Amplified Fragment Length Polymorphism Variation Research Articles

Adaptive Divergence without Distinct Species Relationships Indicate Early Stage Ecological Speciation in Species of the Rhododendronpseudochrysanthum Complex Endemic to Taiwan.

The testing association of environmental variables with genetic and epigenetic variation could be crucial to deciphering the effects of environmental factors playing roles as selective drivers in ecological speciation. Although ecological speciation may occur in closely related species, species boundaries may not be established over a short evolutionary timescale. Here, we investigated the genetic and epigenetic variations using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP), respectively, and tested their associations with environmental variables in populations of four closely related species in the R. pseudochrysanthum complex. No distinctive species relationships were found using genetic clustering analyses, neighbor-joining tree, and neighbor-net tree based on the total AFLP variation, which is suggestive of the incomplete lineage sorting of ancestral variation. Nonetheless, strong isolation-by-environment and adaptive divergence were revealed, despite the significant isolation-by-distance. Annual mean temperature, elevation, normalized difference vegetation index, and annual total potential evapotranspiration were found to be the most important environmental variables explaining outlier genetic and epigenetic variations. Our results suggest that the four closely related species of the R. pseudochrysanthum complex share the polymorphism of their ancestor, but reproductive isolation due to ecological speciation can occur if local environmental divergence persists over time.

Ecological Factors Generally Not Altitude Related Played Main Roles in Driving Potential Adaptive Evolution at Elevational Range Margin Populations of Taiwan Incense Cedar (Calocedrus formosana).

Population diversification can be shaped by a combination of environmental factors as well as geographic isolation interacting with gene flow. We surveyed genetic variation of 243 samples from 12 populations of Calocedrus formosana using amplified fragment length polymorphism (AFLP) and scored a total of 437 AFLP fragments using 11 selective amplification primer pairs. The AFLP variation was used to assess the role of gene flow on the pattern of genetic diversity and to test environments in driving population adaptive evolution. This study found the relatively lower level of genetic diversity and the higher level of population differentiation in C. formosana compared with those estimated in previous studies of conifers including Cunninghamia konishii, Keteleeria davidiana var. formosana, and Taiwania cryptomerioides occurring in Taiwan. BAYESCAN detected 26 FST outlier loci that were found to be associated strongly with various environmental variables using multiple univariate logistic regression, latent factor mixed model, and Bayesian logistic regression. We found several environmentally dependent adaptive loci with high frequencies in low- or high-elevation populations, suggesting their involvement in local adaptation. Ecological factors, including relative humidity and sunshine hours, that are generally not altitude related could have been the most important selective drivers for population divergent evolution in C. formosana. The present study provides fundamental information in relation to adaptive evolution and can be useful for assisted migration program of C. formosana in the future conservation of this species.

Levels of Intra-specific AFLP Diversity in Tuber-Bearing Potato Species with Different Breeding Systems and Ploidy Levels.

DNA-based marker analysis of plant genebank material has become a useful tool in the evaluation of levels of genetic diversity and for the informed use and maintenance of germplasm. In this study, we quantify levels of amplified fragment length polymorphism (AFLP) in representative accessions of wild and cultivated potato species of differing geographic origin, ploidy, and breeding system. We generated 449 polymorphic AFLP fragments in 619 plants, representing multiple plants (16–23) from 17 accessions of 14 potato taxa as well as single plants sampled from available accessions (from 3 to 56) of the same 14 taxa. Intra-accession diversities were compared to those of a synthetic ‘taxon-wide’ population comprising a single individual from a variable number of available accessions of each sampled taxon. Results confirm the expected considerably lower levels of polymorphism within accessions of self-compatible as compared to self-incompatible taxa. We observed broadly similar levels of ‘taxon-wide’ polymorphism among self-compatible and self-incompatible species, with self-compatible taxa showing only slightly lower rates of polymorphism. The most diverse accessions were the two cultivated potato accessions examined, the least diverse being the Mexican allohexaploids Solanum demissum and S. iopetalum. Generally allopolyploid self-compatible accessions exhibited lower levels of diversity. Some purported self-incompatible accessions showed relatively low levels of marker diversity, similar to the more diverse self-compatible material surveyed. Our data indicate that for self-compatible species a single plant is highly representative of a genebank accession. The situation for self-incompatible taxa is less clear, and sampling strategies used will depend on the type of investigation. These results have important implications for those seeking novel trait variation (e.g., disease resistance) in gene banks as well as for the selection of individuals for genomics studies. We also show that AFLPs, despite having been largely replaced by other marker types, is highly suitable for the evaluation of within and between accession diversity in genebanks.

Analyses between Reproductive Behavior, Genetic Diversity and Pythium Responsiveness in Zingiber spp. Reveal an Adaptive Significance for Hemiclonality.

Mode of reproduction is generally considered to have long-range evolutionary implications on population survival. Because sexual reproduction produces genetically diverse genotypes, this mode of reproduction is predicted to positively influence the success potential of offspring in evolutionary arms race with parasites (Red queen) whereas, without segregation and recombination, the obligate asexual multiplication may push a species into extinction due to the steady accumulation of deleterious mutations (Muller’s ratchet). However, the extent of linearity between reproductive strategies, genetic diversity and population fitness, and the contributions of different breeding strategies to population fitness are yet to be understood clearly. Genus Zingiber belonging to the pan-tropic family Zingiberaceae represents a good system to study contributions of different breeding behavior on genetic diversity and population fitness, as this genus comprises species with contrasting breeding systems. In this study, we analyzed breeding behavior, amplified fragment length polymorphism diversity and response to the soft-rot pathogen Pythium aphanidermatum in 18 natural populations of three wild Zingiber spp.: Z. neesanum, Z. nimmonii, and Z. zerumbet, together with the obligately asexual cultivated congener, ginger (Z. officinale). Ginger showed an exceptionally narrow genetic base, and adding to this, all the tested cultivars were uniformly susceptible to soft-rot. Concordant with the postulates of Muller’s ratchet, the background selection may be continuously pushing ginger into the ancestral state, rendering it inefficient in host-pathogen coevolution. Z. neesanum and Z. nimmonii populations were sexual and genetically diverse; however, contrary to Red Queen expectations, the populations were highly susceptible to soft-rot. Z. zerumbet showed a hemiclonal breeding behavior. The populations inhabiting forest understory were large and continuous, sexual and genetically diverse, but were susceptible, whereas populations inhabiting the revenue land were fragmented and monoclonal, but were resistant. It may be possible that, when genetic recombination becomes at a premium due to the genetic constraints imparted by habitat fragmentation or pathogen pressure, Z. zerumbet

English

Warburgia ugandensis Sprague (Canellaceae) occurs in East and Central Africa and is an important multipurpose tree species. Over-exploitation of natural forests for medicinal purposes and clearance for farming threaten the species survival. Cultivation of the tree species would ensure sustainable medicinal source and its conservation. However, on-farm genetic diversity of the species is currently unknown. The genetic diversity of the on-farm W. ugandensis populations and their proximate natural populations were analyzed using the amplified fragment length polymorphism (AFLP). Four primer combinations produced a total of 223 polymorphic bands. Both the natural and on-farm populations had high genetic diversity ranging from H = 0.2892 to H = 0.1278. Principal co-ordinates analysis and dendrogram separated the ten populations into two major groups corresponding to Kenyan and Tanzanian populations, respectively. Ugandan populations were shared between the two major groups; this is probably because Uganda is believed to be the centre of diversity for W. ugandensis. Close genetic relationships between the on-farm and their proximate natural population were revealed. Analysis of molecular variance (AMOVA) revealed that a total of 54% AFLP variation resided within populations with 46% reside among populations. The high genetic diversity of W. ugandensis on-farm populations could be useful in germplasm collection and conservation strategies. Key words: Warburgia ugandensis, amplified fragment length polymorphism (AFLP), domestication, genetic diversity, on-farm, natural.

Spatial patterns of AFLP diversity in Bulbophyllum occultum (Orchidaceae) indicate long-term refugial isolation in Madagascar and long-distance colonization effects in La Réunion.

Bulbophyllum occultum, an epiphytic orchid mainly distributed in the rainforests of (north)eastern Madagascar and La Réunion, represents an interesting model case for testing the effects of anthropogenic vs historical (e.g., climate induced) habitat isolation and long-distance colonization on the genetic structure of plant species with disjunct distributions in the Madagascan region. To this aim, we surveyed amplified fragment length polymorphisms (AFLPs) across 13 populations in Madagascar and nine in La Réunion (206 individuals in total). We found overall high levels of population subdivision (Φ(PT)=0.387) and low within-population diversity (H(E), range: 0.026-0.124), indicating non-equilibrium conditions in a mainly selfing species. There was no impact of recent deforestation (Madagascar) or habitat disturbance (La Réunion) detectable on AFLP diversity. K-means clustering and BARRIER analyses identified multiple gene pools and several genetic breaks, both within and among islands. Inter-island levels of population genetic diversity and subdivision were similar, whereby inter-individual divergence in flower colour explained a significant part of gene pool divergence in La Réunion. Our results suggest that (i) B. occultum persisted across multiple isolated ('refugial') regions along the eastern rainforest corridor of Madagascar over recent climatic cycles and (ii) populations in La Réunion arose from either single or few independent introductions from Madagascar. High selfing rates and sufficient time for genetic drift likely promoted unexpectedly high population genetic and phenotypic (flower colour) differentiation in La Réunion. Overall, this study highlights a strong imprint of history on the genetic structure of a low-gene-dispersing epiphytic orchid from the Madagascan region.

The regional species richness and genetic diversity ofArctic vegetation reflect both past glaciations and current climate

AbstractAimTheArctic has experienced marked climatic differences between glacial and interglacial periods and is now subject to a rapidly warming climate. Knowledge of the effects of historical processes on current patterns of diversity may aid predictions of the responses of vegetation to future climate change. We aim to test whether plant species and genetic diversity patterns are correlated with time since deglaciation at regional and local scales. We also investigate whether species richness is correlated with genetic diversity in vascular plants.LocationCircumarctic.MethodsWe investigated species richness of the vascular plant flora of 21 floristic provinces and examined local species richness in 6215 vegetation plots distributed across theArctic. We assessed levels of genetic diversity inferred from amplified fragment length polymorphism variation across populations of 23 commonArctic species. Correlations between diversity measures and landscape age (time since deglaciation) as well as variables characterizing current climate were analysed using spatially explicit simultaneous autoregressive models.ResultsRegional species richness of vascular plants and genetic diversity were correlated with each other, and both showed a positive relationship with landscape age. Plot species richness showed differing responses for vascular plants, bryophytes and lichens. At this finer scale, the richness of vascular plants was not significantly related to landscape age, which had a small effect size compared to the models of bryophyte and lichen richness.Main conclusionOur study suggests that imprints of past glaciations inArctic vegetation diversity patterns at the regional scale are still detectable today. SinceArctic vegetation is still limited by post‐glacial migration lag, it will most probably also exhibit lags in response to current and future climate change. Our results also suggest that local species richness at the plot scale is more determined by local habitat factors.

The impact of study design and life history traits on genetic variation of plants determined with AFLPs

In this study, we analysed the impact of study design and life history traits on genetic variation of plants determined with amplified fragment length polymorphisms (AFLPs), a technique widely applied in all fields of molecular plant ecology. For the proper interpretation and comparison of genetic variation based upon AFLPs, a meta-analysis based upon a large number of studies of the relationship between study design and plant life history traits on the one hand and of AFLP variation on the other hand is needed but is lacking. To bridge this gap, we extracted data on study design and genetic variation from 115 AFLP studies comprising a total of 152 species. Subsequently, we ascribed the life history traits taxonomic status, life span, frequency, mating system and pollination vector to each of the species. Then, we used linear models to analyse the impact of study design and life history traits on genetic variation. In our data set genetic variation within and among populations depended neither on the number of analysed populations nor the number of analysed individuals per population. However, maximum geographic distance between populations strongly affected genetic variation. Variation within populations decreased while variation among populations increased with maximum geographic distance. Concerning the impact of life history traits, both genetic variation within and among populations depended with increasing strength on the life span, the frequency and the mating system of the species. Following the results of this study, the number of analysed populations or individuals per population is not necessarily a problem when comparing results of different studies, at least when not very low sample sizes are used. However, corresponding study ranges would be highly recommendable, since the maximum geographic distance between populations strongly affects genetic variation.

Species boundaries in <italic>Philaethria</italic> butterflies: an integrative taxonomic analysis based on genitalia ultrastructure, wing geometric morphometrics, DNA sequences, and amplified fragment length polymorphisms

Neotropical passion-vine butterflies in the tribe Heliconiini (Lepidoptera: Nymphalidae) are a major focus of research in ecology and evolution because of their diverse, aposematic wing patterns, extensive Müllerian mimicry, and coevolution with their Passifloraceae host-plants. However, the basic taxonomy of this group, which is essential to evolutionary ecology research, has been built over the last two centuries using primarily gross morphological comparisons, with most species identification being based on wing colour pattern variation. For some taxa, such as the genus Philaethria Billberg, even the most basic information, such as species limits and geographical distributions, remains uncertain. Furthermore, descriptions of new species, within Philaethria and beyond, have generally been based on small sample sizes collected from a restricted area of the full geographical distribution. To address these issues in the genus Philaethria, here we used an integrative taxonomic approach involving both morphology (genitalia ultrastructure; linear and geometric morphometric analyses of wing shape) and molecular data (multilocus DNA sequence data and amplified fragment length polymorphisms). Specifically, we tested the taxonomic validity of two Philaethria species, Philaethria pygmalion and Philaethria wernickei, described in the literature as having disjunct distributions, corresponding to the Amazon Basin and the Atlantic Rain Forest of Brazil, respectively. Our analyses revealed that these two Philaethria species cannot be delimited and diagnosed using metric and nonmetric morphological characters. Furthermore, they occur in sympatry in the Cerrado biome of central Brazil, and appear to form a latitudinal cline in wing colour variation across their combined distribution. These results are further supported by limited genetic differentiation and a lack of reciprocal monophyly between Amazon and Atlantic Rain Forest populations based on DNA sequence data, and unstructured amplified fragment length polymorphism variation. Our combined results allow us to clarify species-level limits within the genus Philaethria, whereby we propose that P. pygmalion is conspecific with P. wernickei (new synonym), and reassess the spatial range of P. wernickei by providing a refined mapping of its geographical distribution. Beyond clarifying the taxonomy of Philaethria, our results provide a solid, integrative framework that could be applied to fully characterize the taxonomy of other species in the Heliconiini and beyond.

Species boundaries inPhilaethriabutterflies: an integrative taxonomic analysis based on genitalia ultrastructure, wing geometric morphometrics, DNA sequences, and amplified fragment length polymorphisms

Neotropical passion-vine butterflies in the tribe Heliconiini (Lepidoptera: Nymphalidae) are a major focus of research in ecology and evolution because of their diverse, aposematic wing patterns, extensive Mullerian mimicry, and coevolution with their Passifloraceae host-plants. However, the basic taxonomy of this group, which is essential to evolutionary ecology research, has been built over the last two centuries using primarily gross morphological comparisons, with most species identification being based on wing colour pattern variation. For some taxa, such as the genus Philaethria Billberg, even the most basic information, such as species limits and geographical distributions, remains uncertain. Furthermore, descriptions of new species, within Philaethria and beyond, have generally been based on small sample sizes collected from a restricted area of the full geographical distribution. To address these issues in the genus Philaethria, here we used an integrative taxonomic approach involving both morphology (genitalia ultrastructure; linear and geometric morphometric analyses of wing shape) and molecular data (multilocus DNA sequence data and amplified fragment length polymorphisms). Specifically, we tested the taxonomic validity of two Philaethria species, Philaethria pygmalion and Philaethria wernickei, described in the literature as having disjunct distributions, corresponding to the Amazon Basin and the Atlantic Rain Forest of Brazil, respectively. Our analyses revealed that these two Philaethria species cannot be delimited and diagnosed using metric and nonmetric morphological characters. Furthermore, they occur in sympatry in the Cerrado biome of central Brazil, and appear to form a latitudinal cline in wing colour variation across their combined distribution. These results are further supported by limited genetic differentiation and a lack of reciprocal monophyly between Amazon and Atlantic Rain Forest populations based on DNA sequence data, and unstructured amplified fragment length polymorphism variation. Our combined results allow us to clarify species-level limits within the genus Philaethria, whereby we propose that P. pygmalion is conspecific with P. wernickei (new synonym), and reassess the spatial range of P. wernickei by providing a refined mapping of its geographical distribution. Beyond clarifying the taxonomy of Philaethria, our results provide a solid, integrative framework that could be applied to fully characterize the taxonomy of other species in the Heliconiini and beyond. © 2014 The Linnean Society of London

Genetic diversity of side-oats grama [Bouteloua curtipendula(Michx.) Torr.] populations as revealed by amplified fragment length polymorphism markers

Biligetu, B., Schellenberg, M. P. and Fu, Y.B. 2013. Genetic diversity of side-oats grama [Bouteloua curtipendula (Michx.) Torr.] populations as revealed by amplified fragment length polymorphism markers. Can. J. Plant Sci. 93: 1105–1114. Side-oats grama [Bouteloua curtipendula (Michx.) Torr.] is a warm-season grass widely distributed in North America and is considered as an important grass for reclamation and summer forage production in drier regions. This study assessed genetic diversity of nine wild populations of side-oats grama grass, their corresponding balanced multiple-site composite (BMSC), and a population selected for forage type (FT), using the amplified fragment length polymorphism (AFLP) technique. Five AFLP primer pairs were employed to genotype 157 plants, and 312 polymorphic AFLP bands were detected. The frequencies of AFLP bands ranged from 0.01 to 0.99, and averaged 0.39. The AFLP analysis revealed 6% of the total AFLP variation present among the nine wild populations and 94% of variation within populations. The Minto population had the largest within-population diversity, while the FT population the lowest based on AFLP band richness and polymorphic loci. The BMSC population displayed significant genetic differentiation from the wild populations, but still captured substantial genetic diversity. Bayesian cluster analysis using BAPS and STRUCTURE programs revealed three and four optimal clusters of populations that explained 9.8 and 9.5% of the total AFLP variation, respectively. A genetic clustering of individual plants displayed no clear genetic separations among wild, FT, and BMSC populations, but the FT population showed some level of genetic shift, indicating the initial impact of artificial selection. These findings are significant for our understanding of the genetic diversity of side-oats grama. Large genetic variation present within populations provides a potential for further genetic enhancement.

Evaluating genetic variation and relationships amongPuccinellia nuttallianapopulations using amplified fragment length polymorphism markers

Liu, Y., Fu, Y.-B. and Coulman, B. E. 2013. Evaluating genetic variation and relationships among Puccinellia nuttalliana populations using amplified fragment length polymorphism markers. Can. J. Plant Sci. 93: 1097–1104. Nuttall's salt-meadow, or alkali grass [Puccinellia nuttalliana (Shultes) Hitchc.], is a native grass species in North America, well known for its salt tolerance. Little information is available about the genetic diversity of natural populations of this species. Amplified fragment length polymorphism (AFLP) markers were used to examine the inter-population relationships and to compare variances within and among 23 populations collected from the Canadian Great Plains. Five AFLP primer pairs were employed to screen 15 genotypes (five sets of three half-sib plants) from each population, and 185 polymorphic AFLP bands were scored for each sample. The frequencies of these scored bands ranged from 0.02 to 0.99 with a mean of 0.60. The analysis of molecular variance revealed more than 96% of the total AFLP variation resided within populations. Populations were not highly differentiated with only 4% of the total AFLP variation residing among populations. A Mantel test revealed a significant but low correlation between genetic and geographic distances (r=0.29, P=0.024). Implications for P. nuttalliana conservation, germplasm sampling, and cultivar development are discussed.

Phylogeography of the heathers Erica arborea and E. trimera in the afro-alpine ‘sky islands’ inferred from AFLPs and plastid DNA sequences

The ericaceous vegetation zone of the unique and highly fragmented afro-alpine environment in the eastern African high mountains is typically dominated by the heather Erica arborea, often in combination with its close relative E. trimera. Both species are shrubs or small trees with tiny seeds, potentially capable of dispersal by wind over long distances. While E. arborea is widely distributed in Africa, the Middle East and Europe, E. trimera is endemic to the afro-alpine region where it is restricted to higher altitudes than E. arborea. We used Amplified Fragment Length Polymorphisms (AFLPs) and variation in non-coding plastid DNA sequences to test whether these two morphologically and ecologically very similar species display similar phylogeographic patterns in the afro-alpine region. We predict that the more high-altitudinal E. trimera shows more distinct genetic structuring than E. arborea, because dispersal of the latter may have been facilitated by formation of interglacial forest bridges between mountains. Based on extensive field sampling in most of the high mountains of Ethiopia and East Africa, we show that the two species are clearly distinct at AFLP and plastid DNA loci. Both showed low levels of overall AFLP diversity, suggesting bottlenecking in small refugial populations during unfavourable climatic periods. However, their genetic structuring and inferred phylogeographic histories were conspicuously different. The more high-altitudinal E. trimera consisted of three to four distinct AFLP groups, which also had different plastid DNA haplotypes and different geographic distributions, suggesting long-term restriction to several refugia (at least one in Ethiopia and two in East Africa). In contrast, E. arborea showed little geographic structuring at AFLP loci and only a single, widespread plastid DNA haplotype, which may suggest recent colonization of the entire study area from a single source population, likely via a combination of gradual expansion via forest bridges and long-distance dispersals. The source population of E. arborea may be situated in (or north of) Ethiopia, which harbours most genetic diversity.

Wide‐Scale Population Sampling Identifies Three Phylogeographic Races of Basin Wildrye and Low‐Level Genetic Admixture with Creeping Wildrye

ABSTRACTBasin wildrye [Leymus cinereus (Scribn. & Merr.) Á. Löve] and creeping wildrye [Leymus triticoides (Buckley) Pilg.] are outcrossing perennial grasses native to western North America. These divergent species are generally adapted to different habitats but can form fertile hybrids. Cultivars of both species are used in agriculture and conservation, but little is known about genetic diversity and gene flow among these species. Therefore, multilocus amplified fragment length polymorphism (AFLP) genotypes and chloroplast DNA sequences were evaluated from 536 L. cinereus and 43 L. triticoides plants from 224 locations of western United States and Canada. Bayesian‐cluster analysis detected three L. cinereus races corresponding to the Columbia, Rocky Mountain, and Great Basin regions. Possible admixture between species was detected in specific areas, but only 2.2% of the plants showed more than 10% introgression. The Columbia and Great Basin races were predominantly octoploid whereas most of the Rocky Mountain accessions were tetraploid. Approximately 36 and 7% of the AFLP variation was apportioned among species and races, respectively, but no discrete marker differences were detected among these groups. Although species can be distinguished using a relatively small set of AFLP markers showing divergent allele frequencies, at least 30 markers were needed to classify plants by race. Approximately 8 and 11% of the chloroplast DNA sequence variation was apportioned among species and races, but these markers were not practically useful for species or race identification.

Spatial scales of genetic structure and gene flow in Calochortus albus (Liliaceae)

Calochortus (Liliaceae) displays high species richness, restriction of many individual taxa to narrow ranges, geographic coherence of individual clades, and parallel adaptive radiations in different regions. Here we test the first part of a hypothesis that all of these patterns may reflect gene flow at small geographic scales. We use amplified fragment length polymorphism variation to quantify the geographic scales of spatial genetic structure and apparent gene flow in Calochortus albus, a widespread member of the genus, at Henry Coe State Park in the Coast Ranges south of San Francisco Bay. Analyses of 254 mapped individuals spaced 0.001–14.4 km apart show a highly significant decline in genetic identity with ln distance, implying a root-mean-square distance of gene flow σ of 5–43 m. STRUCTURE analysis implies the existence of 2–4 clusters over the study area, with frequent reversals among clusters over short distances (<200 m) and a relatively high frequency of admixture within individuals at most sampling sites. While the intensity of spatial genetic structure in C. albus is weak, as measured by the Sp statistic, that appears to reflect low genetic identity of adjacent plants, which might reflect repeated colonizations at small spatial scales or density-dependent mortality of individual genotypes by natural enemies. Small spatial scales of gene flow and spatial genetic structure should permit, under a variety of conditions, genetic differentiation within species at such scales, setting the stage ultimately for speciation and adaptive radiation as such scales as well.

Genetic Variation, Biogeographical History, and Conservation ofAnthyllis montanaL. Ssp.jacquinii(Kern.) Hayek (Fabaceae) at Its Northern Distribution Limit

Genetic patterns in Anthyllis montana ssp. jacquinii populations from the species’ central Balkan distribution toward its northern distribution limit in Austria were analyzed against the background of their evolution, biogeography, and conservation. Genetic structure and diversity were assessed by DNA sequencing (ITS, trnH/psbA) and amplified fragment length polymorphisms (AFLPs). Provisionally, seed mass data were added. Four homoplastic mutations were observed within the trnH/psbA intergenic spacer, while ITS sequences were identical across populations. Population differentiation, as assessed by AFLPs, was moderate (). Isolation by distance was absent within the peripheral and central regions. Genetic-structure analyses revealed two groups, separating the two northernmost Austrian populations from the remaining populations. Population sizes were not correlated with the overall rather low genetic-diversity values, but AFLP diversity was not further reduced in peripheral populations. Seed mass was similar across populations. The unexpected diversity level of the peripheral populations might be best explained by persistence of ancient genetic diversity and accumulation of new mutations. The main evolutionary process acting is probably genetic drift, as confirmed by AFLP outlier analysis. Biogeographical and conservation perspectives emphasize the importance of the peripheral Austrian populations representing an independent gene pool within A. montana ssp. jacquinii.

Assessment of Genetic Diversity in Zoysia Species using Amplified Fragment Length Polymorphism Markers

ABSTRACTZoysia spp. are warm‐season turfgrass species widely used in the transition zone and southern regions of the United States for their superior heat tolerance and relatively low input requirements. A better understanding of the levels of genetic diversity present in Zoysia germplasm could aid in the utilization of these materials in future breeding efforts. The objective of this study was to compare levels of molecular diversity within and among Zoysia spp. {Z. japonica Steud., Z. matrella (L.) Merr., Z. machrostachya Franch. &amp; Sav., Z. minima (Colenso) Zotov, Z. pacifica (Goudswaard) M. Hotta &amp; Kuroki [syn. Z. matrella (L.) Merr. var. pacifica Goudswaard], and Z. sinica Hance} and germplasm types (cultivars, plant introductions, and collections). Two hundred and forty‐one Zoysia accessions from public and private sources were genotyped with 12 amplified fragment length polymorphism (AFLP) primer pairs. Five hundred and fifty‐two polymorphic AFLP loci were scored. Genetic similarity values (Sij) among genotypes ranged from 0.60 to 0.81, with the average being 0.68. Analysis of molecular variance (AMOVA) results indicated that 28 and 54% of the total AFLP variation could be explained by differences among and within species, respectively. Unweighted pair group method with arithmetic averaging (UPGMA) cluster analysis and principal coordinate analysis (PCO) identified high levels of similarity among all genotypes. Analyses revealed that individuals from the same species type occasionally failed to cluster into one distinct group, reflecting a predominantly outcrossing mating system and high levels of gene flow among the species types studied. Understanding the distribution of genetic diversity within and among Zoysia spp. may enhance cultivar development efforts.

Genetic Risk Assessment of a Threatened Remnant Population of Hairy Prairie-Clover (Dalea villosa var. villosa) in the Canadian Prairie

Hairy prairie-clover [Dalea villosa (Nutt.) Spreng. var. villosa] is a threatened Canadian wildflower. To facilitate the efforts of conserving this threatened plant, amplified fragment length polymorphism (AFLP) technique was applied to assess genetic diversity in a remnant hairy prairie-clover population in the Canadian Prairie. Three AFLP primer pairs were employed to genotype 610 individual plants from the population and 15 plants from a North Dakota composite population, and 100 polymorphic AFLP bands were analyzed. The assayed plants displayed 23% AFLP variation present between the remnant population and the North Dakota composite population, but maintained a high level (91%) of AFLP variation within patches of the remnant population. The individual genetic distinctiveness measured by average AFLP dissimilarity was positively associated with latitude and negatively with elevation. The among-patch AFLP variation was significantly related to inter-patch distance, indicating local genetic differentiation within the remnant population. However, the proportions of within-patch AFLP variation were not associated with any patch characteristics assessed (i.e., patch size, perimeter, nearest neighbor distance, mean inter-patch distance). No fine-scale genetic structure was found within three large patches, suggesting little genetic correlations present for plants five meters apart. Some genetically distinctive and diverse patches were also identified. These findings indicate that the genetic risk of the remnant hairy prairie-clover population in the Canadian Prairie is low.

Quaternary range dynamics of ecologically divergent species (Edraianthus serpyllifolius and E. tenuifolius, Campanulaceae) within the Balkan refugium

Aim We investigated Quaternary range dynamics of two closely related but ecologically divergent species (cold-tolerant Edraianthus serpyllifolius and thermophilic Edraianthus tenuifolius) with overlapping distribution ranges endemic to the western Balkan Peninsula, an important yet understudied Pleistocene refugium. Our aims were: to test predictions of the ‘refugia-withinrefugia’ model of strong genetic subdivisions due to population isolation in separate refugia; to explore whether two ecologically divergent species reacted differently to Pleistocene climatic fluctuations; and to test predictions of the displacement refugia model of stronger differentiation among populations in the thermophilic E. tenuifolius compared with the cold-tolerant E. serpyllifolius. Location The western Balkan Peninsula. Methods We gathered amplified fragment-length polymorphism (AFLP) data and plastid DNA sequences from two to five individuals from 10 populations of E. serpyllifolius and 22 populations of E. tenuifolius, spanning their entire respective distribution areas. AFLP data were analysed using a Bayesian clustering approach and a distance-based network approach. Plastid sequences were used to depict relationships among haplotypes in a statistical parsimony network, and to obtain age estimates in a Bayesian framework. Results In E. serpyllifolius, both AFLP and plastid sequence data showed clear geographic structure. Western populations showed high AFLP diversity and a high number of rare fragments. In E. tenuifolius, both markers congruently identified a majorphylogeographicsplitalongthelowerNeretvavalleyincentralDalmatia.The most distinct and earliest diverging chloroplast DNA (cpDNA) haplotypes were found further south in the south-easternmost populations. North-western populations, identified as a separate cluster by Bayesian clustering, were characterized by low genetic diversity and a low number of rare AFLP markers. Main conclusions Clear evidence for multiple Pleistocene refugia is found not only in the high-elevation E. serpyllifolius, but also in the lowland E. tenuifolius, despite the lack of obvious dispersal barriers, in line with the refugia-withinrefugia model. Genealogical relationships and genetic diversity patterns support the hypothesis that cold-adapted E. serpyllifolius responded to climatic oscillations mostly by elevational range shifts, whereas thermophilic E. tenuifolius did so mainly by latitudinal range shifts, with different phases (and probably extents) of range expansion. In contrast to the displacement refugia hypothesis, the two elevationally differentiated species do not differ in their genetic diversity.

Genetic evidence for the origins of range disjunctions in the Australian dry sclerophyll plant Hardenbergia violacea

Aim The distribution of genetic variation in the Australian dry sclerophyll plant Hardenbergia violacea (Fabaceae) is examined in the context of Pleistocene climate change in order to identify likely refugia. Particular consideration is given to the origin of range disjunctions in South Australia and Tasmania, and to determining whether the Tasmanian population is indigenous or recently introduced from mainland Australia. Location Southeastern Australian mainland and Tasmania. Methods A combination of chloroplast polymerase chain reaction–restriction fragment length polymorphism and genomic amplified fragment length polymorphism (AFLP) marker systems was used to examine the genetic structure of 292 individuals from 13 populations across the range of H. violacea in southeastern Australia. Results Hardenbergia violacea populations in Tasmania and southern Victoria were characterized by low, almost monotypic chloroplast diversity. New South Wales showed higher haplotype diversity and haplotype sharing among widely distributed populations. Principal coordinates analysis (PCoA) of the AFLP data found a strong latitudinal cline in AFLP variation from northern New South Wales south to Tasmania. The Tasmanian population formed an isolated and somewhat disjunct genetic cluster at one end of this cline. However, the South Australian population was an exception to the clinal variation shown by all other populations, forming a highly disjunct cluster in the PCoA. Within-population genetic diversity was low in both disjunct populations. Main conclusions The genetic evidence indicates that the Tasmanian population is likely to be indigenous and probably the product of vicariance, which was followed by range contraction at the Last Glacial Maximum or an earlier glacial event. The deep phylogenetic disjunction in South Australia is evidence of a much earlier separation on mainland Australia. The chloroplast structure indicates that, during the Pleistocene, H. violacea underwent broad-scale recolonization in southern Victoria and Tasmania, possibly from a large continental refugium in eastern New South Wales. We conclude that H. violacea, and presumably the sclerophyll communities in which it occurs, have undergone multiple range contractions to large continental refugia during different Pleistocene glaciations in southeastern Australia.