The uncharacterized gene 1700093K21Rik and flanking regions are correlated with reproductive isolation in the house mouse,Mus musculus

Reproductive barriers exist between the house mouse subspecies, Mus musculus musculus and M. m. domesticus, members of the Mus musculus species complex, primarily as a result of hybrid male infertility, and a hybrid zone exists where their ranges intersect in Europe. Using single nucleotide polymorphisms (SNPs) diagnostic for the two taxa, the extent of introgression across the genome was previously compared in these hybrid populations. Sixty-nine of 1316 autosomal SNPs exhibited reduced introgression in two hybrid zone transects suggesting maladaptive interactions among certain loci. One of these markers is within a region on chromosome 11 that, in other studies, has been associated with hybrid male sterility of these subspecies. We assessed sequence variation in a 20 Mb region on chromosome 11 flanking this marker, and observed its inclusion within a roughly 150 kb stretch of DNA showing elevated sequence differentiation between the two subspecies. Four genes are associated with this genomic subregion, with two entirely encompassed. One of the two genes, the uncharacterized 1700093K21Rik gene, displays distinguishing features consistent with a potential role in reproductive isolation between these subspecies. Along with its expression specifically within spermatogenic cells, we present various sequence analyses that demonstrate a high rate of molecular evolution of this gene, as well as identify a subspecies amino acid variant resulting in a structural difference. Taken together, the data suggest a role for this gene in reproductive isolation.     

Genetic dissection of a key reproductive barrier between nascent species of house mice

Reproductive isolation between species is often caused by deleterious interactions among loci in hybrids. Finding the genes involved in these incompatibilities provides insight into the mechanisms of speciation. With recently diverged subspecies, house mice provide a powerful system for understanding the genetics of reproductive isolation early in the speciation process. Although previous studies have yielded important clues about the genetics of hybrid male sterility in house mice, they have been restricted to F1 sterility or incompatibilities involving the X chromosome. To provide a more complete characterization of this key reproductive barrier, we conducted an F2 intercross between wild-derived inbred strains from two subspecies of house mice, Mus musculus musculus and Mus musculus domesticus. We identified a suite of autosomal and X-linked QTL that underlie measures of hybrid male sterility, including testis weight, sperm density, and sperm morphology. In many cases, the autosomal loci were unique to a specific sterility trait and exhibited an effect only when homozygous, underscoring the importance of examining reproductive barriers beyond the F1 generation. We also found novel two-locus incompatibilities between the M. m. musculus X chromosome and M. m. domesticus autosomal alleles. Our results reveal a complex genetic architecture for hybrid male sterility and suggest a prominent role for reproductive barriers in advanced generations in maintaining subspecies integrity in house mice.     

The genomics of speciation: investigating the molecular correlates of X chromosome introgression across the hybrid zone between Mus domesticus and Mus musculus

Understanding the genetic details of reproductive isolation is a key goal in the study of speciation. Hybrid zones, geographical regions where two species meet and exchange genes, can provide insight into the genetic basis of reproductive isolation. This is especially true in species with mapped molecular markers because patterns of gene flow can be compared among different genomic regions. Even greater insight can be obtained in species with complete genome sequences because gene identity, gene number and other features of interest can be assessed for genomic regions with different patterns of introgression. Here, we review recent studies on the well-characterized hybrid zone between Mus domesticus and M. musculus , including a detailed survey of patterns of introgression for 13 markers on the X chromosome. We then compare levels of introgression for these 13 regions to a number of genomic attributes inferred from the complete sequence of the X chromosome, with two purposes. First, we identify candidate genes for reproductive isolation by finding genes that map to an X-linked region of reduced introgression and that are only expressed in the male germ line or that show high rates of protein evolution in comparison with rat. Second, we ask whether patterns of gene flow are correlated with recombination rate, gene density, base composition, CpG island density, mutation rate and the rate of protein evolution, as might be expected if many genes contribute to reproductive isolation. We identify seven candidate genes for reproductive isolation between M. domesticus and M. musculus , and our analyses reveal no general correlations between levels of introgression and other measured sequence characteristics. These results underline the utility of the house mouse as a model system for the study of speciation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 523–534.     

Higher differentiation among subspecies of the house mouse (Mus musculus) in genomic regions with low recombination

In the early stages of reproductive isolation, genomic regions of reduced recombination are expected to show greater levels of differentiation, either because gene flow between species is reduced in these regions or because the effects of selection at linked sites within species are enhanced in these regions. Here, we study the patterns of DNA sequence variation at 27 autosomal loci among populations of Mus musculus musculus, M. m. domesticus, and M. m. castaneus, three subspecies of house mice with collinear genomes. We found that some loci exhibit considerable shared variation among subspecies, while others exhibit fixed differences. We used an isolation-with-gene-flow model to estimate divergence times and effective population sizes (N(e) ) and to disentangle ancestral variation from gene flow. Estimates of divergence time indicate that all three subspecies diverged from one another within a very short period of time approximately 350,000 years ago. Overall, N(e) for each subspecies was associated with the degree of genetic differentiation: M. m. musculus had the smallest N(e) and the greatest proportion of monophyletic gene genealogies, while M. m. castaneus had the largest N(e) and the smallest proportion of monophyletic gene genealogies. M. m. domesticus and M. m. musculus were more differentiated from each other than either were from M. m. castaneus, consistent with greater reproductive isolation between M. m. domesticus and M. m. musculus. F(ST) was significantly greater at loci experiencing low recombination rates compared to loci experiencing high recombination rates in comparisons between M. m. castaneus and M. m. musculus or M. m. domesticus. These results provide evidence that genomic regions with less recombination show greater differentiation, even in the absence of chromosomal rearrangements.     

Genetics and evolution of hybrid male sterility in house mice

Comparative genetic mapping provides insights into the evolution of the reproductive barriers that separate closely related species. This approach has been used to document the accumulation of reproductive incompatibilities over time, but has only been applied to a few taxa. House mice offer a powerful system to reconstruct the evolution of reproductive isolation between multiple subspecies pairs. However, studies of the primary reproductive barrier in house mice-hybrid male sterility-have been restricted to a single subspecies pair: Mus musculus musculus and Mus musculus domesticus. To provide a more complete characterization of reproductive isolation in house mice, we conducted an F(2) intercross between wild-derived inbred strains from Mus musculus castaneus and M. m. domesticus. We identified autosomal and X-linked QTL associated with a range of hybrid male sterility phenotypes, including testis weight, sperm density, and sperm morphology. The pseudoautosomal region (PAR) was strongly associated with hybrid sterility phenotypes when heterozygous. We compared QTL found in this cross with QTL identified in a previous F(2) intercross between M. m. musculus and M. m. domesticus and found three shared autosomal QTL. Most QTL were not shared, demonstrating that the genetic basis of hybrid male sterility largely differs between these closely related subspecies pairs. These results lay the groundwork for identifying genes responsible for the early stages of speciation in house mice.     

Differential patterns of introgression across the X chromosome in a hybrid zone between two species of house mice

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.     

Searching the genomes of inbred mouse strains for incompatibilities that reproductively isolate their wild relatives

Identification of the genes that underlie reproductive isolation provides important insights into the process of speciation. According to the Dobzhansky-Muller model, these genes suffer disrupted interactions in hybrids due to independent divergence in separate populations. In hybrid populations, natural selection acts to remove the deleterious heterospecific combinations that cause these functional disruptions. When selection is strong, this process can maintain multilocus associations, primarily between conspecific alleles, providing a signature that can be used to locate incompatibilities. We applied this logic to populations of house mice that were formed by hybridization involving two species that show partial reproductive isolation, Mus domesticus and Mus musculus. Using molecular markers likely to be informative about species ancestry, we scanned the genomes of 1) classical inbred strains and 2) recombinant inbred lines for pairs of loci that showed extreme linkage disequilibria. By using the same set of markers, we identified a list of locus pairs that displayed similar patterns in both scans. These genomic regions may contain genes that contribute to reproductive isolation between M. domesticus and M. musculus. This hypothesis can now be tested using laboratory crosses and surveys of introgression in the wild.     

Signatures of reproductive isolation in patterns of single nucleotide diversity across inbred strains of mice

Reproductive isolation is often caused by the disruption of genic interactions that evolve in geographically separate populations. Identifying the genomic regions and genes involved in these interactions, known as "Dobzhansky-Muller incompatibilities," can be challenging but is facilitated by the wealth of genetic markers now available in model systems. In recent years, the complete genome sequence and thousands of single nucleotide polymorphisms (SNPs) from laboratory mice, which are largely genetic hybrids between Mus musculus and M. domesticus, have become available. Here, we use these resources to locate genomic regions that may underlie reproductive isolation between these two species. Using genotypes from 332 SNPs that differ between wild-derived strains of M. musculus and M. domesticus, we identified several physically unlinked SNP pairs that show exceptional gametic disequilibrium across the lab strains. Conspecific alleles were associated in a disproportionate number of these cases, consistent with the action of natural selection against hybrid gene combinations. As predicted by the Dobzhansky-Muller model, this bias was differentially attributable to locus pairs for which one hybrid genotype was missing. We assembled a list of potential Dobzhansky-Muller incompatibilities from locus pairs that showed extreme associations (only three gametic types) among conspecific alleles. Two SNPs in this list map near known hybrid sterility loci on chromosome 17 and the X chromosome, allowing us to nominate partners for disrupted interactions involving these genomic regions for the first time. Together, these results indicate that patterns produced by speciation between M. musculus and M. domesticus are visible in the genomes of lab strains of mice, underscoring the potential of these genetic model organisms for addressing general questions in evolutionary biology.     

THE VARIABLE GENOMIC ARCHITECTURE OF ISOLATION BETWEEN HYBRIDIZING SPECIES OF HOUSE MICE

Studies of the genetics of hybrid zones can provide insight into the genomic architecture of species boundaries. By examining patterns of introgression of multiple loci across a hybrid zone, it may be possible to identify regions of the genome that have experienced selection. Here, we present a comparison of introgression in two replicate transects through the house mouse hybrid zone through central Europe, using data from 41 single nucleotide markers. Using both genomic and geographic clines, we found many differences in patterns of introgression between the two transects, as well as some similarities. We found that many loci may have experienced the effects of selection at linked sites, including selection against hybrid genotypes, as well as positive selection in the form of genotypes introgressed into a foreign genetic background. We also found many positive associations of conspecific alleles among unlinked markers, which could be caused by epistatic interactions. Different patterns of introgression in the two transects highlight the challenge of using hybrid zones to identify genes underlying isolation and raise the possibility that the genetic basis of isolation between these species may be dependent on the local population genetic make-up or the local ecological setting.     

Bayesian estimation of genomic clines

We developed a Bayesian genomic cline model to study the genetic architecture of adaptive divergence and reproductive isolation between hybridizing lineages. This model quantifies locus‐specific patterns of introgression with two cline parameters that describe the probability of locus‐specific ancestry as a function of genome‐wide admixture. ‘Outlier’ loci with extreme patterns of introgression relative to most of the genome can be identified. These loci are potentially associated with adaptive divergence or reproductive isolation. We simulated genetic data for admixed populations that included neutral introgression, as well as loci that were subject to directional, epistatic or underdominant selection, and analysed these data using the Bayesian genomic cline model. Under many demographic conditions, underdominance or directional selection had detectable and predictable effects on cline parameters, and ‘outlier’ loci were greatly enriched for genetic regions affected by selection. We also analysed previously published genetic data from two transects through a hybrid zone between Mus domesticus and M. musculus. We found considerable variation in rates of introgression across the genome and particularly low rates of introgression for two X‐linked markers. There were similarities and differences in patterns of introgression between the two transects, which likely reflects a combination of stochastic variability because of genetic drift and geographic variation in the genetic architecture of reproductive isolation. By providing a robust framework to quantify and compare patterns of introgression among genetic regions and populations, the Bayesian genomic cline model will advance our understanding of the genetics of reproductive isolation and the speciation process.     

Genetic analysis of the hybridization zone between two subspecies Mus musculus domesticus and Mus musculus musculus in Bulgaria

The hybrid zone between the two subspecies of mice Mus musculus domesticus and Mus musculus musculus, which has been studied extensively in Denmark, crosses Europe to the Black Sea through the Alps and the Balkans. Two hundred and seventy-nine animals were captured in 22 localities along a transect across the Balkans. The animals were characterized for seven diagnostic nuclear loci by protein electrophoresis and by restriction pattern analysis of their mitochondrial DNA. The nuclear data show a sharp transition between the two subspecies, most of the variations in allele frequencies (from 0.9 to 0.1) occurring within a 36-km section of the transect. The introgression varies from one locus to the other and is more pronounced, in terms of distance, in M. m. musculus territory. Mitochondrial DNA introgression is important but occurs in one direction only, i.e. from M. m. musculus to M. m. domesticus, while a cytoplasmic transfer from M. m. domesticus to M. m. musculus has been reported. A previous study showed that no Y chromosome introgression occurs. The different behaviour of these three types of markers could be due to the interaction between selection against hybrid genomes and meiotic recombination. Objectively, it would appear that the genes that can introgress are neutral or nearly so and have been separated from deleterious genes they were linked to by recombination. This could explain the differential introgression between autosomal loci. The mitochondrial and Y chromosomes undergo no or very little recombination and each is transmitted as a whole. Their degree of introgression is thus indicative of the intensity of selection resulting from the amount of functional differentiation between the two taxa, which seems to be strong for the Y chromosome and weak for mitochondrial DNA. We propose that the asymmetry of nuclear introgression is due to different population structures. As M. m. musculus is relatively less structured, the rapid spreading of introgressed genes would be favoured. Such a scheme, however, can hardly account for the unidirectionality of the mitochondrial flow, which could be due to sex-dependent behaviour.     

Using the Mus musculus hybrid zone to assess covariation and genetic architecture of limb bone lengths

Two subspecies of the house mouse, Mus musculus domesticus and Mus musculus musculus, meet in a narrow contact zone across Europe. Mice in the hybrid zone are highly admixed, representing the full range of mixed ancestry from the two subspecies. Given the distinct morphologies of these subspecies, these natural hybrids can be used for genomewide association mapping at sufficiently high resolution to directly infer candidate genes. We focus here on limb bone length differences, which is of special interest for understanding the evolution of developmentally correlated traits. We used 172 first‐generation descendants of wild‐caught mice from the hybrid zone to measure the length of stylopod (humerus/femur), zeugopod (ulna/tibia) and autopod (metacarpal/metatarsal) elements in skeletal CT scans. We find phenotypic covariation between limb elements in the hybrids similar to patterns previously described in Mus musculus domesticus inbred strains, suggesting that the hybrid genotypes do not influence the covariation pattern in a major way. Mapping was performed using 143,592 SNPs and identified several genomic regions associated with length differences in each bone. Bone length was found to be highly polygenic. None of the candidate regions include the canonical genes known to control embryonic limb development. Instead, we are able to identify candidate genes with known roles in osteoblast differentiation and bone structure determination, as well as recently evolved genes of, as yet, unknown function.     

Studies of the species barrier between Drosophila subobscura and D. madeirensis V: the importance of sex-linked inversion in preserving species identity

The X chromosome is known to exert a disproportionately large effect on characters related to post-zygotic reproductive isolation. There is also growing evidence about the important role of the chromosomal regions with reduced recombination (such as inversions) in maintaining the identity of closely related species. Using molecular markers, we examine the effect of different regions of the X chromosome on determination of hybrid traits (viability, testes size, sperm motility and morphological anomalies) in hybrid males between Drosophila madeirensis and Drosophila subobscura. The preponderant effect of a region localized inside the A2 inversion in the X chromosome in all hybrid traits is identified. Other marked regions exert a weaker influence or only influence some of the hybrid trait. Our results confirm the crucial role of sex-linked chromosomal inversion in preserving the identity of species with incomplete reproductive isolation. The specific genomic make-up of parental lines used to perform crosses has a great effect on hybrid fitness.     

Asymmetry and polymorphism of hybrid male sterility during the early stages of speciation in house mice

House mice offer a powerful system for dissecting the genetic basis of phenotypes that isolate species in the early stages of speciation. We used a series of reciprocal crosses between wild-derived strains of Mus musculus and M. domesticus to examine F(1) hybrid male sterility, one of the primary phenotypes thought to isolate these species. We report four main results. First, we found significantly smaller testes and fewer sperm in hybrid male progeny of most crosses. Second, in some crosses hybrid male sterility was asymmetric and depended on the species origin of the X chromosome. These observations confirm and extend previous findings, underscoring the central role that the M. musculus X chromosome plays in reproductive isolation. Third, comparisons among reciprocal crosses revealed polymorphism at one or more hybrid incompatibilities within M. musculus. Fourth, the spermatogenic phenotype of this polymorphic interaction appears distinct from previously described hybrid incompatibilities between these species. These data build on previous studies of speciation in house mice and show that the genetic basis of hybrid male sterility is fairly complex, even at this early stage of divergence.     

Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone

In this paper, we present results of the first comprehensive study of the introgression of both autosomal and sex-chromosome markers across the central European portion of the hybrid zone between two house mouse subspecies, Mus musculus musculus and M. m. domesticus. More than 1800 individuals sampled from 105 sites were analyzed with a set of allozyme loci (hopefully representing neutral or nearly neutral markers) and X-linked loci (which are assumed to be under selection). The zone center is best modeled as a single straight line independent of fine-scale local geographic or climatic conditions, being maintained by a balance between dispersal and selection against hybrids. The width (w) of the multilocus autosomal cline was estimated as 9.6 km whereas the estimate for the compound X-chromosome cline was about 4.6 km only. As the former estimate is comparable to that of the Danish portion of the zone (assumed to be much younger than the central European one), zone width does not appear to be related to its age. The strength (B) of the central barrier was estimated as about 20 km; with dispersal (sigma) of about 1 km/gen(1/2), this means effective selection (s*) is approximately 0.06-0.09 for autosomal loci and about 0.25 for X-linked loci. The number of loci under selection was estimated as N= 56-99 for autosomes and about 380 for X-linked loci. Finally, we highlight some potential pitfalls in hybrid zone analyses and in comparisons of different transects. We suggest that conclusions about parts of the mouse genome involved in reproductive isolation and speciation should be drawn with caution and that analytical approaches always providing some estimates should not be used without due care regarding the support or confidence of such estimates, especially if conclusions are based on the difference between these estimates. Finally, we recommend that analysis in two-dimensional space, dense sampling, and rigorous treatment of data, including inspection of likelihood profiles, are essential for hybrid zone studies.     

Development of unique house mouse resources suitable for evolutionary studies of speciation

Two house mouse subspecies, Mus musculus domesticus and Mus musculus musculus, form a hybrid zone in Europe and represent a suitable model for inferring the genes contributing to isolation barriers between parental taxa. Despite long-term intensive studies of this hybrid zone, we still know relatively little about the causes and mechanisms maintaining the 2 taxa as separate subspecies; therefore, to gain insight into this process, we developed 8 wild-derived inbred house mouse strains. In order to produce strains as pure domesticus or musculus genomes as possible, the individuals used to establish the breeding colony for the 3 domesticus and 2 of the musculus strains were captured in the Czech Republic from wild populations at extreme western and eastern edges of the subspecific contact zone, respectively. The remaining 3 musculus strains were bred from mice captured about 250 km east of the hybrid zone. Genetic analysis based on 361 microsatellite loci showed that 82% of these markers are diagnostic for either the musculus or the domesticus strains. In order to demonstrate the potential utility of this genetic differentiation in such strains, phenotypic variation was scored for 2 strains from opposite edges of the hybrid zone and significant differences in morphology, reproductive performance, in vitro immune responses, mate choice based on urinary signals, and aggressiveness were found. In addition, the 3 strains derived from musculus populations far from the hybrid zone display significant differences in polymorphism in hybrid male sterility when crossed with the laboratory strains C57BL/6 or C57BL/10, which have a predominantly domesticus genome. Although further studies will be necessary to demonstrate intersubspecific differences, all analyses presented here indicate that these newly developed house mouse strains represent a powerful tool for elucidating the genetic basis of isolation barriers in hybrid zones and for studying speciation in general.     

Testing for selection on the androgen-binding protein in the Danish mouse hybrid zone

The three peripheral subspecies of the house mouse Mus musculus have fixed specific variants of the androgen-binding protein (ABP) that have been proposed to be part of a recognition mechanism that could participate in sexual isolation between the subspecies. We tested for selection on Abpa by characterizing the pattern of Abpa introgression across a transect of the hybrid zone between M. m. musculus and M. m. domesticus in Jutland. On the musculus side, the cline for Abpa resembled that of a nearly neutral allozyme more than that of strongly selected X and Y chromosome markers. However, the high central step which displaces the tail of introgression of Abpa to higher frequencies was best accounted for by linkage to a locus under strong selection against hybrids. Still, we cannot exclude that this pattern results from weak selection against Abpa in the tail of introgression, which would be compatible with an assortative choice mechanism. On the domesticus side there was little introgression close to the hybrid zone, presumably due to a geographical barrier to migration. However, substantial frequencies of musculus alleles occurred further away, suggesting mixed colonization patterns as well as flow across the hybrid zone.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 447–459.     

Reduced X-linked diversity in derived populations of house mice

Contrasting patterns of X-linked vs. autosomal diversity may be indicative of the mode of selection operating in natural populations. A number of observations have shown reduced X-linked (or Z-linked) diversity relative to autosomal diversity in various organisms, suggesting a large impact of genetic hitchhiking. However, the relative contribution of other forces such as population bottlenecks, variation in reproductive success of the two sexes, and differential introgression remains unclear. Here, we survey 13 loci, 6 X-linked and 7 autosomal, in natural populations of the house mouse (Mus musculus) subspecies complex. We studied seven populations of three different subspecies, the eastern house mouse M. musculus castaneus, the central house mouse M. m. musculus, and the western house mouse M. m. domesticus, including putatively ancestral and derived populations for each. All populations display lower diversity on the X chromosomes relative to autosomes, and this effect is most pronounced in derived populations. To assess the role of demography, we fit the demographic parameters that gave the highest likelihood of the data using coalescent simulations. We find that the reduction in X-linked diversity is too large to be explained by a simple demographic model in at least two of four derived populations. These observations are also not likely to be explained by differences in reproductive success between males and females. They are consistent with a greater impact of positive selection on the X chromosome, and this is supported by the observation of an elevated K(A) and elevated K(A)/K(S) ratios on the rodent X chromosome. A second contribution may be that the X chromosome less readily introgresses across subspecies boundaries.     

Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus-M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (~30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.