Isolation and characterization of polymorphic microsatellite markers in the black spiny tailed iguana (Ctenosaura pectinata) and their cross-utility in other Ctenosaura

We isolated and characterized 10 polymorphic microsatellite loci from the Mexican black iguana (Ctenosaura pectinata) and assessed levels of polymorphism in sampling sites located in the northern areas of the species' distribution range. Two to 19 alleles per locus and observed heterozygosity ranging from 0.15 to 0.96 were detected. These markers will be useful to describe population genetic structure, the extent of gene flow in contact zones, to study the mating system of the species and to address conservation genetics issues. Additionally, we evaluated the potential utility of these markers for studies of other species within the genus Ctenosaura (i.e. C. hemilopha, C. similis and C. oaxacana).     

Diversification in the northern neotropics: mitochondrial and nuclear DNA phylogeography of the iguana Ctenosaura pectinata and related species

While Quaternary climatic changes are considered by some to have been a major factor promoting speciation within the neotropics, others suggest that much of the neotropical species diversity originated before the Pleistocene. Using mitochondrial and nuclear sequence data, we evaluate the relative importance of Pleistocene and pre-Pleistocene events within the evolutionary history of the Mexican iguana Ctenosaura pectinata , and related species. Results support the existence of cryptic lineages with strong mitochondrial divergence (> 4%) among them. Some of these lineages form zones of secondary contact, with one of them hybridizing with C. hemilopha . Evolutionary network analyses reveal the oldest populations of C. pectinata to be those of the northern and southern Mexican coastal regions. Inland and mid-latitudinal coastal populations are younger in age as a consequence of a history of local extinction within these regions followed by re-colonization. Estimated divergence times suggest that C. pectinata originated during the Pliocene, whereas geographically distinct mitochondrial DNA lineages first started to diverge during the Pliocene, with subsequent divergence continuing through the Pleistocene. Our results highlight the influence of both Pliocene and Pleistocene events in shaping the geographical distribution of genetic variation within neotropical lowland organisms. Areas of high genetic diversity in southern Mexico were detected, this finding plus the high levels of genetic diversity within C. pectinata , have implications for the conservation of this threatened species.     

Effect of feed type and sex on digestibility and feed efficiency utilization in black spiny‐tailed iguana (Ctenosaura pectinata)

Digestibility, feed efficiency, and the effect of sex were evaluated in black iguanas (Ctenosaura pectinata) using two commercial pellets (rabbit and chicken). The experiment was performed in 80 iguanas in a completely randomized design with a factorial arrangement 2×2 over 105 days. No differences were detected by food type in weight gain (chicken vs. rabbit: 121 vs. 154 mg/d) and daily intake (chicken vs. rabbit: 524 vs. 551 mg/d), but differences were detected (P<0.05) in feed conversion (chicken vs. rabbit: 6.45 vs. 4.47). Rabbit pellets showed higher digestibility than chicken food (P<0.01) in dry matter (59.8 vs. 41.4%) and NDF (55.4 vs. 43.6%), respectively. Sex had no effect in any of the variable responses. Black iguanas can be raised since 6 months old in captivity with commercial food designed for rabbit or broiler. No special physiological adaptations occur in black iguanas correlated with change in feeding habits during ontogeny. Zoo Biol 30:349–354, 2011. © 2010 Wiley‐Liss, Inc.     

Nuclear markers,mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni)

The smooth and the Montandon's newts (Triturus vulgaris and T. montandoni) are genetically similar sister species with highly divergent male secondary sexual traits involved in complex courtship behaviour. Their parapatric ranges overlap at moderate elevations in the Carpathian Mountains where they hybridize readily. Here we present a detailed study of genetic and morphological variation in populations from the area of sympatry. Analysis of variation at seven nuclear markers, mtDNA and male sexual secondary traits was complemented with an ecological survey of breeding sites characteristics. Extensive hybridization was revealed with back-cross individuals similar to either parental species predominating among hybrids. The hybrid zone exhibited a mosaic pattern: the genetic composition of the populations was correlated only weakly with their geographical position. No association with habitat type was found. Departures from Hardy-Weinberg proportions, significant linkage disequilibria and bimodal distribution of genotypes suggest strongly that assortative mating is an important factor shaping the genetic composition of hybrid populations. The pattern of cytonuclear disequilibria did not indicate much asymmetry in interspecific matings. Changes in the frequency of nuclear markers were highly concordant, whereas mtDNA showed much wider bidirectional introgression with 14% excess of T. montandoni haplotype. We argue that the mosaic structure of the newt hybrid zone results mainly from stochastic processes related to extinction and recolonization. Microgeographical differences in mtDNA introgression are explained by historical range shifts. Since morphologically intermediate males were underrepresented when compared to hybrid males identified by genetic markers, sexual selection acting against the morphological intermediates is implied. We discuss the implications of these findings in the context of reinforcement of prezygotic isolation in newts.     

Molecular and morphological patterns of introgression between two large white-headed gull species in a zone of recent secondary contact

Incomplete reproductive isolation promotes gene flow between diverging taxa. However, any gene encoding for traits involved in the reproductive barriers will be less prone to introgression than neutral markers. Comparing introgression rates among loci is thus informative of the number and functions of loci involved in the reproductive barriers. This study aimed at identifying possible mechanisms of restriction to gene flow across a zone of recent secondary contact between Larus argentatus and Larus cachinnans by comparing introgression patterns for nine microsatellite loci, a fragment of mitochondrial DNA and a set of phenotypic traits. The low linkage disequilibrium between neutral nuclear markers indicated introgression without any barrier to gene flow. However, asymmetric introgression of mitochondrial DNA suggested that interspecific crosses may be more successful in one direction. The introgression rate for phenotypic traits was variable and low compared to neutral molecular markers. This was particularly evident in colouration of bare parts: individuals with intermediate colouration were scarcer in sympatry than expected if the genomes recombined freely. We hypothesized that one of these variables, the orbital ring colour, may play a role in mate choice, acting as an incomplete premating barrier through assortative mating. This study emphasizes that multilocus approaches are useful to discriminate among possible mechanisms responsible for the maintenance of hybrid zones.     

Evidence for historical introgression along a contact zone between two species of char (Pisces: Salmonidae) in northwestern North America

Phylogeographic analyses can yield valuable insights into the geographic and historical contexts of contact and hybridization between taxa. Two species of char (Salmonidae), Dolly Varden (Salvelinus malma) and bull trout (S. confluentus) have largely parapatric distributions in watersheds of northwestern North America. They are, however, sympatric in several localities and hybridization and some introgression occurs across a broad area of contact. We conducted a comparative phylogenetic analysis of Dolly Varden and bull trout to gain a historical perspective of hybridization between these species and to test for footprints of historical introgression. We resolved two major Dolly Varden mitochondrial DNA (mtDNA) clades (with 1.4-2.2% sequence divergence between haplotypes) that had different geographical distributions. Clade N is distributed across most of the range of Dolly Varden, from southern British Columbia through to the Kuril Islands in Asia. Clade S had a much more limited distribution, from Washington state, at the southern limit of the Dolly Varden range, to the middle of Vancouver Island. The distribution and inferred ages of the mtDNA clades suggested that Dolly Varden survived the Wisconsinan glaciation in a previously unsuspected refuge south of the ice sheet, and that Dolly Varden and bull trout were probably in continuous contact over most of the last 100,000 years. When bull trout were included in the phylogenetic analysis, however, the mtDNA of neither species was monophyletic: Clade S Dolly Varden clustered within the bull trout mtDNA clade. This pattern was discordant with two nuclear phylogenies produced (growth hormone 2 and rRNA internal transcribed sequence 1), in which Dolly Varden and bull trout were reciprocally monophyletic. This discordance between mtDNA- and nDNA-based phylogenies indicates that historical introgression of bull trout mtDNA into Dolly Varden occurred. Percent sequence divergence within these introgressed Dolly Varden (clade S) was 0.2-0.6%, implying that the introgression occurred prior to the most recent glaciation. Our analysis and other evidence of contact between divergent lineages in northwestern North America strongly suggests that the area may be the site of previously unsuspected suture zones of aquatic biotas.     

Estimating the temporal and spatial extent of gene flow among sympatric lizard populations (genus Sceloporus) in the southern Mexican highlands

Interspecific gene flow is pervasive throughout the tree of life. Although detecting gene flow between populations has been facilitated by new analytical approaches, determining the timing and geography of hybridization has remained difficult, particularly for historical gene flow. A geographically explicit phylogenetic approach is needed to determine the overlap of ancestral populations. In this study, we performed population genetic analyses, species delimitation, simulations and a recently developed approach of species tree diffusion to infer the phylogeographic history, timing and geographic extent of gene flow in lizards of the Sceloporus spinosus group. The two species in this group, S. spinosus and S. horridus, are distributed in eastern and western portions of Mexico, respectively, but populations of these species are sympatric in the southern Mexican highlands. We generated data consisting of three mitochondrial genes and eight nuclear loci for 148 and 68 individuals, respectively. We delimited six lineages in this group, but found strong evidence of mito‐nuclear discordance in sympatric populations of S. spinosus and S. horridus owing to mitochondrial introgression. We used coalescent simulations to differentiate ancestral gene flow from secondary contact, but found mixed support for these two models. Bayesian phylogeography indicated more than 60% range overlap between ancestral S. spinosus and S. horridus populations since the time of their divergence. Isolation–migration analyses, however, revealed near‐zero levels of gene flow between these ancestral populations. Interpreting results from both simulations and empirical data indicate that despite a long history of sympatry among these two species, gene flow in this group has only recently occurred.     

Limited hybridization along a large contact zone between two genetic lineages of the butterfly Erebia medusa (Satyrinae, Lepidoptera) in Central Europe

Genetic lineages evolving during glacial isolation frequently come into contact as the result of postglacial range expansions. Hybridization often occurs along these contact zones. In Europe, the high mountain systems of the Alps and Pyrenees are well known for their hybrid belts. This article studies the contact zone of the Woodland Ringlet Erebia medusa in a Hercynian mountain area in the Czech-German border region not exceeding 1500 m a.s.l. Hybrid populations between an eastern and a western genetic lineage were detected by amova -based tests, principal component analysis and neighbour joining analysis. Over most of the range of the Czech-German Border Mts, the ridges separate the western and the eastern genetic lineage from each other. However, two important hybrid areas were detected: (1) the watershed of the Ohře river in the north-west of that area, a major valley system passing through these mountains and (2) the high plateaux of the Šumava Mts in the south-east, an extended area of high elevation. The location of hybrid populations in geographical vicinity to non-hybrid populations and the generally low F _IS (2.1%) make reduced fitness of hybrid individuals little likely. The hybrid populations have intermediate genetic diversity between the genetically poor western and the genetically rich eastern lineage populations.     

Geographic variation in hybridization across a reinforcement contact zone of chorus frogs (Pseudacris)

Reinforcement contact zones, which are secondary contact zones where species are diverging in reproductive behaviors due to selection against hybridization, represent natural laboratories for studying speciation‐in‐action. Here, we examined replicate localities across the entire reinforcement contact zone between North American chorus frogs Pseudacris feriarum and P. nigrita to investigate geographic variation in hybridization frequencies and to assess whether reinforcement may have contributed to increased genetic divergence within species. Previous work indicated these species have undergone reproductive character displacement (RCD) in male acoustic signals and female preferences due to reinforcement. We also examined acoustic signal variation across the contact zone to assess whether signal characteristics reliably predict hybrid index and to elucidate whether the degree of RCD predicts hybridization rate. Using microsatellites, mitochondrial sequences, and acoustic signal information from >1,000 individuals across >50 localities and ten sympatric focal regions, we demonstrate: (1) hybridization occurs and (2) varies substantially across the geographic range of the contact zone, (3) hybridization is asymmetric and in the direction predicted from observed patterns of asymmetric RCD, (4) in one species, genetic distance is higher between conspecific localities where one or both have been reinforced than between nonreinforced localities, after controlling for geographic distance, (5) acoustic signal characters strongly predict hybrid index, and (6) the degree of RCD does not strongly predict admixture levels. By showing that hybridization occurs in all sympatric localities, this study provides the fifth and final line of evidence that reproductive character displacement is due to reinforcement in the chorus frog contact zone. Furthermore, this work suggests that the dual action of cascade reinforcement and partial geographic isolation is promoting genetic diversification within one of the reinforced species.     

Congruent signals of population history but radically different patterns of genetic diversity between mitochondrial and nuclear markers in a mountain lizard

Historical factors, current population size, population connectivity and selective processes at linked loci contribute to shaping contemporary patterns of neutral genetic diversity. It is now widely acknowledged that nuclear and mitochondrial markers react differently to current demography as well as to past history, so the use of both types of markers is often advocated to gain insight on both historical and contemporary processes. We used 12 microsatellite loci genotyped in 13 populations of a mountain lizard (Iberolacerta bonnali) to test whether the historical scenario favoured by a previous mitochondrial study was also supported by nuclear markers and thereby evaluated the consequences of postglacial range movements on nuclear diversity. Congruent signals of recent history were revealed by nuclear and mitochondrial markers using an Approximate Bayesian computation approach, but contemporary patterns of mtDNA and nuclear DNA diversity were radically different. Although dispersal in this species is probably highly restricted at all spatial scales, colonization abilities have been historically good, suggesting capability for reestablishment of locally extinct populations except in fully disconnected habitats.     

Spatial genetic features of eastern oysters (Crassostrea virginica Gmelin) in the Gulf of Mexico: northward movement of a secondary contact zone

The eastern oyster (Crassostrea virginica Gmelin) is an economically and ecologically valuable marine bivalve occurring in the Gulf of Mexico. This study builds upon previous research that identified two divergent populations of eastern oysters in the western Gulf of Mexico. Allelic and genotypic patterns from 11 microsatellite markers were used to assess genetic structure and migration between the previously described oyster populations in Texas. The main findings are as follows: (1) there are two distinct populations (F_ST = 0.392, P < 0.001) of oysters that overlap in the Corpus Christi/Aransas Bay estuarine complex in Texas, (2) the distribution of genotypes among individuals in the contact zone suggests limited hybridization between populations, (3) the variables of salinity, temperature, dissolved oxygen, turbidity and depth are not correlated with allele frequencies on reefs in the contact zone or when analyzed across Texas, and (4) there is little evidence of directional selection acting on the loci assayed here, although patterns at four markers suggested the influence of balancing selection based on outlier analyses. These results are consistent with long‐term historical isolation between populations, followed by secondary contact. Recent hydrological changes in the area of secondary contact may be promoting migration in areas that were previously inhospitable to eastern oysters, and observed differences in the timing of spawning may limit hybridization between populations. Comparison of these findings with the results of an earlier study of oysters in Texas suggests that the secondary contact zone has shifted approximately 27 km north, in as little as a 23‐year span.     

Allozyme and mitochondrial DNA analysis of a hybrid zone between white-tailed deer and mule deer (Odocoileus) in west texas

Thirty allozyme loci and 35 mitochondrial DNA (mtDNA) restriction sites were examined in 24 white-tailed deer and 46 mule deer from a hybrid zone in West Texas. A common mtDNA genotype is shared by all of the mule deer with 67% of the white-tailed deer. At the albumin locus, 13% of the white-tailed deer and 24% of the mule deer are heterozygous, sharing alleles that are otherwise species-specific in allopatric populations; 7% of the mule deer are homozygous for the allele that is characteristic of allopatric white-tailed deer. Gene flow appears to have been bidirectional, with greater genetic introgression into mule deer. The mtDNA data suggest that matings between white-tailed and mule deer have occurred in the past. Despite evidence of genetic introgression, analysis of multilocus genotypes indicates that none of the deer examined is an F₁ hybrid. Production of such hybrids appears to be generally uncommon in North American deer; management plans that assume otherwise should be reconsidered.This work was supported by an NIH Biomedical Research Support Grant, Texas Agricultural Experiment Station Program Development and Expanded Research Awards, the Caesar Kleberg Research Program in Wildlife Ecology, and a Natural Sciences and Engineering Research Council Operating Grant.     

Intraspecific genetic variation in the common midwife toad (Alytes obstetricans): subspecies assignment using mitochondrial and microsatellite markers

The common midwife toad (Alytes obstetricans), widely distributed in the northern half of the Iberian Peninsula and part of Western Europe, is currently subdivided into four subspecies: A. o. obstetricans, A. o. boscai, A. o. pertinax and A. o. almogavarii. However, the delimitation of these subspecies and their ranges are still under discussion because strong discordances have been found between morphological and molecular data, and especially among different genetic markers. Here, we screen a set of novel microsatellite loci and mtDNA sequences of A. obstetricans populations representative of all currently recognized subspecies to investigate the correspondence between genetic groupings inferred from clustering analysis of microsatellite genotypes and the described subspecies and test whether patterns of mtDNA variation are concordant with those genetic clusters. Our results confirm previous expectations of extremely high intraspecific diversity in A. obstetricans in Iberia. Analyses of microsatellite and mtDNA data were concordant in recovering five well‐defined groups, of which three correspond to previously defined subspecies, while the two additional clusters correspond to populations of subspecies A. o. boscai separated by the Douro River. Our results suggest the occurrence of two distinct genetic units within A. o. boscai that likely result from a long independent evolutionary history, thus deserving special attention from a conservation point of view.     

Differential patterns of hybridization and introgression between the swallowtails Papilio machaon and P. hospiton from Sardinia and Corsica islands (Lepidoptera,Papilionidae)

Proportions of hybridization and introgression between the swallowtails Papilio hospiton, endemic to Sardinia and Corsica, and the holarctic Papilio machaon, were characterized using nine fully diagnostic and two differentiated allozyme loci and a mitochondrial DNA marker. Very low frequencies of F1 hybrids were detected in both Sardinia (0-4%, average 1.4%) and Corsica (0-3%, average 0.5%), as well as of first generation backcrosses (B1). No F2 were observed, in agreement with the hybrid breakdown detected in laboratory crosses. In spite of this minimal current gene exchange, specimens carrying introgressed alleles were found in high proportions in P. machaon but in lower proportions in P. hospiton. Introgression apparently occurred through past hybridization and repeated backcrossing, as evidenced by hybrid index scores and Bayesian assignment tests. Levels of introgression were low (0-1%) at two sex-linked loci and mitochondrial DNA, limited (0.4-2%) at three autosomal loci coding for dimeric enzymes, and high (up to 43%) at four autosomal loci coding for monomeric enzymes. Accordingly, selective filters are acting against foreign alleles, with differential effectiveness depending on the loci involved. The low levels of introgression at sex-linked loci and mitochondrial DNA are in agreement with Haldane's rule and suggest that introgression in P. machaon proceeds mainly through males, owing to a lower fitness of hybrid females. Papilio machaon populations showed higher levels of introgression in Sardinia than in Corsica. The role of reinforcement in the present reproductive isolation between P. machaon and P. hospiton is examined, as well as the evolutionary effects of introgressive hybridization between the two species.     

Mosaic variation in allozyme and plastid DNA markers in the European ranges of Silene vulgaris and its partially sympatric relative S. uniflora (Caryophyllaceae)

The perennial herbs Silene vulgaris and S. uniflora are closely related, partially sympatric and interfertile, yet morphologically distinct. We used nuclear (allozyme) and plastid (polymerase chain reaction–restriction fragment length polymorphism) DNA markers to investigate whether these species have a shared history of postglacial colonization and hybridization in Europe, as inferred from large‐scale patterns of geographic variation. The majority of plastid haplotypes and allozyme alleles were widespread and patchily distributed within both species and there was no geographic structure in the distributions of shared allozymes or haplotypes. The mosaic variation is consistent with a scenario in which repeated episodes of interspecific hybridization pre‐dated the largely allopatric range expansion of the two species during the postglacial period. Our overall results are not consistent with a scenario of extensive hybridization and introgression during the postglacial range expansion of the species or within their current areas of sympatry, but we found some evidence for local, postglacial evolution and hybridization in the Baltic region. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166 , 127–148.     

Geographical patterns of morphological variation and environmental correlates in contact zones: a multi-scale approach using two Mediterranean vipers (Serpentes)

Adaptation to similar selective pressures can explain morphological convergence between closely related species in contact zones. Geostatistics and Geographical Information System were used to identify multi-scale patterns of morphological variability and test the hypothesis of morphological convergence due to local environmental pressures in the contact areas between Vipera aspis and Vipera latastei in the Iberian Peninsula. Nine morphological traits from 630 and 362 vipers at regional and local scale, respectively, were interpolated by Kriging to generate surfaces of morphological variation. Kriging is a geostatistical algorithm that allows investigating the spatial structure of data with statistical models. At both scales, a convergent north–south pattern in morphological variability was observed and the contact areas were identified as integration zones where intermediate vipers are found. Significant correlations were found between surfaces of univariate and multivariate traits, with precipitation and temperature seasonality. Thus, several morphological traits were apparently under local environmental selection. Nevertheless, the influence of biotic pressures and gene flow on morphological convergence of vipers in contact zones deserves further study.     

Ecological, morphological and allozymic differentiation between diploid and tetraploid knapweeds (Centaurea jacea) from a contact zone in the Belgian Ardennes

In the northeastern part of Belgium, the Centaurea jacea complex shows extensive morphological variation and is represented by a diploid (2 n = 22) and a tetraploid (2 n = 44) cytotype. Polysomic inheritance of allozyme markers in the tetraploids, suggesting autopolyploidy, is here demonstrated for the first time. In order to test whether the two cytotypes occupy distinct habitats and possess different gene pools, patterns of allozymic and morphological variation were investigated in relation to ploidy level and site characteristics in 26 populations from the Belgian Ardennes. The two cytotypes showed a parapatric distribution, the diploids occurring at higher elevations (mostly above 500 m) than the tetraploids (mostly below 500 m). Three mixed populations were found near the contact zone of the two cytotypes. Within the mixed populations no triploid plant and no evidence for gene flow between cytotypes were found, despite widely overlapping flowering periods. The two cytotypes can be distinguished on the basis of morphological traits and enzymatic gene pools. The congruence of morphological and allozymic variation with chromosome numbers suggests a secondary contact between the two cytotypes with limited gene flow between them. The origin and persistence of the parapatric distribution are discussed.