Genetic diversity in fragmented populations of the critically endangered spider orchid Caladenia huegelii: implications for conservation

The Orchidaceae is characterised by a diverse range of life histories, reproductive strategies and geographic distribution, reflected in a variety of patterns in the population genetic structure of different species. In this study, the genetic diversity and structure was assessed within and among remnant populations of the critically endangered sexually deceptive orchid, Caladenia huegelii. This species has experienced severe recent habitat loss in a landscape marked by ancient patterns of population fragmentation within the Southwest Australian Floristic Region, a global biodiversity hotspot. Using seven polymorphic microsatellite loci, high levels of within-population diversity (mean alleles/locus = 6.73; mean H _E = 0.690), weak genetic structuring among 13 remnant populations (F _ST = 0.047) and a consistent deficit of heterozygotes from Hardy–Weinberg expectation were found across all populations (mean F _IS = 0.22). Positive inbreeding coefficients are most likely due to Wahlund effects and/or inbreeding effects from highly correlated paternity and typically low fruit set. Indirect estimates of gene flow (Nm = 5.09 using F _ST; Nm = 3.12 using the private alleles method) among populations reflects a historical capacity for gene flow through long distance pollen dispersal by sexually deceived wasp pollinators and/or long range dispersal of dust-like orchid seed. However, current levels of gene flow may be impacted by habitat destruction, fragmentation and reduced population size. A genetically divergent population was identified, which should be a high priority for conservation managers. Very weak genetic differentiation indicates that the movement and mixing of seeds from different populations for reintroduction programs should result in minimal negative genetic effects.     

Depleted genetic variation of the European ground squirrel in Central Europe in both microsatellites and the major histocompatibility complex gene: implications for conservation

Habitat fragmentation may influence the genetic make-up and adaptability of endangered populations. To facilitate genetic monitoring of the endangered European ground squirrel (EGS), we analyzed 382 individuals from 16 populations in Central Europe, covering almost half of its natural range. We tested how fragmentation affects the genetic architecture of presumably selectively neutral (12 microsatellites) and non-neutral (the major histocompatibility class II DRB gene) loci. Spatial genetic analyses defined two groups of populations, “western” and “eastern”, with a significantly higher level of habitat fragmentation in the former group. The highly fragmented western populations had significantly lower genetic diversity in both types of markers. Only one allele of the DRB gene predominated in populations of the western group, while four alleles were evenly distributed across the eastern populations. Coefficient of inbreeding values (F _IS) calculated from microsatellites were significantly higher in the western (0.27–0.79) than in eastern populations (−0.060–0.119). Inter-population differentiation was very high, but similar in both groups (western F _ST = 0.23, eastern F _ST = 0.25). The test of isolation by distance was significant for the whole dataset, as well as for the two groups analyzed separately. Comparison of genetic variability and structure on microsatellites and the DRB gene does not provide any evidence for contemporary selection on MHC genes. We suggest that genetic drift in small bottlenecked and fragmented populations may overact the role of balancing selection. Based on the resulting risk of inbreeding depression in the western populations, we support population management by crossbreeding between the western and eastern populations.     

Genetic structure of Brandt’s vole (<Emphasis Type="Italic">Lasiopodomys brandtii</Emphasis>) populations in Inner Mongolia,China, based on microsatellite analysis

Brandt’s vole (Lasiopodomys brandtii) distribution is discontinuous in Inner Mongolia with some populations isolated from others. Recently, some isolated populations have suffered extinction, and the factors responsible remain elusive. Genetic drift is one of the processes affecting population genetic differentiation, and can play a substantial role in the divergence of small, isolated populations. Using seven microsatellite markers, we genotyped four geographically isolated populations of Brandt’s vole, all of which exhibit episodic fluctuations in population density. The results showed a strong genetic differentiation among the geographically distinct populations (total F _ST = 0.124) and in particular, one population (Zhengxiangbaiqi) was isolated from all others (F _ST values were greatest between Zhengxiangbaiqi and other populations). Furthermore, high levels of inbreeding (F _IS values ranged from 0.205 to 0.290) within each distinct population suggest that inbreeding has and is likely occurring in Brandt’s vole populations. These processes can decrease average individual fitness and consequently increase the risk of extinction of the species.     

RECENT EVOLUTION AND DIVERGENCE AMONG POPULATIONS OF A RARE MEXICAN ENDEMIC,CHIHUAHUA SPRUCE,FOLLOWING HOLOCENE CLIMATIC WARMING

Fragmentation and reduction in population size are expected to reduce genetic diversity. However, examples from natural populations of forest trees are scarce. The range of Chihuahua spruce retreated northward and fragmented coincident with the warming climate that marked the early Holocene. The isolated populations vary from 15 to 2441 trees, which provided an opportunity to test whether census number is a good predictor of genetic diversity. Mean expected heterozygosity, H_e, based on 24 loci in 16 enzyme systems, was 0.093 for 10 sampled populations, which is within the range reported for conifers. However, estimates varied more than twofold among populations and H_e was closely related to the logarithm of the number of mature trees in the population (r_He,N = 0.93). Diversity among populations, F_ST, was 24.8% of the total diversity, which is higher than that observed in almost all conifer species studied. Nei's genetic distance, D, was not related to geographic distance between populations, and D? was 0.033, which is higher than estimates for most wide-ranging species. Most populations had excess homozygosity and the fixation index, F_IS, was higher than that reported for all but one species of conifer. Nm, the number of migrants per generation, was 0.43 to 0.76, depending on estimation procedure, and is the smallest observed in conifers. The data suggest that populations of Chihuahua spruce have differentiated by drift and that they are effectively isolated. The results illustrate how a combination of paleontological observation and molecular markers can be used to illuminate recent evolutionary events. Multilocus estimates of outcrossing for two small populations were zero (complete selfing) and 0.153, respectively, which are in striking contrast to the near complete outcrossing observed in most conifers. The high fixation index and a high proportion of empty seeds (45%) suggest that inbreeding may be a serious problem for conservation of Chihuahua spruce.     

Genic diversity, genetic structure, and biogeography of Pinus sabiniana Dougl.

Pinus sabiniana Dougl. (grey pine) forms savanna forests in the foothills surrounding California's Great Central Valley. However, its fossil record, which dates from the late Miocene through the Pliocene and Pleistocene, is found exclusively in southern California, south of the species’ present range. A total of twenty-nine isozyme loci, representing eighteen enzyme systems, was assayed to analyse the genetic structure in eight populations of grey pine and attempt to track its migration history from southern to northern California. Expected heterozygosity in the two southernmost samples was 0.128 and 0.150, and heterozygosity tended to decrease with increasing latitude, suggesting the loss of diversity as grey pine dispersed northward. However, genetic distances between populations were very small, even on opposite sides of the treeless Great Central Valley; and estimated time since divergence was 900 to 9000 years at a maximum. Wright's F_ST, the proportion of total genetic diversity among populations, was only 0.057, which is similar to values found in many conifers with continuous distributions. Nm, the number of migrants among populations per generation, was 4.1 to 6.7, depending on estimator, and indicates that gene flow is extensive, or was so in the recent past. In every population, observed heterozygosity was less than expected heterozygosity, and the fixation index, F_IS, for the progeny was 0.128, which indicates a fairly high rate of inbreeding. The genetic similarity of disjunct populations, in combination with paleogeographic and paleoclimatic evidence, suggests that grey pine formed a continuous population throughout the Great Central Valley, perhaps between 12,000 and 8000 yrs BP . Its range became fragmented during the Xerothermic, when it ascended into the foothills. Gaps in its range correlate with late Pleistocene–early Holocene lakes in adjacent basins and with the Sacramento–San Joaquin Delta.     

Estimation and adjustment of microsatellite null alleles in nonequilibrium populations

Nonamplified (null) alleles are a common feature of microsatellite genotyping and can bias estimates of allele and genotype frequencies, thereby hindering population genetic analyses. The frequency of microsatellite null alleles in diploid populations can be estimated for populations that are in Hardy–Weinberg equilibrium. However, many microsatellite data sets are from nonequilibrium populations, often with known inbreeding coefficients (F) or fixation indices (F_IS or F_ST). Here, we propose a novel null allele estimator that can be used to estimate the null allele frequency and adjust visible allele frequencies in populations for which independent estimates of F, F_IS or F_ST are available. The algorithm is currently available as an Excel macro that can be downloaded at no cost from http://www.microchecker.hull.ac.uk/ and will be incorporated into the software micro ‐checker .     

Comparison of quantitative and molecular variation in agroforestry populations of the shea tree (Vitellaria paradoxa C.F. Gaertn) in Mali

In this study we investigated the within- and between-population genetic variation using microsatellite markers and quantitative traits of the shea tree, Vitellaria paradoxa, an important agroforestry tree species of the Sudano–Sahelian region in Africa. Eleven populations were sampled across Mali and in northern Côte d’Ivoire. Leaf size and form and growth traits were measured in a progeny test at the nursery stage. Eight microsatellites were used to assess neutral genetic variation. Low levels of heterozygosity were recorded (1.6–3.0 alleles/locus; H_E = 0.25–0.42) and the fixation index (F_IS = −0.227–0.186) was not significantly different from zero suggesting that Hardy–Weinberg equilibrium is encountered in all populations sampled. Quantitative traits exhibited a strong genetic variation between populations and between families within populations. The degree of population differentiation of the quantitative traits (Q_ST = 0.055–0.283, Q_STmean = 0.189) strongly exceeds that in eight microsatellite loci (F_ST = −0.011–0.142, F_STmean = 0.047). Global and pairwise F_ST values were very low and not significantly different from zero suggesting agroforestry practices are amplifying gene flow (Nm = 5.07). The population means for quantitative traits and the rainfall variable were not correlated, showing variation was not linked with this climatic cline. It is suggested that this marked differentiation for quantitative traits, independent of environmental clines and despite a high gene flow, is a result of local adaptation and human selection of shea trees. This process has induced high linkage disequilibrium between underlying loci of polygenic characters.     

Evidence of recent population bottlenecks and inbreeding in British populations of Bechstein’s bat, Myotis bechsteinii

We investigated the population genetics of seven maternity roosts of Bechstein’s bats widely distributed across the south of England. Across all of the populations sampled, two mitochondrial DNA microsatellite loci were fixed for single haplotypes. Genetic diversity across eight nuclear microsatellite loci was similar in all seven populations, with a mean He of 0.727. However, six of the populations showed substantial homozygote excess, with F _IS estimates greater than zero, indicative of recent inbreeding. Bottleneck tests also implied that six of the populations have experienced recent declines. Genetic differentiation among the populations was low, with a mean intersite F _ST estimate of 0.041. There was no significant isolation by distance using allele frequency-based criteria (F _ST and genetic distances), however, a weak correlation was found using the allele size-based R _ST criterion. Assignment tests were unable to distinguish the seven sampling sites as distinct clusters. Mean intra-roost relatedness (r) was 0.079, indicative of recent inbreeding relative to German populations. All but one of the bats had one or more half or full siblings in its maternity roost. In addition, family relationships of individuals within a colony were significantly commoner than family relationships among four proximal roosts <8 km apart. The results are discussed in the context of conservation requirements for this rare British bat.     

Drift happens: Molecular genetic diversity and differentiation among populations of jewelweed (Impatiens capensis Meerb.) reflect fragmentation of floodplain forests

Landscape features often shape patterns of gene flow and genetic differentiation in plant species. Populations that are small and isolated enough also become subject to genetic drift. We examined patterns of gene flow and differentiation among 12 floodplain populations of the selfing annual jewelweed (Impatiens capensis Meerb.) nested within four river systems and two major watersheds in Wisconsin, USA. Floodplain forests and marshes provide a model system for assessing the effects of habitat fragmentation within agricultural/urban landscapes and for testing whether rivers act to genetically connect dispersed populations. We generated a panel of 12,856 single nucleotide polymorphisms and assessed genetic diversity, differentiation, gene flow, and drift. Clustering methods revealed strong population genetic structure with limited admixture and highly differentiated populations (mean multilocus F_ST = 0.32, F_ST’ = 0.33). No signals of isolation by geographic distance or environment emerged, but alleles may flow along rivers given that genetic differentiation increased with river distance. Differentiation also increased in populations with fewer private alleles (R² = 0.51) and higher local inbreeding (R² = 0.22). Populations varied greatly in levels of local inbreeding (F_IS = 0.2–0.9) and F_IS increased in more isolated populations. These results suggest that genetic drift dominates other forces in structuring these Impatiens populations. In rapidly changing environments, species must migrate or genetically adapt. Habitat fragmentation limits both processes, potentially compromising the ability of species to persist in fragmented landscapes.     

High population differentiation and genetic variation in the endangered Mexican pine Pinus Rzedowskii (Pinaceae)

Pinus rzedowskii is an endangered pine species from Michoaca´n (central Me´xico), which has been previously reported from only three localities. Classified within the subgenus Strobus, it exhibits intermediate morphological characters between subgenera Strobus and Pinus. We analyzed genetic aspects that could shed light on the evolution and conservation of this species. The genetic structure of nine populations was examined using 14 isozyme loci. Pinus rzedowskii has a relatively high level of genetic variation with 46.8% of the loci assayed being polymorphic, a total of 35 alleles, and a mean heterozygosity per population of 0.219. We calculated Wright's F_ST statistic to estimate gene flow indirectly and to evaluate whether or not there was genetic structuring among populations. We found a marked differentiation among populations (F_ST = 0.175) and significant inbreeding (F_IS = 0.247). No pattern of isolation by distance was found. We also constructed a dendrogram based on a genetic distance matrix to obtain an overview of the possible historical relationships among populations. Finally, we found a convex relationship between the genetic distance among populations and the number of ancestral lineages, suggesting that demographically this species has not been at risk recently. Although endangered, with small and fragmented populations, P. rzedowskii shows higher levels of genetic variation than other conifer species with larger populations or similar conservation status.     

Population genetic structure of the gynodioecious Thymus vulgaris L. (Labiatae) in southern France

In a self-compatible gynodioecious species, the abundance of female plants (which are obligate outcrossers) relative to hermaphrodites (which may self and outcross) may be a critical factor influencing genetic diversity and population structure. In the gynodioecious Thymus vulgaris L., female frequency varies from 5 to 95%, providing a suitable model to examine this issue. In this study, we use allozyme markers to (1) evaluate the relationship between female frequency, genetic diversity and population structure, (2) determine whether females and hermaphrodites vary in heterozygote deficiency and (3) examine whether other factors such as plant density are related to heterozygote deficiency. Twenty three natural populations, with female frequencies ranging from 11 to 92%, were sampled in and around the St-Martin-de-Londres basin in southern France. Based on four polymorphic allozyme loci, we found no significant correlation between female frequency and heterozygote deficiency. A significant (P < 0.05) F_IS value over loci and over populations of 0.11 was detected. The F_IS value per population showed a significant heterozygote deficiency in 11 of the 23 populations. However, no significant difference between female and hermaphrodite F_IS values was found. A significant heterozygote deficiency only occurred in populations of intermediate density. There was little differentiation among populations (F_ST = 0.038) nor among subpopulations within each population. The significant F_IS values are thus mostly due to inbreeding effects. The lack of a correlation between F_IS values and female frequency may be due to outcrossing in hermaphrodites and/or restoration of male fertility which may occur to a greater extent at low female frequency. The similarity of female and hermaphrodite F_IS values indicates that females may occasion high levels of biparental inbreeding.     

Inbreeding variability and population structure in the invasive haplodiploid palm‐seed borer (Coccotrypes dactyliperda)

We investigated the mating system and population genetic structure of the invasive haplodiploid palm‐seed borer Coccotrypes dactyliperda in California. We focused on whether these primarily inbreeding beetles have a ‘mixed‐breeding’ system that includes occasional outbreeding, and whether local inbreeding coefficients (F_IS) varied with dominant environmental factors. We also analysed the genetic structure of C. dactyliperda populations across local and regional scales. Based on the analysis of genetic variation at seven microsatellite loci in 1034 individual beetles from 59 populations, we found both high rates of inbreeding and plentiful evidence of mixed‐breeding. F_IS ranged from ?0.56 to 0.90, the highest variability reported within any animal species. There was a negative correlation between F_IS and latitude, suggesting that some latitude‐associated factor affecting mating decisions influenced inbreeding rates. Multiple regressions suggested that precipitation, but not temperature, may be an important correlate. Finally, we found highly significant genetic differentiation among sites, even over short geographic distances (< 1000 m).     

Genetic diversity and population differentiation in the endangered Siberian flying squirrel (<Emphasis Type="Italic">Pteromys volans</Emphasis>) in a fragmented landscape

Siberian flying squirrel (Pteromys volans) has declined in Finland and it is considered an endangered species. We studied microsatellite variation in four flying squirrel populations in a fragmented landscape in Finland to determine the amount of gene flow and genetic diversity in the populations. Demographic data from these areas suggest that the populations are declining. All the populations are significantly differentiated (F _ST = 0.23). The most notable result is the high degree of differentiation between two adjacent populations (F _ST = 0.11) and low genetic variability (number of alleles 3.0) in one of the populations. These findings suggest problems in dispersal and possible fragmentation effects in the landscape where only 10–20% of habitat favorable for the flying squirrel is left. Conservation ensuring dispersal should be urgently considered. Future studies should concentrate on the modeling of the population viability and on the effects of inbreeding in these small populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conservation genetics of the annual hemiparasitic plant <Emphasis Type="Italic">Melampyrum sylvaticum</Emphasis> (Orobanchaceae) in the UK and Scandinavia

Melampyrum sylvaticum is an endangered annual hemiparasitic plant that is found in only 19 small and isolated populations in the United Kingdom (UK). To evaluate the genetic consequences of this patchy distribution we compared levels of diversity, inbreeding and differentiation from ten populations from the UK with eight relatively large populations from Sweden and Norway where the species is more continuously distributed. We demonstrate that in both the UK and Scandinavia, the species is highly inbreeding (global F _IS = 0.899). Levels of population differentiation were high (F’_ST = 0.892) and significantly higher amongst UK populations (F’_ST = 0.949) than Scandinavian populations (F’_ST = 0.762; P < 0.01). The isolated populations in the UK have, on average, lower genetic diversity (allelic richness, proportion of loci that are polymorphic, gene diversity) than Scandinavian populations, and this diversity difference is associated with the smaller census size and population area of UK populations. From a conservation perspective, the naturally inbreeding nature of the species may buffer the species against immediate effects of inbreeding depression, but the markedly lower levels of genetic diversity in UK populations may represent a genetic constraint to evolutionary change. In addition, the high levels of population differentiation suggest that gene flow among populations will not be effective at replenishing lost variation. We thus recommend supporting in situ conservation management with ex situ populations and human-mediated seed dispersal among selected populations in the UK.     

Conservation Genetics of the Endemic Mexican Heliconia uxpanapensis in the Los Tuxtlas Tropical Rain Forest

Heliconia uxpanapensis (Heliconiaceae) is an outcrossing endemic herb that grows within continuous and fragmented areas of the tropical rain forest of southeast Veracrúz (México). The genetic diversity, population differentiation, and genetic structure of seven populations of the studied species were assessed using inter‐simple sequence repeat) markers. Population differentiation was moderately high (F_ST range: 0.18–0.22) and indirect estimates of gene flow were rather low (Nm=0.65–0.83). Analysis of molecular variance indicated that the populations explained 22.2 percent of the variation, while individuals within the populations accounted for 77.8 percent. The similar and high level of genetic diversity found within populations of the continuous and fragmented forest suggests that H. uxpanapensis has not suffered yet the expected negative effect of fragmentation. Genetic structure analyses indicated the presence of fewer genetic clusters (K=4) than populations (N=7). Three of the four fragmented forest populations were assigned each to one of the clusters found within the continuous forest, suggesting the absence of a negative fragmentation effect on the amount and distribution of genetic variation. Given the significant genetic structure combined with high genetic diversity and low levels of gene flow, theoretical simulations indicated that H. uxpanapensis might be highly susceptible to changes in the mating system, which promotes inbreeding within fragmented populations. Thus, future conservation efforts in this species should be directed to ensure that levels of gene flow among populations are sufficient to prevent an increment in the magnitude of inbreeding within fragments.     

Genetic variability and structure of natural and domesticated populations of Caribbean pine (Pinus caribaea Morelet)

 Isozyme analysis of seed samples derived from natural and managed populations of the tropical pine Pinus caribaea vars ‘bahamensis’ and ‘caribaea’ was used to assess population genetic structure in its native range and to detect changes occurring during early domestication of the species. Baseline data from natural populations of the two varieties showed that populations sampled as seed are characterized by high gene diversity (mean H_e=0.26) and a low level of inbreeding ( mean F_is=0.15). A UPGMA tree of genetic relatedness among populations indicates that the two varieties represent distinct evolutionary units. Within each variety there is significant differentiation among populations, and this is greater for the more fragmented populations of var ‘bahamensis’ (F_st=0.08) than for var ‘caribaea’ (F_st=0.02). Seed from a seed orchard population of var ‘caribaea’ established within its natural range showed no change in genetic diversity but did show a reduced inbreeding coefficient (F_is=0.09) compared with its progenitor populations, suggesting a decrease in selfing and/or biparental inbreeding. A bulked seed sample from an exotic plantation of var ‘bahamensis’ in Australia displayed a large increase in the inbreeding coefficient (F_is=0.324) compared with that found in natural populations, possibly due to elevated self-fertilization. Finally, a bulked seed sample from an exotic plantation population of var ‘caribaea’ from China showed enhanced genetic diversity, an increase in the inbreeding coefficient and more linkage disequilibrium than its presumed progenitor populations. It was also genetically divergent from them. RFLP analysis of chloroplast DNA variation in the Chinese sample suggested that seeds of the related taxa P. elliottii and P. taeda, or seeds derived from hybridization with these taxa growing in the seed production area, had been included in the seed crop during harvesting. We conclude that monitoring of appropriate genetic markers may be an effective means of identifying potentially deleterious genetic changes occurring during forest tree domestication. Received: 10 August 1998 / Accepted: 8 September 1998     

Inbreeding shapes the evolution of marine invertebrates

Inbreeding is a potent evolutionary force shaping the distribution of genetic variation within and among populations of plants and animals. Yet, our understanding of the forces shaping the expression and evolution of nonrandom mating in general, and inbreeding in particular, remains remarkably incomplete. Most research on plant mating systems focuses on self-fertilization and its consequences for automatic selection, inbreeding depression, purging, and reproductive assurance, whereas studies of animal mating systems have often assumed that inbreeding is rare, and that natural selection favors traits that promote outbreeding. Given that many sessile and sedentary marine invertebrates and marine macroalgae share key life history features with seed plants (e.g., low mobility, modular construction, and the release of gametes into the environment), their mating systems may be similar. Here, we show that published estimates of inbreeding coefficients (F_IS) for sessile and sedentary marine organisms are similar and at least as high as noted in terrestrial seed plants. We also found that variation in F_IS within invertebrates is related to the potential to self-fertilize, disperse, and choose mates. The similarity of F_IS for these organismal groups suggests that inbreeding could play a larger role in the evolution of sessile and sedentary marine organisms than is currently recognized. Specifically, associations between traits of marine invertebrates and F_IS suggest that inbreeding could drive evolutionary transitions between hermaphroditism and separate sexes, direct development and multiphasic life cycles, and external and internal fertilization.     

Genetic structure of refugial populations of the temperate plant Shortia rotundifolia (Diapensiaceae) on a subtropical island

Continental island systems harbour relict biota and populations that might have migrated during glacial periods due to the formation of landbridges. Here we analysed the genetic structure of relict populations of the temperate plant Shortia rotundifolia on the subtropical island of Iriomotejima, Japan. This plant, which inhabits riparian environments, is designated “near threatened”. Only five extant populations have been found on the island. Our analyses of 10 nuclear microsatellite loci detected genetic diversity of H _E = 0.488 and H _O = 0.358 for all populations of S. rotundifolia on the island. A high inbreeding coefficient for all populations together (F _IS = 0.316) and each population separately (F _IS = 0.258–0.497) might be attributable to crossing among closely related descendants within a population, an idea that is supported by the relatedness coefficient. These results and an examination of the populations’ demographic histories suggest that the extant populations on Iriomotejima have not experienced a recent population bottleneck. The five extant populations were genetically differentiated (F _ST = 0.283; P < 0.001), suggesting low seed dispersal by gravity and/or low pollen flow via pollinators in the riparian environment. In addition, population differentiation was not related to genetic distance, implying that at one time, ancestral populations might have been distributed over a wider area of the island. However, population fragmentation and range contraction might have occurred at random during the postglacial period.     

Genetic diversity and molecular differentiation of Chinese toad based on microsatellite markers

Genetic diversity and population structure of 9 populations of Bufo gargarizans with total 111 samples in China were assessed using seven microsatellite loci. The analysed microsatellite markers produced 161 alleles, varied from 9 to 38 alleles each locus. The number of alleles per population per locus ranged from 4.43 to 10.29. Polymorphic information content showed that all seven loci were highly informative (mean = 0.810 ± 0.071). The average observed heterozygosity was less than the expected (0.353 ± 0.051 and 0.828 ± 0.067, respectively). All tested populations gave significant departures from Hardy–Weinberg equilibrium. Genetic differentiation among the populations was considerably high with the overall and pairwise F _ST values (mean = 0.160 ± 0.039), and showed fairly high level of inbreeding (indicated by a mean F _IS value of 0.504 ± 0.051) and global heterozygote deficit. In comparison to other amphibian studies; however, our results suggested that the level of genetic structuring in B. gargarizans was relatively low in the geographical scale of the study area. Interestingly, the speculated population bottleneck was found to be absent and the analyses provide only weak evidence for a recent contraction in size even though there was severe inbreeding (indicated by the F _IS value) in the Chinese toad populations.     

Genetic diversity in adult and seedling populations of <Emphasis Type="Italic">Primula vulgaris</Emphasis> in a fragmented agricultural landscape

Habitat fragmentation is known to generally reduce the size of plant populations and increase their isolation, leading to genetic erosion and increased between-population genetic differentiation. In Flanders (northern Belgium) Primula vulgaris is very rare and declining. Populations have incurred strong fragmentation for the last decades and are now restricted to a few highly fragmented areas in an intensively used agricultural landscape. Previous studies showed that small populations of this long-lived perennial herb still maintained high levels of genetic variation and low genetic differentiation. This pattern can either indicate recent gene flow or represent historical variation. Therefore, we used polymorphic microsatellite loci to investigate genetic variation and structure in adult (which may still reflect historical variation) and seedling (recent generation, thus affected by current processes) life stages. The recent generation (seedlings) showed a significant loss of observed heterozygosity (H _o) together with lower expected heterozygosity (H _e), a trend for higher inbreeding levels (F _IS) and higher differentiation (F _ST) between populations compared to the adult generation. This might result from (1) a reduction in effective population size, (2) higher inbreeding levels in the seedlings, (3) a higher survival of heterozygotes over time due to a higher fitness of heterozygotes (heterosis) and/or a lower fitness of homozygotes (inbreeding depression), (4) overlapping generations in the adult life stage, or (5) a lack of establishment of new (inbred) adults from seedlings due to degraded habitat conditions. Combining restoration of both habitat quality and gene flow between populations may be indispensable to ensure a sustainable conservation of fragmented populations.