Strong isolation by distance among local populations of an endangered butterfly species (Euphydryas aurinia)

The marsh fritillary (Euphydryas aurinia) is a critically endangered butterfly species in Denmark known to be particularly vulnerable to habitat fragmentation due to its poor dispersal capacity. We identified and genotyped 318 novel SNP loci across 273 individuals obtained from 10 small and fragmented populations in Denmark using a genotyping‐by‐sequencing (GBS) approach to investigate its population genetic structure. Our results showed clear genetic substructuring and highly significant population differentiation based on genetic divergence (F _ST) among the 10 populations. The populations clustered in three overall clusters, and due to further substructuring among these, it was possible to clearly distinguish six clusters in total. We found highly significant deviations from Hardy–Weinberg equilibrium due to heterozygote deficiency within every population investigated, which indicates substructuring and/or inbreeding (due to mating among closely related individuals). The stringent filtering procedure that we have applied to our genotype quality could have overestimated the heterozygote deficiency and the degree of substructuring of our clusters but is allowing relative comparisons of the genetic parameters among clusters. Genetic divergence increased significantly with geographic distance, suggesting limited gene flow at spatial scales comparable to the dispersal distance of individual butterflies and strong isolation by distance. Altogether, our results clearly indicate that the marsh fritillary populations are genetically isolated. Further, our results highlight that the relevant spatial scale for conservation of rare, low mobile species may be smaller than previously anticipated.     

gesp: A computer program for modelling genetic effective population size,inbreeding and divergence in substructured populations

The genetically effective population size (N_e) is of key importance for quantifying rates of inbreeding and genetic drift and is often used in conservation management to set targets for genetic viability. The concept was developed for single, isolated populations and the mathematical means for analysing the expected N_e in complex, subdivided populations have previously not been available. We recently developed such analytical theory and central parts of that work have now been incorporated into a freely available software tool presented here. gesp (Genetic Effective population size, inbreeding and divergence in Substructured Populations) is R‐based and designed to model short‐ and long‐term patterns of genetic differentiation and effective population size of subdivided populations. The algorithms performed by gesp allow exact computation of global and local inbreeding and eigenvalue effective population size, predictions of genetic divergence among populations (G_ST) as well as departures from random mating (F_IS, F_IT) while varying (i) subpopulation census and effective size, separately or including trend of the global population size, (ii) rate and direction of migration between all pairs of subpopulations, (iii) degree of relatedness and divergence among subpopulations, (iv) ploidy (haploid or diploid) and (v) degree of selfing. Here, we describe gesp and exemplify its use in conservation genetics modelling.     

Population Genetic Structure of Pacific White Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>) from Mexico to Panama: Microsatellite DNA Variation

Genetic variation and population structure of wild white shrimp (Litopenaeus vannamei) from 4 geographic locations from Mexico to Panama were investigated using 5 microsatellite DNA loci. The genetic diversity between populations was indicated by the mean number of alleles per locus and mean observed heterozygosity, which ranged from 7.4 to 8.6 and from 0.241 to 0.388, respectively. Significant departures from Hardy-Weinberg equilibrium were found at most locations at each locus, with the exception Guatemala at Pvan0013, and were caused by high heterozygote deficiencies. Genetic differences between localities were detected by pairwise comparison based on allelic and genotypic frequencies, with the exception of locus Pvan1003. Significant pairwise F _ST values between locations and total F _ST showed that the white shrimp population is structured into subpopulations. However, population differentiation does not follow an isolation-by-distance model. Knowledge of the genetic diversity and structure of L.vannamei populations will be of interest for aquaculture and fisheries management to utilize and preserve aquatic biodiversity.     

Calculations of population differentiation based on GST and D: forget GST but not all of statistics!

G _ST‐values and its relatives (F_ST) belong to the most used parameters to define genetic differences between populations. Originally, they were developed for allozymes with very low number of alleles. Using highly polymorphic microsatellite markers it was often puzzling that G_ST‐values were very low but statistically significant. In their papers, Jost (2008) and Hedrick (2005) explained that G_ST‐values do not show genetic differentiation, and Jost suggested calculating D‐values instead. Theoretical mathematical considerations are often difficult to follow; therefore, we chose an applied approach comparing two artificial populations with different number of alleles at equal frequencies and known genetic divergence. Our results show that even for more than one allele per population G_ST‐values do not calculate population differentiation correctly; in contrast, D‐values do reflect the genetic differentiation indicating that data based on G_ST‐values need to be re‐evaluated. In our approach, statistical evaluations remained similar. We provide information about the impact of different sample sizes on D‐values in relation to number of alleles and genetic divergence.     

Genetic variation and population structure ofCarex breviculmis (Cyperaceae) in Korea

We determined the genetic diversity and population structures ofCarex breviculmis (Cyperaceae) populations in Korea, using genetic variations at 23 allozyme loci.C. breviculmis is a long-lived herbaceous species that is widely distributed in eastern Asia. A high level of genetic variation was found in 15 populations. Twelve enzymes revealed 23 loci, of which 11 were polymorphic (47.8%). Genetic diversity at the speciesand population levels were 0.174 and 0.146, respectively. Total genetic diversity (H_T = 0.363) and within-population genetic diversity (H_s = 0.346) were high, whereas the extent of the population divergence was relatively low (G_ST = 0.063). Deviation from random mating (Fis) within the 15 populations was 0.206. An indirect estimate of the number of migrants per generation(Nm = 3.69) indicated that gene flow was extensive among Korean populations of this species. Analysis of fixation indices revealed a substantial heterozygote deficiency in some populations and at some loci. Genetic identity between popu-lations was high, exceeding 0.956.     

Genetic Diversity and Population Structure of Alnus hirsuta (Betulaceae) in Korea

Alnus hirsuta in Korea was measured to estimate the amount and pattern of genetic diversity and population structure. The mean genetic diversity within populations was 0.166. Korean alder populations have slightly high levels of genetic diversity compared to those of two Canadian alder species. The genetic differentiation among populations accounted for 9% of the total variation. The rate of gene flow was estimated high (Nm=2.63). Analysis of inbreeding coefficient, calculated for all polymorphic loci in each population, showed a substantial heterozygote deficiency relative to Hardy-Weinberg expectations. The mean G _ST value of A. hirsuta in Korea was 0.087. The low value of G _ST in this species, reflecting little spatial genetic differentiation, may indicate extensive gene flow. A relationship between the mean heterozygosity and annual rainfall showed a positive relationship (r ²=0.54, F=4.67). Received 8 August 1998/ Accepted in revised form 7 July 1999     

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.     

Quantifying population structure on short timescales

Quantifying the contribution of the various processes that influence population genetic structure is important, but difficult. One of the reasons is that no single measure appropriately quantifies all aspects of genetic structure. An increasing number of studies is analysing population structure using the statistic D, which measures genetic differentiation, next to G_ST, which quantifies the standardized variance in allele frequencies among populations. Few studies have evaluated which statistic is most appropriate in particular situations. In this study, we evaluated which index is more suitable in quantifying postglacial divergence between three‐spined stickleback (Gasterosteus aculeatus) populations from Western Europe. Population structure on this short timescale (10 000 generations) is probably shaped by colonization history, followed by migration and drift. Using microsatellite markers and anticipating that D and G_ST might have different capacities to reveal these processes, we evaluated population structure at two levels: (i) between lowland and upland populations, aiming to infer historical processes; and (ii) among upland populations, aiming to quantify contemporary processes. In the first case, only D revealed clear clusters of populations, putatively indicative of population ancestry. In the second case, only G_ST was indicative for the balance between migration and drift. Simulations of colonization and subsequent divergence in a hierarchical stepping stone model confirmed this discrepancy, which becomes particularly strong for markers with moderate to high mutation rates. We conclude that on short timescales, and across strong clines in population size and connectivity, D is useful to infer colonization history, whereas G_ST is sensitive to more recent demographic events.     

Population Genetic structure ofPotentilla fragariodes var.major (Rosaceae) in Korea

The genetic diversity and population structure of eighteenPotentilla fragariodes var.major (Rosaceae) populations in Korea were determined using genetic variations at 22 allozyme loci. The percent of polymorphic loci within the enzymes was 66.7%. Genetic diversity at the species level and at the population level was high (Hes = 0.203; Hep = 0.185, respectively), whereas the extent of the population divergence was relatively low (G_ST = 0.069). F_IS, a measure of the deviation from random mating within the 18 populations, was 0.075. An indirect estimate of the number of migrants per generation (Nm = 3.36) indicated that gene flow was high among Korean populations of the species. In addition, analysis of fixation indices revealed a slight heterozygote deficiency in some populations and at some loci. Wide geographic ranges, perennial herbaceous nature and the persistence of multiple generations are associated with the high level of genetic variation. AlthoughP. fragariodes var.major usually propagated by asexually-produced ramets, we could not rule out the possibility that sexual reproduction occurred at a low rate because each ramet may produce terminal flowers. Mean genetic identity between populations was 0.983. It is highly probable that directional movement toward genetic uniformity in a relatively homogeneous habitat operates among Korean populations ofP. fragariodes var.major.     

Comparative allozyme genetics and range history of the European river barbel (Teleostei,Cyprinidae: Barbus barbus) in the Rhine/upper Danube contact area1

Enzymes, representing 23 genetic loci, were studied in n = 320 European river barbels (Barbus barbus L. 1758) from the contact zone of the Rhine and the upper Danube. Barbel revealed the highest heterozygosity estimate (H_e = 0.137) of 10 sympatric freshwater fishes (range H_e = 0.017–0.106). The geographical genetic subdivision in barbel (G_ST = 0.1690) surpassed the respective estimates in two other cyprinid species (G_ST ≤ 0.1415) and in three anadromous fishes (range G_ST = 0.0330–0.1290) from the same study area, but it fell short of the spatial genetic fragmentation of two resident salmonid species (G_ST ≥ 0.2678) and of sculpins (G_ST = 0.8489). The theoretical gene flow in the unobstructed Danube was N_em^DAN = 4.98, and across the weirs in the southern Rhine N_em^RHE = 1.84. The Rhine/Danube watershed exceeded any other isolation effect on the gene pool despite a low genetic distance (D^RHE/DAN = 0.0321) among Rhenish and Danubian samples. The range history in central Europe might imply the colonization of the study area from a Danubian and from a Rhenish or a Rhôdanian refuge. The concluded admixture of eastern and western barbels has produced a weakly pronounced cline of increasing allozymic affinity of Rhenish to Danubian barbels in the southern Rhine system. Oberschwaben has served as an important colonization pathway in the phylogeographical exchange between the Danube and west Europe. The influence of stocking for fisheries on the allozymes is not known precisely, but it appears to be of minor importance.     

Population genetic structure of the common warthog (Phacochoerus africanus) in Uganda: evidence for a strong philopatry among warthogs and social structure breakdown in a disturbed population

Fine‐scale genetic structure of large mammals is rarely analysed. Yet it is potentially important in estimating gene flow between the now fragmented wildlife habitats and in predicting re‐colonization following local extinction events. In this study, we examined the extent to which warthog populations from five localities in Uganda are genetically structured using both mitochondrial control region sequence and microsatellite allele length variation. Four of the localities (Queen Elizabeth, Murchison Falls, Lake Mburo and Kidepo Valley) are national parks with relatively good wildlife protection practices and the other (Luwero), not a protected area, is characterized by a great deal of hunting. In the total sample, significant genetic differentiation was observed at both the mtDNA locus (F_ST = 0.68; P < 0.001) and the microsatellite loci (F_ST = 0.14; P < 0.001). Despite the relatively short geographical distances between populations, significant genetic differentiation was observed in all pair‐wise population comparisons at the two marker sets (mtDNA F_ST = 0.21–0.79, P < 0.001; microsatellite F_ST = 0.074–0.191, P < 0.001). Significant heterozygote deficiency was observed at most loci within protected areas while no significant deviation from Hardy–Weinberg expectation was observed in the unprotected Luwero population. We explain these results in terms of: (i) a strong philopatry among warthogs, (ii) a Wahlund effect resulting from the sampling regime and (iii) break down of social structure in the disturbed Luwero population.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

Heterozygote excess through life history stages in Cestrum miradorense Francey (Solanaceae), an endemic shrub in a fragmented cloud forest habitat

Comparisons of genetic diversity and population genetic structure among different life history stages provide important information on the effect of the different forces and micro‐evolutionary processes that mould diversity and genetic structure after fragmentation. Here we assessed genetic diversity and population genetic structure using 32 allozymic loci in adults, seeds, seedlings and juveniles of eight populations of the micro‐endemic shrub Cestrum miradorense in a highly fragmented cloud forest in central–eastern Mexico. We expected that due to its long history or rarity, this species may have endured the negative effects of fragmentation and would show moderate to high levels of genetic diversity. High genetic diversity (H_e = 0.445 ± 0.03), heterozygote excess (F_IT = ?0.478 ± 0.034, F_IS = ?0.578 ± 0.023) and low population differentiation (F_ST = 0.064 ± 0.011) were found. Seeds had higher genetic diversity (H_e = 0.467 ± 0.05) than the later stages (overall mean for adults, seedlings and juveniles H_e = 0.438 ± 0.08). High gene flow was observed despite the fact that the fragmentation process began more than 100 years ago. We conclude that the high genetic diversity was the result of natural selection, which favours heterozygote excess in all stages, coupled with a combination of a reproductive system and seed/pollen dispersal mechanisms that favour gene flow.     

Indicators to assess temporal genetic diversity in the French Catalogue: no losses for maize and peas

The aim of this study, led by the GEVES (Research and Control Group for Varieties and Seeds), was to suggest indicators to assess the diversity available to farmers since the French Official Catalogue for Plant Varieties and Species was initiated. The largest datasets of 1990 inbred maize lines and 578 pea lines from the last 50 years were analysed using morphological and enzymatic parameters. Lines were grouped into three to five periods. Genetic diversity was estimated in each period from morphological and enzymatic markers by computing numerous indices, such as the number of classes of scores for each characteristic, allelic richness or genetic diversity index (H _e ). Population differentiation parameters (G_ST, G_ST′, F_ST, Q_ST) were also estimated between periods. While genetic diversity computed from distinction, uniformity, stability traits was more marked for maize (0.66) than for garden peas (0.35) or feed peas (0.29), the opposite trend was observed with enzymes, resulting in a genetic diversity of 0.43, 0.35 and 0.22 for garden peas, feed peas and maize, respectively. However, no significant changes in genetic diversity were observed over time, and genetic differentiation was slight between periods. All our results demonstrated that no significant reduction in the diversity available to farmers had been observed since initiation of the French Catalogue. The H _e was a good indicator providing a quantitative estimate of genetic diversity, but it should be interpreted alongside a more precise indicator such as allelic richness or the number of classes for morphological characteristics.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Population structure and pattern of geographic variation in Muscari comosum along its range of distribution

Genetic variation for six loci in 37 populations of Muscari comosum L. (Liliaeeae) is surveyed. One locus is monomorphic and identical in all the populations. The remaining loci are polymorphic. Although the GOT-1 and GOT-3 loci show a pronounced heterozygote deficit explained by selection acting upon these loci (or on genes linked to them), the remaining loci nearly conform to Hardy-Weinberg proportions. The overall pattern shows a low level of heterozygote deficit (F_IS=0.08) explained by the mixed mating system. The organization of genetic variation shows a low level of interpopulation differentiation (F_ST or G_ST=0.04). At the same time, autocorrelation analysis shows no pattern of geographical variation. It is concluded that gene flow and selection interact to produce the overall pattern of genetic variation.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.     

GENETIC VARIATION IN BROMUS TECTORUM (POACEAE): POPULATION DIFFERENTIATION IN ITS NORTH AMERICAN RANGE

Allelic variation in seedlings from 60 North American populations of the alien annual grass Bromus tectorum was determined at 25 loci using starch gel electrophoresis. Populations were collected from four regions; east of the Rocky Mountains, Nevada and California, the Intermountain West, and British Columbia. Compared to other diploid seed plants, genetic variation within these populations of B. tectorum is low: 4.60% of loci are polymorphic per population, with an average of 1.05 alleles per locus and a mean expected heterozygosity of 0.012. Although 2,141 individuals were analyzed, no heterozygous individuals were detected, and consequently, mean observed heterozygosity is 0.000. Extensive deviations from Hardy-Weinberg expectations were observed at every polymorphic locus due to heterozygote deficiencies. The mean genetic identity (Nei's I) between population pairs was 0.980 and indicates a high level of overall genetic similarity among populations. The among-population component of the total gene diversity is high (G_ST ⁼ 0.478), indicating substantial genetic differentiation among populations. These results are consistent with previous reports for highly self-pollinating plants of low genetic variation and substantial genetic differentiation among populations. Despite the lack of genetic variation as measured by enzyme electrophoresis, this weedy grass has become exceedingly abundant in a diverse array of arid environments throughout much of western North America, perhaps due to phenotypic plasticity.     

General <Emphasis Type="Italic">G</Emphasis><Subscript>ST</Subscript> and <Emphasis Type="Italic">θ</Emphasis> inflation due to biased intra-population sampling,and its consequences for the conservation of the Canarian Flora

While knowledge of the degree of inter-population genetic differentiation underlies the understanding of microevolutionary processes in any organism, its calculation through G _ST, F _ST, or θ (which, unlike the previous two, was designed to correct for unequal and small sample sizes) is often based in severely restricted intra-population samples, which are nonetheless tacitly assumed adequate to their accurate estimation. Empirical assessment of the influence of the number and intra-population distribution of samples on the values of G _ST and θ for several Canarian endemic plants compellingly suggests that (1) contrary to expectations based on simulated datasets, θ does not account for empirical sampling bias better than G _ST; (2) sample sizes being equal, collections scattered across each population’s occupancy area entail significantly lower over-estimates of G _ST and θ than if they only consider one of the population extremes, especially in narrow allogamous taxa with small populations; (3) in small samples, a scattered sampling strategy is significantly less sensitive to G _ST inflation than sampling in one of the population extremes; and (4) a software-related component of bias should be considered when pooling values of G _ST from different studies to calculate averages. Thus, unlike the sampling methods used for many plant endemics from the Canaries and other regions, collections for a reliable estimation of inter-population differentiation using molecular markers should encompass the whole occupancy area of each population, and include a higher proportion of individuals respect to the total size in narrow endemics than in widespread congeners. Critically, the high average allozyme inter-population differentiation reported for the Canarian endemic Flora is possibly an over-estimate, and could be explained predominantly by the generally biased intra-population sampling associated with G _ST estimates, rather than by specific factors of insularity that restrict gene flow radically, as it has been hitherto assumed.     

Does GST underestimate genetic differentiation from marker data?

The widely applied genetic differentiation statistics F_ST and G_ST have recently been criticized for underestimating differentiation when applied to highly polymorphic markers such as microsatellites. New statistics claimed to be unaffected by marker polymorphisms have been proposed and advocated to replace the traditional F_ST and G_ST. This study shows that G_ST gives accurate estimates and underestimates of differentiation when demographic factors are more and less important than mutations, respectively. In the former case, all markers, regardless of diversity (H_S), have the same G_ST value in expectation and thus give replicated estimates of differentiation. In the latter case, markers of higher H_S have lower G_ST values, resulting in a negative, roughly linear correlation between G_ST and H_S across loci. I propose that the correlation coefficient between G_ST and H_S across loci, r_GH, can be used to distinguish the two cases and to detect mutational effects on G_ST. A highly negative and significant r_GH, when coupled with highly variable G_ST values among loci, would reveal that marker G_ST values are affected substantially by mutations and marker diversity, underestimate population differentiation, and are not comparable among studies, species and markers. Simulated and empirical data sets are used to check the power and statistical behaviour, and to demonstrate the usefulness of the correlation analysis.