conjugations between strains originating from S. latifolia were much higher than conjugation frequencies between strains originating from S. dioica . A significant positive correlation was detected between the relative success of strains in competition and in conjugation, suggesting that success of a strain in competition might be partly determined by its swiftness of mating. In addition, reciprocal differences within heterotypic crosses revealed a significant effect of fungal mating type, with mating type a₁ being the main determinant of mating pace. The observed differences in infection success, conjugation rate, and latency period in favor of strains from S. latifolia relative to strains from S. dioica on both host species are discussed in an evolutionary context of opportunities for the maintenance of differentiation between different formae speciales upon secondary contact.     

Complex hybridization dynamics between golden-winged and blue-winged warblers (Vermivora chrysoptera and Vermivora pinus) revealed by AFLP, microsatellite, intron and mtDNA markers

Blue-winged (Vermivora pinus) and golden-winged warblers (Vermivora chrysoptera) have an extensive mosaic hybrid zone in eastern North America. Over the past century, the general trajectory has been a rapid replacement of chrysoptera by pinus in a broad, northwardly moving area of contact. Previous mtDNA-based studies on these species' hybridization dynamics have yielded variable results: asymmetric and rapid introgression from pinus into chrysoptera in some areas and bidirectional maternal gene flow in others. To further explore the hybridization genetics of this otherwise well-studied complex, we surveyed variation in three nuclear DNA marker types--microsatellites, introns, and a panel of amplified fragment length polymorphisms (AFLPs)--with the goal of generating a multilocus assay of hybrid introgression. All markers were first tested on birds from phenotypically and mitochondrially pure parental-type populations from outside the hybrid zone. Searches for private alleles and assignment test approaches found no combination of microsatellite or intron markers that could separate the parental populations, but seven AFLP characters exhibited significant frequency differences among them. We then used the AFLP markers to examine the extent and pattern of introgression in a population where pinus-phenotype individuals have recently invaded a region that previously supported only a chrysoptera-phenotype population. Despite the low frequency of phenotypic hybrids at this location, the AFLP data suggest that almost a third of the phenotypically pure chrysoptera have introgressed genotypes, indicating the presence of substantial cryptic hybridization in the history of this species. The evidence for extensive cryptic introgression, combined with the lack of differentiation at other nuclear loci, cautions against hybrid assessments based on single markers or on phenotypic traits that are likely to be determined by a small number of loci. Considered in concert, these results from four classes of molecular markers indicate that pinus and chrysoptera are surprisingly weakly differentiated and that far fewer genetically 'pure' populations of chrysoptera may exist than previously assumed, two findings with broad implications for the conservation of this rapidly declining taxon.     

Extensive hybridization and associated geographic trends between two rockfishes Sebastes vulpes and S. zonatus (Teleostei: Scorpaeniformes: Sebastidae)

Interspecific hybridization is an important evolutionary process, which has significant influence on the diversity within and between participating taxa. Although interspecific hybridization in terrestrial and freshwater organisms has been subjected to many detailed studies, studies in marine realm have been limited in terms of both numbers and detail. In this study, the potential for interspecific hybridization between two rockfishes, Sebastes vulpes and S. zonatus, occurring in the western North Pacific, was assessed on the basis of 177 specimens collected from three sampling localities within the main geographic distribution of both species, and analysed using a combination of amplified fragment length polymorphisms (AFLP), mitochondrial DNA (mtDNA) markers and morphometric characters. Bayesian‐based individual genetic assignment based on 364 AFLP loci detected a total of 63 (35.6%) hybrid specimens in the data set, the presence of interspecific hybrids also being rigorously supported by mtDNA analysis using partial sequences from the control region and morphological analysis based on 31 morphometric characters. Hybrids from all three localities were found, showing a common pattern of biased introgression across the localities whereby hybrids were more closely related to S. zonatus than to S. vulpes. Apart from this common pattern, rates of hybridization varied considerably among the localities, being greater in the northern localities. Variations in the local rates of hybridization were associated with variations in habitat segregation and thermal regime, implying that vertical water temperature regimes determined the extent of habitat segregation of the two species and, accordingly, the opportunity for hybridization.     

The final frontier: early-stage genetic introgression and hybrid habitat use in the northwestern extent of the Golden-winged Warbler breeding range

Anthropogenic changes to the landscape and climate have resulted in secondary contact between previously allopatric species. This can result in genetic introgression and reverse speciation when closely related species are able to hybridize. The Golden-winged Warbler has declined or been extirpated across much of its range where it has come into secondary contact with the Blue-winged Warbler. Genetic screening previously showed that introgression had occurred range-wide with the exception of Manitoba, Canada. Our goal was to reassess the genetic status of the Golden-winged Warbler population in Manitoba and to examine the demographics and habitat use of phenotypic and genetic hybrids. From 2011 to 2014, we sampled and screened mtDNA from 205 Golden-winged Warblers and hybrids in southeast Manitoba. In 2012, we monitored all Golden-winged Warbler territories within those sites and measured territory- and landscape-level habitat variables. Of the birds screened, 195 had a phenotype that matched their mtDNA type, two were phenotypic hybrids, and eight showed a phenotypic-mtDNA mismatch (cryptic hybrids). We found no difference in the habitat used by Golden-winged Warblers compared with hybrids at either scale. The low proportion of hybrids found in Manitoba and the lack of a distinguishable difference in habitat use by Golden-winged Warblers and hybrids indicates that the exclusion of hybrid birds from Golden-winged Warbler habitat is unlikely to be a successful conservation strategy. The best way to manage for Golden-winged Warblers is to slow the habitat loss and fragmentation that continues within Manitoba and to actively manage early-successional deciduous forest using tools such as fire and logging.     

Amplified fragment length polymorphism analysis identifies hybrids between two subspecies of warblers

In the western Pyrenees (Southwest France and Northwest Spain), a narrow hybrid zone exists between the common chiffchaff Phylloscopus (collybita) collybita and the Iberian chiffchaff Phylloscopus (c.) brehmii. In this zone, which is approximately 20 km wide, mixed matings and individuals singing the songs of both taxa occur at substantial frequencies (24 and 8.6%, respectively), suggesting frequent hybridization. Previous studies have shown very weak mitochondrial gene flow (Nm = 0.065), whereas four microsatellites suggested much higher nuclear gene flow (Nm = 4.9). In this study we used the amplified fragment length polymorphism (AFLP) method in order to identify hybrids and early backcrosses. We typed 91 birds from both allopatric and sympatric areas for 12 informative AFLP markers (of > 141 polymorphic fragments), obtained by screening 13 AFLP primer combinations. These individuals were previously typed for song (brehmii, collybita or mixed singers), mitochondrial DNA (mtDNA) haplotype and allelic genotypes at four microsatellite loci. Assignment tests demonstrated that in the zone of sympatry, a substantial number of intermediate genotypes existed among the birds previously believed to be pure collybita and brehmii, based on song and mtDNA haplotype. The majority of the mixed singers had intermediate genotypes. Our data suggest that the fraction of the adult population having a hybrid origin (hybrids or backcrosses) is in the order of 10%. With such a frequency of genetic hybrids, there would have been much more mtDNA introgression than observed, had female hybrids been perfectly fertile/viable. This result is consistent with male-biased gene flow and Haldane's rule.     

Mito‐nuclear discordance across a recent contact zone for California voles

To examine the processes that maintain genetic diversity among closely related taxa, we investigated the dynamics of introgression across a contact zone between two lineages of California voles (Microtus californicus). We tested the prediction that introgression of nuclear loci would be greater than that for mitochondrial loci, assuming ongoing gene flow across the contact zone. We also predicted that genomic markers would show a mosaic pattern of differentiation across this zone, consistent with genomes that are semi‐permeable. Using mitochondrial cytochrome b sequences and genome‐wide loci developed via ddRAD‐seq, we analyzed genetic variation for 10 vole populations distributed along the central California coast; this transect included populations from within the distributions of both parental lineages as well as the putative contact zone. Our analyses revealed that (1) the two lineages examined are relatively young, having diverged ca. 8.5–54 kya, (2) voles from the contact zone in Santa Barbara County did not include F1 or early generation backcrossed individuals, and (3) there appeared to be little to no recurrent gene flow across the contact zone. Introgression patterns for mitochondrial and nuclear markers were not concordant; only mitochondrial markers revealed evidence of introgression, putatively due to historical hybridization. These differences in genetic signatures are intriguing given that the contact zone occurs in a region of continuous vole habitat, with no evidence of past or present physical barriers. Future studies that examine specific isolating mechanisms, such as microhabitat use and mate choice, will facilitate our understanding of how genetic boundaries are maintained in this system.     

Dynamics of drift, gene flow, and selection during speciation in Silene

The mechanics of speciation with gene flow are still unclear. Disparity among genes in population differentiation (F(ST)) between diverging species is often interpreted as evidence for semipermeable species boundaries, with selection preventing "key" genes from introgressing despite ongoing gene flow. However, F(ST) can remain high before it reaches equilibrium between the lineage sorting of species divergence and the homogenizing effects of gene flow (via secondary contact). Thus, when interpreting F(ST), the dynamics of drift, gene flow, and selection need to be taken into account. We illustrate this view with a multigenic analyses of gene flow and selection in three closely related Silene species, S. latifolia, S. dioica, and S. diclinis. We report that although S. diclinis appears to have evolved in allopatry, isolation with (bidirectional) gene flow between S. latifolia and S. dioica is likely, perhaps as a result of parapatric speciation followed by more extensive sympatry. Interestingly, we detected the signatures of apparently independent instances of positive selection at the same locus in S. latifolia and S. dioica. Despite gene flow between the species, the adaptive alleles have not crossed the species boundary, suggesting that this gene has independently undergone species-specific (diversifying or parallel) selection.     

Hybridization and asymmetric introgression between Cypripedium tibeticum and C. yunnanense in Shangrila County,Yunnan Province,China

Hybridization and introgression are thought to be important for speciation and adaptation in many plants. However, little is known about the hybridization and introgression among Cypripedium species. To investigate the evidence for hybridization and the pattern of introgression between Cypripedium yunnanense and C. tibeticum in Shangrila County, Yunnan Province, China, morphological characters and amplified fragment length polymorphism (AFLP) data for both the species and their putative hybrids were studied. Hand pollination was also performed to verify the crossability of the putative parents. Principal coordinate analysis based on morphological characters and the AFLP data suggested that the putative hybrids were true hybrids of these two Cypripedium species. Analysis with the NewHybrids software indicated that the putative hybrids were F1 generation individuals and backcrosses to C. yunnanese, but no F2 generation was found. Analysis with the Structure software demonstrated asymmetric introgression from C. tibeticum to C. yunnanense. We conclude that natural hybridization and introgression can occur between these two species and that in situ conservation of the parental species is required before fully assessing the evolutionary potential of hybrids.     

Natural hybridization does not dissolve species boundaries in commercially important sea cucumbers

The study of species boundaries in areas of sympatry provides important insight into speciation processes. We investigated whether (i) two sympatric holothurians, Holothuria scabra and H. s. var. versicolor constituted species, and (ii) specimens of intermediate phenotype hybrids. Results from allozyme and 16S mtDNA sequence analyses indicated these two sea cucumbers to be distinct but young biological and phylogenetic species. Several private allozyme alleles existed and a Bayesian analysis grouped varieties into separate clusters. MtDNA sequences hardly varied within each taxon, and nine single bp changes were diagnostic between these two taxa. Allozyme allele frequencies in individuals of intermediate phenotype were intermediate to those of H. scabra and H. s.  var.  versicolor , most private alleles were present and heterozygote frequencies were higher than in either species. Ancestry coefficients modelled for these individuals were close to 0.5, indicating that the two taxa contributed equally to their genome. MtDNA sequences were identical to those of either species. We conclude that individuals of intermediate phenotype represent F1 hybrids. The presence of hybrids demonstrates that the opportunity for introgression exists, but is not realized, as backcrossing and introgression were not supported by the data. Thus, the genetic integrity of either holothurian species remains intact through an unknown postzygotic mechanism, possibly hybrid sterility.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 261–270.     

Inbreeding and outbreeding depression in Stylidium hispidum: implications for mixing seed sources for ecological restoration

The benefits of composite rather than local seed provenances for ecological restoration have recently been argued, largely on the basis of maximizing evolutionary potential. However, these arguments have downplayed the potentially negative consequences of outbreeding depression once mixed provenances interbreed. In this study, we compared intraspecific F1 hybrid performance and molecular marker differentiation among four populations of Stylidium hispidum, a species endemic to Southwestern Australia. Multivariate ordination of 134 AFLP markers analyzed genetic structure and detected two clusters of paired sites that diverged significantly for marker variation along a latitudinal boundary. To test for outbreeding depression and to determine the consequences of molecular population divergence for hybrid fitness, we conducted controlled pollinations and studied germination and survival for three cross categories (within‐population crosses, short‐ and long‐distance F1 hybrids) for paired sites distributed within and between the two genetically differentiated regions. We found evidence of outbreeding depression in long‐distance hybrids (111–124 km), and inbreeding depression among progeny of within‐population crosses, relative to short‐distance (3–10 km) hybrids, suggesting an intermediate optimal outcrossing distance in this species. These results are discussed in light of the evolutionary consequences of mixing seed sources for biodiversity restoration.     

A selective sweep in the chloroplast DNA of dioecious silene (section Elisanthe)

Gene flow occurs predominantly via pollen in angiosperms, leading to stronger population subdivision for maternally inherited markers, relative to paternally or biparentally inherited genes. In contrast to this trend, population subdivision within Silene latifolia and S. dioica, as well as subdivision between the two species, is substantially lower in maternally inherited chloroplast genes compared to paternally inherited Y-linked genes. A significant frequency spectrum bias toward rare polymorphisms and a significant loss of polymorphism in chloroplast genes compared to Y-linked and autosomal genes suggest that intra- and inter-specific subdivision in the chloroplast DNA may have been eroded by a selective sweep that has crossed the S. latifolia and S. dioica species boundary.     

Efficient molecular sexing in dioecious Silene latifolia and S. dioica and paternity analysis in F(1) hybrids

Two polymerase chain reaction-based assays have been developed that work in combination with an efficient DNA extraction protocol to rapidly and reliably determine sex in the dioecious plant species Silene latifolia and S. dioica. In addition, one of the assays allows assessing paternity in the F(1) generation of intra- and interspecific matings involving the two species.     

Can hybridization cause local extinction: a case for demographic swamping of the Australian native Senecio pinnatifolius by the invasive Senecio madagascariensis?

Hybridization between native and invasive species can have several outcomes, including enhanced weediness in hybrid progeny, evolution of new hybrid lineages and decline of hybridizing species. Whether there is a decline of hybridizing species largely depends on the relative frequencies of parental taxa and the viability of hybrid progeny. Here, the individual- and population-level consequences of hybridization between the Australian native Senecio pinnatifolius and the exotic Senecio madagascariensis were investigated with amplified fragment length polymorphism (AFLP) markers, and this information was used to estimate the annual loss of viable seeds to hybridization. A high frequency (range 8.3-75.6%) of hybrids was detected in open pollinated seeds of both species, but mature hybrids were absent from sympatric populations. A hybridization advantage was observed for S. madagascariensis, where significantly more progeny than expected were sired based on proportional representation of the two species in sympatric populations. Calculations indicated that S. pinnatifolius would produce less viable seed than S. madagascariensis, if hybridization was frequency dependent and S. madagascariensis reached a frequency of between 10 and 60%. For this native-exotic species pair, prezygotic isolating barriers are weak, but low hybrid viability maintains a strong postzygotic barrier to introgression. As a result of asymmetric hybridization, S. pinnatifolius would appear to be under threat if S. madagascariensis increases numerically in areas of contact.     

Exploring species limits in two closely related Chinese oaks

BACKGROUND: The species status of two closely related Chinese oaks, Quercus liaotungensis and Q. mongolica, has been called into question. The objective of this study was to investigate the species status and to estimate the degree of introgression between the two taxa using different approaches. METHODOLOGY/PRINCIPAL FINDINGS: Using SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) markers, we found that interspecific genetic differentiation is significant and higher than the differentiation among populations within taxa. Bayesian clusters, principal coordinate analysis and population genetic distance trees all classified the oaks into two main groups consistent with the morphological differentiation of the two taxa rather than with geographic locations using both types of markers. Nevertheless, a few individuals in Northeast China and many individuals in North China have hybrid ancestry according to Bayesian assignment. One SSR locus and five AFLPs are significant outliers against neutral expectations in the interspecific FST simulation analysis, suggesting a role for divergent selection in differentiating species. MAIN CONCLUSIONS/SIGNIFICANCE: All results based on SSRS and AFLPS reached the same conclusion: Q. liaotungensis and Q. mongolica maintain distinct gene pools in most areas of sympatry. They should therefore be considered as discrete taxonomic units. Yet, the degree of introgression varies between the two species in different contact zones, which might be caused by different population history or by local environmental factors.     

Host-related genetic differentiation in the anther smut fungus Microbotryum violaceum in sympatric, parapatric and allopatric populations of two host species Silene latifolia and S. dioica

We investigated genetic diversity in West European populations of the fungal pathogen Microbotryum violaceum in sympatric, parapatric and allopatric populations of the host species Silene latifolia and S. dioica, using four polymorphic microsatellite loci. In allopatric host populations, the fungus was highly differentiated by host species, exhibiting high values of F(ST) and R(ST), and revealed clear and distinct host races. In sympatric and parapatric populations we found significant population differentiation as well, except for one sympatric population in which the two host species grew truly intermingled. The mean number of alleles per locus for isolates from each of the host species was significantly higher in sympatric/parapatric than in allopatric populations. This suggests that either gene flow between host races in sympatry, or in case of less neutral loci, selection in a more heterogeneous host environment can increase the level of genetic variation in each of the demes. The observed pattern of host-related genetic differentiation among these geographically spread populations suggest a long-term divergence between these host races. In sympatric host populations, both host races presumably come in secondary contact, and host-specific alleles are exchanged depending on the amount of fungal gene flow.     

Geographic variability in mitochondrial introgression among hybridizing populations of Golden-winged (<Emphasis Type="Italic">Vermivora chrysoptera</Emphasis>) and Blue-winged (<Emphasis Type="Italic">V.opinus</Emphasis>) Warblers

The rapidly declining Golden-winged Warbler (Vermivora chrysoptera) is of conservation concern owing in part to hybridization with the closely related Blue-winged Warbler (V. pinus). These species hybridize extensively in eastern North America and over the past century the Blue-winged Warbler has displaced the Golden-winged Warbler from substantial regions of its historic breeding range. A previous study suggested that these genetic interactions result in rapid and asymmetric introgression of Blue-winged Warbler mitochondrial DNA (mtDNA) into Golden-winged phenotype populations within the zones of contact, but more recent and extensive surveys have documented a more complex pattern of genetic interchange between these taxa. We surveyed mtDNA/phenotype associations in 104 individuals of known phenotype drawn from two locations with different histories of contact and found substantial variation between sites in the extent of introgression. Where both species have co-existed for more than a century, we found evidence of bi-directional introgression and the long-term persistence of Golden-winged mtDNA haplotypes. At the leading edge of the northward expansion of Blue-winged Warblers, we found predominantly Golden-winged Warbler mtDNA haplotypes in both Golden-winged and hybrid-phenotype individuals. Across both sites, genetic swamping does not appear to be occurring via the early immigration of Blue-winged Warbler females into populations dominated by Golden-winged Warbler phenotypes. Instead, the differing patterns of mitochondrial introgression may be driven by the relative local population sizes of the parental species coupled with subtle between-species differences in mate choice and habitat preferences.