Large geographic range size reflects a patchwork of divergent lineages in the long‐toed salamander (Ambystoma macrodactylum)

For northern taxa, persistence in multiple vs. single Pleistocene refugia may have been an important determinant of contemporary range size, with larger ranges achieved by species that colonized the north from several glacial refugia. Under this hypothesis, widespread species are expected to demonstrate marked phylogeographic structure in previously glaciated regions. We use a genome‐wide survey to characterize genetic structure and evaluate this hypothesis in the most widely distributed salamander in the Pacific Northwest, the long‐toed salamander (Ambystoma macrodactylum). Patterns of variation based on 751 amplified fragment length polymorphism (AFLP) loci and mitochondrial sequence data were concordant and support the recognition of at least four distinct lineages of long‐toed salamander. The distributions of these lineages indicate that multiple refugia contributed to the species' large contemporary range. At the same time, with up to 133 AFLP bands differing between lineages and levels of sequence divergence ranging from 2.5 to 5.8%, these lineages would be considered separate species by some definitions. Such splitting would partition the large geographic range of the long‐toed salamander into several relatively restricted ranges. Our results thus also underscore the potential for estimates of geographic range size to vary considerably depending on the taxonomic treatment of cryptic lineages.     

Biogeographical patterns of genetic differentiation in dung beetles of the genus Trypocopris (Coleoptera, Geotrupidae) inferred from mtDNA and AFLP analyses

Aim To examine the phylogeography and population structure of three dung beetle species of the genus Trypocopris (Coleoptera, Geotrupidae). We wanted to test whether genetic differences and genealogies among populations were in accordance with morphologically described subspecies and we aimed to establish times of divergence among subspecies to depict the appropriate temporal framework of their phylogeographical differentiation. We also wished to investigate the historical demographic events and the relative influences of gene flow and drift on the distribution of genetic variability of the different populations. Location Europe (mostly Italy). Methods We collected adult males from dung pats from 15 Italian localities over the period 2000–2002. For sequence analysis, some dried specimens from Albania, Croatia, Slovakia and Spain were also used. We applied cytochrome oxidase I mitochondrial DNA sequencing and the amplified fragment length polymorphism (AFLP) technique to determine whether phylogeographical patterns within the three species support the proposed hypotheses of subspecies designations, and to detect further structure among populations that might mediate diversification. Results and main conclusions The results show a high concordance between the distribution of mtDNA variation and the main morphological groups recognized as subspecies, which thus may represent independent evolutionary units. The degree of mitochondrial divergence suggests that speciation events occurred during the Pliocene, while diversification of the main subspecific lineages took place in the Pleistocene, from c. 0.3 to 1.5 Ma. Mitochondrial and nuclear data also reveal that there is phylogeographical structuring among populations within each of the main groups and that both contemporary and historical processes determined this pattern of genetic structure. Geographical populations form monophyletic clades in both phylogenetic and network reconstructions. Despite the high levels of intrapopulational diversity, F_ST values indicate moderate but significant genetic differentiation among populations, and a Bayesian clustering analysis of the AFLP data clearly separates the geographical populations. Nucleotide and gene diversity estimates reveal interspecific differences in the degree of diversification among populations that may be related to the different ecological requirements of the three species.     

Genetic structure of a hybrid zone between two violets,Viola rossii Hemsl. and V. bissetii Maxim.: dominance of F1 individuals in a narrow contact range

The genetic composition of a hybrid zone can provide insight into the evolution of diversification in plants. We carried out morphological and amplified fragment length polymorphism analyses to investigate the genetic composition of a hybrid zone between two violets, Viola bissetii Hemsl. and Viola rossii Maxim. Our aim was to clarify the formation and maintenance of hybrids between these Viola species. We found that most hybrid individuals (V. bissetii × V. rossii) were of the F₁ generation, with a few of the F₂ generation. We found no backcrosses. The scarcity of post‐F₁ hybrids indicates that a species barrier is established between the parental species. The F₁‐dominated hybrid zone occupied only a narrow, intermediate ecotone between the parental habitats, suggesting that selection by environmental factors against hybrids may help to maintain the current conditions in this hybrid zone.     

Rapid genetic delineation of local provenance seed‐collection zones for effective rehabilitation of an urban bushland remnant

Abstract The rehabilitation of native plant communities in urban bushland remnants is an increasingly important activity requiring the collection of large amounts of seed. Best practice generally identifies that local seed are best, but how far does the local provenance extend? Using the DNA fingerprinting technique amplified fragment length polymorphism, we assessed genetic differentiation between potential seed source populations and the target population, Bold Park, a large and significant bushland remnant in Perth, Western Australia. For each of 15 species, analysis of molecular variance was used to partition genetic variation within and among populations. Genetic differentiation between Bold Park and potential seed source populations was assessed by non‐metric multidimensional scaling ordination, and statistically by Fisher’s exact tests. The partitioning of variation among populations (Φ_ST) varied from 0.66 for Santalum acuminatum to 0.04 for Mesomelaena pseudostygia. For eight of 15 species, Bold Park plants were completely or largely non‐overlapping with other populations in ordinations, suggesting genetic differentiation and a narrow provenance. Five species showed overlap between Bold Park and some other, but not all, populations sampled, with geographically closest populations generally undifferentiated. Only two species, Acanthocarpus preissii and Mesomeleana pseudostygia, showed little genetic differentiation between Bold Park and all other populations, suggesting a regional genetic provenance. These species can be classified into three broad provenance classes – narrow, local and regional – to help guide decisions about appropriate seed‐collection zones for the rehabilitation of urban bushland remnants.     

Nuisance growth of Juncus bulbosus: the roles of genetics and environmental drivers tested in a large‐scale survey

1. Invasive species can transform aquatic ecosystems, and the nuisance growth of the freshwater macrophyte Juncus bulbosus has become a problem in many lakes and rivers in northern Europe. It affects biodiversity strongly and conflicts with human uses, not least compromising the generation of hydroelectricity. The causes of the proliferation of these massive stands of J. bulbosus are not finally resolved, however. 2. In this study, a wide range of catchment, lake and sediment parameters (n = 34) were assessed for 139 lakes in Southern Norway, with the aim of explaining the presence or absence of J. bulbosus and to assess potential drivers behind its prolific growth. 3. Juncus bulbosus was more often present in lakes with lower pH and phosphate concentrations, and a higher element ratio of dissolved inorganic nitrogen (DIN) to total phosphorus (DIN : TotP). 4. Despite the many parameters measured across substantial environmental gradients, none explained nuisance growth. Genetic screening (amplified fragment length polymorphism fingerprinting) of plants from a subset of lakes and additional river sites also showed no genetic differences between the various growth forms. A macrophyte trophic index, however, suggested that the most problematic growth occurred in the most oligotrophic lakes. 5. The lack of consistent patterns may reflect either factors not assessed in our survey, or that the current extension of stands represents a gradual cumulative response over time, not characterised effectively in our snapshot survey. Nevertheless, we can now exclude some putative causes of nuisance growth, including in particular genetics and N‐deposition.     

Adaptive plasticity and epigenetic variation in response to warming in an Alpine plant

Environmentally induced phenotypic plasticity may be a critical component of response to changing environments. We examined local differentiation and adaptive phenotypic plasticity in response to elevated temperature in half‐sib lines collected across an elevation gradient for the alpine herb, Wahlenbergia ceracea. Using Amplified Fragment Length Polymorphism (AFLP), we found low but significant genetic differentiation between low‐ and high‐elevation seedlings, and seedlings originating from low elevations grew faster and showed stronger temperature responses (more plasticity) than those from medium and high elevations. Furthermore, plasticity was more often adaptive for plants of low‐elevation origin and maladaptive for plants of high elevation. With methylation sensitive‐AFLP (MS‐AFLP), we revealed an increase in epigenetic variation in response to temperature in low‐elevation seedlings. Although we did not find significant direct correlations between MS‐AFLP loci and phenotypes, our results demonstrate that adaptive plasticity in temperature response to warming varies over fine spatial scales and suggest the involvement of epigenetic mechanisms in this response.     

Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism (AFLP)

The utility of the new polymerase chain reaction (PCR)-based multilocus DNA fingerprinting technique amplified fragment length polymorphism (AFLP) for paternity analysis in natural plant populations was assessed. In a natural population of 25 plants of Persoonia mollis (Proteaceae), three AFLP primer pairs generated 147 dominant loci. Of these, 125 (85%) were polymorphic, with a mean recessive allele frequency of 0.735. The theoretical expected percentage of offspring for which all males except the true father can be excluded ( P _ET) was 99.9% for this population. The estimates of P _ET drop marginally to 99.6% and 97.6% for larger populations of 100 and 1000 individuals, respectively. A preliminary investigation confirmed the power of AFLP for paternity analysis by assigning paternity, or excluding all known potential sires, for 242 of 252 (96.0%) naturally pollinated seeds. Ambiguous paternity for the remaining 10 seeds was quickly resolved by utilizing two further AFLP primer pairs, ultimately generating over 200 polymorphic loci and resulting in the exclusion of all nonsires for all 252 (100%) seeds. This study highlights the utility of AFLP for paternity analysis because: (i) it generates sufficiently large numbers of highly reproducible polymorphic loci, that are (ii) quickly and accurately scored using an automated DNA sequencer and dedicated software, and (iii) unlike microsatellites, requires no sequence knowledge so it is more easily applied to new study species.     

Clarification of the genetic component of hybrids between Phyllodoce caerulea and Phyllodoce aleutica (Ericaceae) in Hokkaido,northern Japan

Natural hybridization provides great opportunities to understand the interaction of genetics and ecology in determining species boundaries. We examined the genetic relationships of Phyllodoce taxa and revealed that most hybrids were fertile F₁s and an extremely small number of backcross and no F₂ plants were established in natural conditions. Because this trend was irrespective of regions, we conclude that negative endogenous selection may act after the germination of F₁ seeds and prevent the establishment of later‐generation hybrids. Based on these results, we discuss the ecological and evolutionary significance of natural hybridization in Phyllodoce taxa.     

High genetic differentiation in the alpine plant Campanula alpina Jacq. (Campanulaceae): evidence for glacial survival in several Carpathian regions and long-term isolation between the Carpathians and the Alps

A survey of amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) variation was conducted to elucidate the phylogeography of Campanula alpina , a key species of silicicolous alpine grasslands in the Carpathians with a disjunct distribution in the Eastern European Alps. The Carpathians experienced a different glacial history from the Alps: local glaciers were present only in the highest massifs, while alpine habitats extended over larger areas related to their present distribution in this region. We asked: (i) whether in the Carpathians a high-mountain plant exhibits a complex phylogeographical structure or rather signatures of recent migrations, and (ii) whether the disjunct part of the species' distribution in the Alps resulted from a recent colonization from the Carpathians or from a restricted expansion from separate Eastern Alpine refugia. Our study revealed a clear phylogeographical pattern in AFLPs supported by congruent groups of distinct cpDNA haplotypes. Highest genetic differentiation was observed between the Alps and the Carpathians, indicating a long-term isolation between populations from these two mountain ranges. Further genetic division within the Carpathians suggests that current species' distribution is composed of several groups which have been isolated from each other for a long period. One genetic break separates Western from Southeastern Carpathian material, which is in line with a classical biogeographical boundary. A further, strongly supported genetic group was identified at the southwestern edge of the Carpathian arch. In the Eastern Alps, genetic traces of glacial survival in separate refugial areas in the calcareous northern part and the siliceous central part were found.     

Variability of the Ramularia collo‐cygni Population in Central Europe

The genetic structure of the fungal barley pathogen Ramularia collo‐cygni (Rcc) population in Central Europe involving the isolates from the Czech Republic, the Slovak Republic, Germany and Swiss was determined using amplified fragment length polymorphism (AFLP) analysis. One hundred and eighty‐four markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation of Rcc isolates. Among the 337 isolates collected, the overall gene diversity was moderate ( = 0.216). The level of multilocus genotypic diversity was higher within populations than among them. All individuals had unique multilocus genotypes. Genetic differentiation was significant among populations in localities, but at a moderate level (θ = 0.12; P < 0.001), suggesting that gene flow is occurring among populations. The isolates from all twelve clusters produced by Structure were found in all local populations, although at different frequencies. Therefore, the inferred clusters did not represent geographical populations. Although the null hypothesis of random mating in Rcc populations was rejected, the high level of genotypic diversity suggests that the Rcc population structure appears to be generated by a mixed reproductive system including both asexual and sexual reproduction, along with a rather high migration rate.     

Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus complex in north‐eastern Ukraine

Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north‐eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus–P. esculentus without triploids; II) P. ridibundus–P. esculentus without diploid hybrids; and III) P. ridibundus–P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids.     

Genetic variation of two species with different life‐history traits in the endangered renosterveld of South Africa – a comparative analysis of Eriocephalus africanus and Hemimeris racemosa

We tested the effects of life‐history traits on genetic variation and conducted a comparative analysis of two plant species with differing life‐history traits co‐occurring in the highly endangered renosterveld of South Africa. We selected eighteen renosterveld remnants with varying degrees of size and isolation where populations of the herbaceous, annual and insect‐pollinated Hemimeris racemosa and the shrubby perennial and both wind‐ and insect‐pollinated Eriocephalus africanus occurred. We postulated a lower genetic variation within populations and increased genetic variation between populations in the annual than in the perennial species. Genetic variation was lower within populations of H. racemosa than within E. africanus, as is typical for annual compared to perennial species. Variation within populations was, however, not correlated with fragment size or distance in either of the two species and genetic variation between populations of the two species was comparable (Φ_ST = 0.10, 0.09).     

Selection on outlier loci and their association with adaptive phenotypes in Littorina saxatilis contact zones

A fundamental issue in speciation research is to evaluate phenotypic variation and the genomics driving the evolution of reproductive isolation between sister taxa. Above all, hybrid zones are excellent study systems for researchers to examine the association of genetic differentiation, phenotypic variation and the strength of selection. We investigated two contact zones in the marine gastropod Littorina saxatilis and utilized landmark‐based geometric morphometric analysis together with amplified fragment length polymorphism (AFLP) markers to assess phenotypic and genomic divergence between ecotypes under divergent selection. From genetic markers, we calculated the cline width, linkage disequilibrium and the average effective selection on a locus. Additionally, we conducted an association analysis linking the outlier loci and phenotypic variation between ecotypes and show that a proportion of outlier loci are associated with key adaptive phenotypic traits.     

Population structure and cryptic speciation in bonnethead sharks Sphyrna tiburo in the south‐eastern U.S.A. and Caribbean

Population structure and lineage diversification within a small, non‐dispersive hammerhead shark species, the bonnethead shark Sphyrna tiburo, was assessed. Sphyrna tiburo is currently described as one continuously distributed species along the Atlantic continental margins of North, Central and South America, but recent genetic analysis of an insular population (Trinidad) suggests the possibility of cryptic speciation. To address this issue S. tiburo were sampled at six sites along c. 6200 km of continuous, continental coastline and from one island location (Grand Bahama) across a discontinuity in their distribution (the Straits of Florida), in order to test if they constitute a single lineage over this distribution. A total of 1030 bp of the mitochondrial control region (CR) was obtained for 239 S. tiburo, revealing 73 distinct haplotypes, high nucleotide diversity (0·01089) and a pair of highly divergent lineages estimated to have separated 3·61–5·62 million years ago. Mitochondrial cytochrome oxidase I and nuclear internal transcribed spacer loci show the same pattern. Divergence is similar within S. tiburo to that observed between established elasmobranch sister species, providing further evidence of cryptic speciation. A global AMOVA based on CR confirms that genetic diversity is primarily partitioned among populations (Φ_ST = 0·828, P < 0·001) because the divergent lineages are almost perfectly segregated between Belize and North America–The Bahamas. An AMOVA consisting solely of the North American and Bahamian samples is also significantly different from zero (Φ_ST = 0·088, P < 0·001) and pairwise F_ST is significantly different between all sites. These findings suggest that S. tiburo comprises a species complex and supports previous research indicating fine population structure, which has implications for fisheries management and biodiversity conservation.     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Founder effects and phenotypic variation in Adelges cooleyi, an insect pest introduced to the eastern United States

Introduced organisms experience founder effects including genetic bottlenecks that result in significant reductions in genetic variation. Genetic bottlenecks may constrain the evolution of phenotypic traits that facilitate success in novel habitats. We examined the effect of introduction into novel environments on genetic diversity of an insect pest, Adelges cooleyi, which was introduced into the eastern United States during the mid nineteenth century. We compared variation in mitochondrial and nuclear genomes in native and introduced samples to determine the effect of introduction on genetic variation experienced by this insect. We also measured an ecologically important phenotype, variation in host preference, in both native and introduced samples to compare variation in that trait with molecular genetic variation. To further investigate the relationship between genetic and phenotypic variation, we examined the degree to which mtDNA haplotypes provide information about host preference. Adelges cooleyi in eastern North America has significantly reduced genetic and phenotypic variation, but this low variation does not appear to have prevented persistence in a novel environment. Introduced insects appear to have retained host preference phenotypes similar to those of insects found where introductions likely originated.     

The role of local ecology during hybridization at the initial stages of ecological speciation in a marine snail

Hybrid zones of ecologically divergent populations are ideal systems to study the interaction between natural selection and gene flow during the initial stages of speciation. Here, we perform an amplified fragment length polymorphism (AFLP) genome scan in parallel hybrid zones between divergent ecotypes of the marine snail Littorina saxatilis, which is considered a model case for the study of ecological speciation. Ridged‐Banded (RB) and Smooth‐Unbanded (SU) ecotypes are adapted to different shore levels and microhabitats, although they present a sympatric distribution at the mid‐shore where they meet and mate (partially assortatively). We used shell morphology, outlier and nonoutlier AFLP loci from RB, SU and hybrid specimens captured in sympatry to determine the level of phenotypic and genetic introgression. We found different levels of introgression at parallel hybrid zones and nonoutlier loci showed more gene flow with greater phenotypic introgression. These results were independent from the phylogeography of the studied populations, but not from the local ecological conditions. Genetic variation at outlier loci was highly correlated with phenotypic variation. In addition, we used the relationship between genetic and phenotypic variation to estimate the heritability of morphological traits and to identify potential Quantitative Trait Loci to be confirmed in future crosses. These results suggest that ecology (exogenous selection) plays an important role in this hybrid zone. Thus, ecologically based divergent natural selection is responsible, simultaneously, for both ecotype divergence and hybridization. On the other hand, genetic introgression occurs only at neutral loci (nonoutliers). In the future, genome‐wide studies and controlled crosses would give more valuable information about this process of speciation in the face of gene flow.