Isolation and characterization of eight dinucleotide microsatellite loci from two closely related clupeid species (Alosa alosa and A. fallax)

Eight dinucleotide microsatellite loci were developed through an enrichment protocol for allis shad (Alosa alosa) and twaite shad (A. fallax). Cross‐species amplification was successful for all loci isolated. The number of alleles per locus ranged from three to nine for A. alosa and from two to seven for A. fallax, while the observed heterozygosity ranged from 0.267 to 0.926 and from 0.240 to 0.727, respectively. These markers will constitute useful tools for studies of population structure and gene flow between two closely related hybridizing species.     

Genetic monitoring of two decades of hybridization between allis shad (Alosa alosa) and twaite shad (Alosa fallax)

Habitat alteration has been implicated in driving hybridization between the sympatric migratory shads Alosa alosa and Alosa fallax. Morphological and molecular evidence is consistent with hybridization across the overlapping range of these species, but the temporal extent of hybrid occurrence and genetic consequences for populations have not been explored. Using eight nuclear microsatellite loci and samples collected between 1989 and 2008 in the Solway Firth (UK), we genetically identified hybrids, studied temporal changes in their frequency, and explored changes in allele frequencies of parental populations. These molecular data confirmed the hybrid status of individuals identified using morphology (number of rakers on the outer gill arch), and enabled separation of hybrids from purebred individuals. Mitochondrial cytochrome-b sequencing revealed the presence of two haplogroups, each predominantly occurring in one species. Heterospecific haplotypes were found in 22.3 and 12.8% of A. alosa and A. fallax individuals, respectively, consistent with backcrossing and suggesting that hybrids are fertile. On average, microsatellite-identified hybrids comprised 12.7% of all samples, but when individuals with cytonuclear discordance were also considered introgressed on average 25.4% of individuals were of hybrid ancestry. Overall, allelic richness remained largely unchanged within species, but there were declines in the inbreeding coefficient (F _IS) of both species and episodes of significant temporal allelic frequency change. Hybrids sampled between 2004 and 2008 showed no evidence of lower fecundity relative to purebred individuals. Together, results suggest that hybridization between shad species in northern Europe is prevalent, and has been ongoing over at least two decades. The challenge is now to understand the extent to which observed patterns are linked to immigration from other populations, and the mechanisms that have prevented species collapse despite apparent hybrid fertility and longstanding introgression of neutral markers.     

From hybridization to introgression between two closely related sympatric ant species

Interspecific hybridization is becoming more frequent worldwide due to increasing global changes and translocations of organisms. For individual organisms, the most significant negative consequences are sterility or inviability of hybrid offspring. However, hybridization sometimes leads to fertile offspring, promoting introgression from one species into another. In such situations, hybridization can play a key role in evolution and speciation. Combining hypervariable DNA (microsatellites) and mitochondrial DNA markers with the use of several modeling methods allow an efficient detection of hybridization processes. The present study therefore investigates hybridization between two ant species, Tetramorium immigrans and T. caespitum, using multiple methods, and systematically comparing results with simulated data to ensure accurate identification of hybrids. Introgression was revealed both by backcross detection based on 14 nuclear microsatellite loci and by mitochondrial‐nuclear discordance based on comparison with mitochondrial DNA (cytochrome c oxidase subunit I). Results were spatially consistent, with hybrids located at latitudes where parental species are sympatric. The causes and consequences of hybridization and introgression between T. caespitum and T. immigrans remain to be further investigated, especially because T. immigrans could be an invasive species in France.     

Asymmetric hybridization between two species of sunfishes (Lepomis: Centrarchidae)

Many of the sunfishes (Centrarchidae) hybridize in natural systems, yet little is known about the interactions among hybridizing individuals in nature. We used allozyme electrophoresis to identify interspecific hybrids between bluegill (Lepomis macrochirus) and pumpkinseed (L. gibbosus) in Lake Opinicon, Ontario, and restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) to identify the maternal parentage of those individuals. All 44 individuals collected and identified electrophoreti-cally as F₁ hybrids had a pumpkinseed mtDNA haplotype, indicating that hybridization between these species in Lake Opinicon is asymmetrical; F₁ hybrids result only from matings between female pumpkinseed and male bluegill.     

Ecology of Allis Shad Alosa alosa and Twaite Shad Alosa fallax in the Solway Firth, Scotland

The basis of this study were 132 adult Allis Shad and 150 Twaite Shad collected as bycatches from salmon stake nets in Scotland on the north side of the Solway Firth. Most (60%) of the Allis Shad were immature fish 2–3 years of age (mean length 305 mm). Mature males were younger (3–5 years) and smaller (mean length 421 mm) than females (4–6 years and 481 mm mean length). The largest Allis Shad was a female of 515 mm and 2183 g. In contrast, almost all the Twaite Shad were mature, the males younger (3–5 years) and smaller (mean length 341 mm) than the females (4–6 years and 364 mm mean length). The largest Twaite Shad was a female of 400 mm and 1213 g. The food of Allis Shad consisted mainly of small zooplankton with some fish and larger Crustacea. Fine vegetable fragments were common in the stomachs and attributed to filter feeding. The food of Twaite Shad was mainly small fish with some Crustacea. Mature Allis Shad of both sexes with large gonads (maximum female GSI: 20.63) were found throughout the spring and summer but no definite evidence of local spawning was obtained. Mature Twaite Shad of both sexes with large gonads (maximum female GSI: 23.32) were found until early July, thereafter most of the fish were spent. It appears that Twaite Shad spawn locally in June. Some hybrids between the two species were found.     

Primary and secondary oocyte growth dynamics in anadromous semelparous Allis shad Alosa alosa

We analysed the ovarian dynamics of the anadromous semelparous allis shad Alosa alosa for which our working hypothesis was that mature pre-spawning females would have very low or even exhausted primary growth (PG) oocyte reserves; semelparity has been linked with the depletion of the pool of PG oocytes. To test this hypothesis, the PG oocytes were enumerated, their recruitment pattern to the secondary growth (SG) phase was analysed and their potential replenishment from the pool of oogonia was examined in females caught very close to the Mondego River mouth, in central Portugal and along the river. The development of the SG oocytes was also analysed, the fecundity (batch, total and annual) values were estimated and the intensity of atresia was quantified. Ovarian samples and histological sections were investigated in parallel. A dynamic recruitment pattern of PG oocytes to the SG phase was revealed, where all PG oocytes were recruited and were not replenished by oogonia. Annual fecundity was subject to down-regulation due to atresia prior to spawning and its size was multiple times higher than the size of batch fecundity. Lack of population synchronicity in ovarian development and spawning migration was also observed. This multifaceted analysis of the ovarian dynamics of this species will contribute to management efforts for this critically endangered and economically important fish throughout its geographical distribution. The results reported in this study will also assist in unravelling the complexity of the early processes of oogenesis in fish.     

Rare and asymmetrical hybridization of the endemic Barbus carpathicus with its widespread congener Barbus barbus

In endemic species that co-occur with widespread congeners, hybridization can lead to an influx of novel and beneficial genetic variation, but high rates of introgression may cause genetic swamping of the endemic species and have detrimental effects on its survival potential. This study examines hybridization between sympatric populations of the Carpathian barbel Barbus carpathicus , a recently discovered cryptic species with a restricted range, and the widespread common barbel Barbus barbus . Based on six diagnostic allozyme loci, a microsatellite locus and mtDNA, hybrids were found to be present at multiple localities within the Vistula River drainage (Baltic Sea) as well as in the Tisza River system of the Danube River drainage (Black Sea). However, the numbers of hybrids were very low; four individuals of 230 fish sampled from the Vistula drainage. Bayesian assessment of their nuclear genotypes suggested that two hybrids in the Vistula drainage and nine in the Tisza system were F₁ generation, and one in the Vistula drainage and one in the Tisza system were backcrosses (BC) to B. barbus , while no F₂ or BC to B. carpathicus were detected. No hybrid carried B. carpathicus mtDNA and cytonuclear linkage disequilibria showed significant positive associations between hybrid genotypes and B. barbus mtDNA, suggesting unidirectionality in the interspecific mating with a disproportionate contribution of B. barbus mothers. Despite geographically broad occurrence of hybrids, these data provide evidence of strong constraints on hybridization in the native breeding habitats and the lack of introgression towards B. carpathicus .     

Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed-species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) x Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.     

Morphological variation in hybrids between Salmo marmoratus, and alien Salmo species in the Volarja stream, Soca River basin, Slovenia

There were significant correlations between colour pattern, LDH -5*, genotype and certain meristic characters in 59 hybrid trout Salmo , sp. from the Volarja stream, Soca River basin, Slovenia. It is concluded that panmixia between native Salmo marmoratus , and introduced S. trutta , of Atlantic, Danubian and Mediterranean origin had not been reached in this zone, despite the long period of introgression. The result is in agreement with other studies dealing with introgression in Salmo , and for management purposes certain morphological characters, especially colour pattern, can be a valuable tool in restoring the marble trout population in the Soca River.     

Genetic polymorphism of adenosine deaminase (ADA; E.C. 3.5.4.4.) in allis shad, Alosa alosa and twaite shad, Alosa fallax

A genetic polymorphism of adenosine deaminase (ADA), is described for the first time in European shad species, Alosa alosa and Alosa fallax . Significant heterogeneity in gene frequencies was found, both between species and within species.     

Ecology and genetics of interspecific hybridization in the swallowtails, Papilio hospiton Gene and P. machaon L., in Corsica (Lepidoptera: Papiliomdae)

The Corsican swallowtail butterfly, Papilio hospiton , is endemic to Corsica and Sardinia (France and Italy) and included in the list of endangered species by the Washington Convention, It is spread all over Corsica in scattered populations linked to diverse habitats. A study by enzyme electrophoresis showed that the genetic diversity of the species is of the same order of magnitude as that of Papilio machaon from continental France and Corsica. The differentiation between populations is rather low, which is consistent with the high vagility of the adults. Natural hybridization between P. hospiton and P. machaon is frequent, and laboratory crosses show that the hybrids are not sterile. However, developmental perturbations impair the viability of further hybrid progenies. Although limited introgression between the two species is likely to take place, enzyme electrophoresis and PCR-RFLP analysis of mitochondrial DNA show that their gene pools remain distincfTtienetic assimilation by P. machaon therefore does not seem to be a threat for P. hospiton.     

Rapid genetic assimilation of native wall lizard populations (Podarcis muralis) through extensive hybridization with introduced lineages

The Common Wall Lizard (Podarcis muralis) has established more than 150 non-native populations in Central Europe, stemming from eight geographically distinct evolutionary lineages. While the majority of these introduced populations are found outside the native range, some of these populations also exist at the northern range margin in southwestern Germany. To (i) infer the level of hybridization in contact zones of alien and native lineages; and (ii) compare the genetic diversity among purebred introduced, native and hybrid populations, we used a combination of maternally inherited markers (mtDNA: cytb) and Mendelian markers (microsatellites). Our results suggest a rapid genetic assimilation of native populations by strong introgression from introduced lineages. Discordant patterns of mtDNA and nDNA variation within hybrid populations may be explained by directed mate choice of females towards males of alien lineages. In contrast to previous studies, we found a nonlinear relationship between genetic diversity and admixture level. The genetic diversity of hybrid populations was substantially higher than in introduced and native populations belonging to a single lineage, but rapidly reaching a plateau of high genetic diversity at an admixture level of two. However, even introduced populations with low founder sizes and from one source population retained moderate levels of genetic diversity and no evidence for a genetic bottleneck was found. The extent of introgression and the dominance of alien haplotypes in mixed populations indicate that introductions of non-native lineages represent a serious threat to the genetic integrity of native populations due to the rapid creation of hybrid swarms.     

Introduction. Extent, processes and evolutionary impact of interspecific hybridization in animals

Since the time of Charles Darwin, studies of interspecific hybridization have been a major focus for evolutionary biologists. Although this phenomenon has often been viewed as problematic in the fields of ecology, taxonomy and systematics, it has become a primary source of data for studies on speciation and adaptation. Effects from genetic/evolutionary processes, such as recombination and natural selection, usually develop over extended periods of time; however, they are accelerated in cases of hybridization. Interspecific hybrids exhibit novel genomes that are exposed to natural selection, thus providing a key to unravel the ultimate causes of adaptation and speciation. Here we provide firstly a historic perspective of hybridization research, secondly a novel attempt to assess the extent of hybridization among animals and thirdly an overview of the reviews and case studies presented in this theme issue.     

Phylogeographic analysis of mitochondrial gene flow and introgression in the salamander, Plethodon shermani

Plethodon shermani comprises a series of geographically disjunct populations occupying high-elevation mountain isolates. These populations hybridize at their borders with salamanders of the Plethodon glutinosus species complex, and past range expansions inferred from Pleistocene climatic cycles may have increased the possible genetic interactions between P. shermani and species of the P. glutinosus complex. Because mitochondrial DNA haplotypes often show introgression across species borders, we survey mtDNA variation for evidence of past and ongoing genetic interactions between P. shermani, its close relative Plethodon cheoah, and species of the P. glutinosus complex. Ongoing hybridization with the P. glutinosus-complex species Plethodon teyahalee is accompanied by extensive mitochondrial introgression in some Unicoi populations of P. shermani, but it has little genetic impact on P. shermani populations outside hybrid zones at three other isolates (Tusquitee, Wayah Bald, Standing Indian). Some Unicoi populations of P. shermani exhibit mtDNA evidence of past hybridization with diverse lineages from P. aureolus and P. glutinosus. The Tusquitee isolate of P. shermani is also characterized by mtDNA haplotypes most closely related to Plethodon aureolus and P. glutinosus, presumably introduced by past genetic contact with these species or with introgressed populations of Unicoi P. shermani. The mtDNA variation in sampled populations of the Wayah Bald and Standing Indian isolates of P. shermani appears largely unaffected by ongoing hybridization. Principal components analyses of allozymic data indicate that P. shermani isolates collectively form a genetically homogeneous unit clearly demarcated from species with which they have had current or past genetic interactions. Rapid mtDNA introgression associated with transient contacts between P. shermani and other species permits a fine-level resolution of evolutionary lineages not evident from allozymic data.     

Interspecific hybridization increases MHC class II diversity in two sister species of newts

Our understanding of the evolutionary mechanisms generating variation within the highly polymorphic major histocompatibility complex (MHC) genes remains incomplete. Assessing MHC variation across multiple populations, of recent and ancient divergence, may facilitate understanding of geographical and temporal aspects of variation. Here, we applied 454 sequencing to perform a large-scale, comprehensive analysis of MHC class II in the closely related, hybridizing newts, Lissotriton vulgaris (Lv) and Lissotriton montandoni (Lm). Our study revealed an extensive (299 alleles) geographically structured polymorphism. Populations at the southern margin of the Lv distribution, inhabited by old and distinct lineages (southern Lv), exhibited moderate MHC variation and strong population structure, indicating little gene flow or extensive local adaptation. Lissotriton vulgaris in central Europe and the northern Balkans (northern Lv) and almost all Lm populations had a high MHC variation. A much higher proportion of MHC alleles was shared between Lm and northern Lv than between Lm and southern Lv. Strikingly, the average pairwise F(ST) between northern Lv and Lm was significantly lower than between northern and southern Lv for MHC, but not for microsatellites. Thus, high MHC variation in Lm and northern Lv may result from gene flow between species. We hypothesize that the interspecific exchange of MHC genes may be facilitated by frequency-dependent selection. A marginally significant correlation between the MHC and microsatellite allelic richness indicates that demographic factors may have contributed to the present-day pattern of MHC variation, but unequivocal signatures of adaptive evolution in MHC class II sequences emphasize the role of selection on a longer timescale.     

Genetic differentiation,polyploidization and hybridization in northern EuropeanDactylorhiza (Orchidaceae): Evidence from allozyme markers

Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.     

Fluctuating asymmetry in the honey bee,Apis mellifera: effects of ploidy and hybridization

We present a new measure of morphological asymmetry that avoids most of the statistical problems inherent in character-by-character analysis of size or shape. The method is an application of Procrustes analysis, which computes best-fitting super-positions of configurations of landmarks to the left and right sides of a single specimen. The Procrustes method combines subtle deviations in all aspects of the landmark configuration into one net asymmetry score. Directional asymmetry is separated from fluctuating asymmetry in a simple partition of a net sum-of-squares, and geometrical details of either component can be inspected by traditional methods of multivariate statistical analysis of landmarks. We demonstrate this method in a comparison of wing venation asymmetry in male (haploid) and female (diploid) honey bees (Apis mellifera). In addition we investigate the effects of ploidy and inter-subspecies hybridization on asymmetry and wing venation abnormalities, using the subspecies A. m. mellifera, A. m. carnica, and the hybrid strain “Nigra”. Results suggest that while the haploid males showed a higher frequency of wing venation abnormalities and greater total asymmetry than the diploid females, most of the asymmetry difference between males and females was in the form of directional, not fluctuating, asymmetry. Hybrid females had a higher frequency of wing venation abnormalities than females of either subspecies, but there were no significant differences in the mean level of asymmetry among females of A. m. mellifera, A. m. carnica and hybrid Nigra. Hybrid males had higher absolute frequency of wing venation abnormalities and asymmetry than males of either subspecies. However the mean frequency of venation abnormalities did not differ significantly between Nigra and A. m. carnica males, and mean asymmetries were not significantly different between Nigra and A. m. mellifera males. We discuss the relationship which is assumed to exist between developmental stability and fluctuating asymmetry in light of our result.     

Differential selection to avoid hybridization in two toad species

Abstract.— The fitness consequences of hybridization critically affect the speciation process. When hybridization is costly, selection favors the evolution of prezygotic isolating mechanisms (e.g., mating behaviors) that reduce heter-ospecific matings and, consequently, enhance reproductive isolation between species (a process termed reinforcement). If, however, selection to avoid hybridization differs between species, reinforcement may be impeded. Here, we examined both the frequency and fitness effects of hybridization between plains spadefoot toads ( Spea bombifrons ) and New Mexico spadefoot toads ( S. multiplicata ). Hybridization was most frequent in smaller breeding ponds that tend to be ephemeral, and heterospecific pairs consisted almost entirely of S. bombifrons females and S. multiplicata males. Moreover, in controlled experimental crosses, hybrid offspring from crosses in which S. multiplicata was maternal had significantly lower survival and longer development time than pure S. multiplicata offspring. By contrast, hybrid offspring from crosses in which S. bombifrons was maternal outperformed pure S. bombifrons offspring by reaching metamorphosis faster. These data suggest that, although S. multiplicata females are under selection to avoid hybridization, selection might favor those S. bombifrons females that hybridize with S. multiplicata if their breeding pond is highly ephemeral. Generally, the strength of selection to avoid hybridization may differ for hybridizing species, possibly impeding reinforcement.     

Interspecific interactions influence contrasting spatial genetic structures in two closely related damselfly species

Spatial genetic structure (SGS) is largely determined by colonization history, landscape and ecological characteristics of the species. Therefore, sympatric and ecologically similar species are expected to exhibit similar SGSs, potentially enabling prediction of the SGS of one species from that of another. On the other hand, due to interspecific interactions, ecologically similar species could have different SGSs. We explored the SGSs of the closely related Calopteryx splendens and Calopteryx virgo within Finland and related the genetic patterns to characteristics of the sampling localities. We observed different SGSs for the two species. Genetic differentiation even within short distances in C. splendens suggests genetic drift as an important driver. However, we also observed indication of previous gene flow (revealed by a negative relationship between genetic differentiation and increasing potential connectivity of the landscape). Interestingly, genetic diversity of C. splendens was negatively related to density of C. virgo, suggesting that interspecific interactions influence the SGS of C. splendens. In contrast, genetic differentiation between C. virgo subpopulations was low and only exhibited relationships with latitude, pointing to high gene flow, colonization history and range margin effects as the drivers of SGS. The different SGSs of the two ecologically similar species caution indirect inferences of SGS based on ecologically similar surrogate species.     

Ambiguous species boundaries: Hybridization and morphological variation in two closely related Rubus species along altitudinal gradients

Although hybridization frequently occurs among plant species, hybrid zones of divergent lineages formed at species boundaries are less common and may not be apparent in later generations of hybrids with more parental‐like phenotypes, as a consequence of backcrossing. To determine the effects of dispersal and selection on species boundaries, we compared clines in leaf traits and molecular hybrid index along two hybrid zones on Yakushima Island, Japan, in which a temperate (Rubus palmatus) and subtropical (Rubus grayanus) species of wild raspberry are found. Leaf sinus depth in the two hybrid zones had narrower clines at 600 m a.s.l. than the molecular hybrid index and common garden tests confirmed that some leaf traits, including leaf sinus depth that is a major trait used in species identification, are genetically divergent between these closely related species. The sharp transition in leaf phenotypic traits compared to molecular markers indicated divergent selection pressure on the hybrid zone structure. We suggest that species boundaries based on neutral molecular data may differ from those based on observed morphological traits.