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.     

Effects of inbreeding on the magnitude of inbreeding depression in a highly self-fertilizing tree, Magnolia obovata

Inbreeding depression is one of the major selective forces driving the evolution of mating systems. Previous theories predict that long-lived plants will show a negative correlation between inbreeding depression and the level of inbreeding (as determined by an inbreeding coefficient) at maturity, but the extent of this correlation may vary among life stages because of variation in the genetic basis for inbreeding depression at different stages. To test this prediction, I used electrophoretic allozyme analysis and pollination experiments to examine the fixation index (F _is) at maturity and inbreeding depression in the early and late life stages of two populations with different outcrossing rates of a highly self-fertilizing tree, Magnolia obovata. The magnitude of inbreeding depression for early survival (δ _e) in an outcrossing population (t _m = 0.51; F _is = −0.015) was higher (δ _e = 0.97) than that in an inbreeding population (t _m = 0.18; F _is = 0.15; δ _e = 0.38). From these results, I estimated that both populations exhibited high inbreeding depression for late survival (δ _l) (0.94 in the outcrossing population and 0.93 in the inbreeding one) and lifetime survival (δ _t) (0.99 and 0.96, respectively). My results and previously published data demonstrate the predicted relationship between inbreeding depression and the level of inbreeding for early survival, but not for late survival. This suggests that there is a differential genetic basis for inbreeding depression at different life stages. The inbreeding depression for late survival appears to play a central role in the maintenance of reproductive traits that promote outcrossing in M. obovata.     

Low levels of genetic variation inPhenakospermum guyannense (Strelitziaceae), a widespread bat-pollinated Amazonian herb

Phenakospermum guyannense is a monotypic, arborescent, long-lived monocot that is widespread in Amazonian South America. This outcrossing species is pollinated primarily by phyllostomid bats. Given these life-history characteristics,P. guyannense is expected to exhibit high levels of genetic variation and gene flow. We used isozyme electrophoresis and randomly amplified polymorphic DNA (RAPD) to characterize genetic variation in populations ofP. guyannense from French Guiana. Both measures detected a surprisingly low level of genetic variation, with only five out of twenty (25%) allozyme loci polymorphic (P), 1.35 alleles per locus (A), and an expected heterozygosity (H_e) of 0.090 at the species level. Isozymic genetic variation was even lower within populations (P = 17.5, A = 1.24, H_e = 0.074), and was corroborated by a RAPD assay that used 26 arbitrary primers (P = 3.61, A = 1.04, H_e = 0.014). Although overall levels of variation were low, the detectable variation was distributed as would be expected for an outcrossing species with extensive gene flow (mean G_ST = 0.230). We suspect thatP. guyannense is depauperate in genetic variation because of a series of bottlenecks that affected the species over this portion of its range.     

Floral trait variation in fourCyclamen (Primulaceae) species

The purpose of this study is to examine floral trait variation between fourCyclamen species that show variation in their ability to reproduce in the absence of pollinators and in levels of inbreeding. Pollen and ovule production, pollen/ovule ratio, pollen volume, petal length and width, diameter of the corolla mouth, pistil and stamen length, and stigma-anther separation varied significantly between the four study species. Flower, pollen and ovule production, pollen volume and corolla size were generally highest inC. hederifolium, the species with the lowest level of inbreeding (mean F_is = 0.329), intermediate in both species with relatively high levels of pollinator-mediated inbreeding,C. repandum (mean F_is = 0.658) andC. creticum (mean F_is = 0.748), and lowest in the highly inbredC. balearicum (mean F_is = 0.930). The two species with the most different inbreeding coefficients,C. hederifolium andC. balearicum, had lower pollen-ovule ratios and shorter longevities of stigma receptivity and pollen viability thanC. creticum andC. repandum. These patterns of variation in floral traits are discussed in relation to the relatedness, pollination ecology and levels of inbreeding of the four species.     

Genetic variation and population structure in Korean populations of sand dune speciesSalsola komarovi (Chenopodiaceae)

Salsola komarovi lljin is a herbaceous annual native to the sand dunes and beaches of Japan, northern China, Sakhalln and Korea. Starch-gel electrophoresis was conducted on leaves and stems collected from 300 plants in eight Korean populations. The mean number of alleles per locus (A _p=1.51), mean expected heterozygosity (He _p=0.116), and total genetic diversity (H _T=0.279) were comparable with those for species with similar life history and ecological traits. A general conformance of genotype frequencies to Hardy-Weinberg expectations (meanF _IS=−0.030) indicates thatS. komarovi is an outcrossing species. Slightly more than 20% of the genetic variation was found among populations (F _ST=0.204). In addition, significant differences in allele frequency were detected between populations at all 11 polymorphic loci (P<0.001). Nei's genetic identities range from 0.885 to 0.985 with a mean of 0.942. However, indirect estimates of the number of migrant per generation (0.97, calculated fromF _ST and 0.31, calculated from seven private alleles) indicate that the levels of gene flow is low among Korean populations. Although the species maintains a moderate level of genetic variation within populations, the small, isolated natural populations of the species have been severely destructed by human activities, particularly in summer season. If this is true, conservation efforts should be focused on those populations that currently maintain the most genetic diversity (e.g., populations of Cheju Island and coast of the southwestern Korean Peninsula).     

Allozyme variation in the diploid (A genome) populations ofScilla scilloides (Hyacinthaceae)

Allozyme variation at eleven loci encoding seven enzyme systems were examined in 20 populations of diploid (genome AA, 2n = 16)Scilla scilloides in China. In comparison with the average species of seed plants studied, populations of this species display a high amount of genetic variation (A = 2.0, P = 58.6%, H_o = 0.172, and H_e = 0.185). Allozyme variation pattern revealed predominant outcrossing within populations and considerable differentiation (F_ST = 0.314) among populations as well as between the subtropic and temperate regions. The wide distribution, long existence and outcrossing are presumably the main factors responsible for the high genetic diversity within populations. But the gravity dispersal of seeds and pollination by small insects set limits to the increase of genetic variation within populations and promote differentiation between populations and regions. In addition, allozyme variation does not distinguishS. scilloides var.albo-viridis and suggests that subtropic populations may be considered as a genetic entity.     

Microsatellite markers reveal spatial genetic structure of Tetranychus urticae (Acari: Tetranychidae) populations along a latitudinal gradient in Europe

The genetic structure of populations of the two-spotted spider mite Tetranychus urticae was investigated along a south–north European transect spanning from southern France to The Netherlands. Mites were collected on Urtica dioica in 6 sampling zones. Microsatellite variation at 5 loci revealed considerable genetic variation with an average heterozygozity of 0.49. Significant heterozygote deficiency was found in 7 populations out of the 18 samples analyzed and one of them was completely monomorphic. Tetranychus urticae populations show some level of genetic structuring. First, genetic differentiation between localities (F _ST estimates) was significant for all comparisons. Second, the analysis of molecular variance, AMOVA, indicates that there is an effect, albeit low (9%), of the locality in accounting for allele frequency variance. Geographic distance emerges as a factor responsible for this genetic structure. The results are discussed in relation to the biological features of the species and the known patterns of migration. Related agronomical issues are addressed.     

Pattern of genetic differentiation in two Isophya species (Orthoptera: Tettigonoidea) in north-east Hungary

Allozyme polymorphism was studied in two populations of Isophya kraussi and Isophya stysi. Both species are flightless and have low dispersal ability. As a consequence, we expected high level of genetic differentiation among their local populations. Samples were collected in three regions of Hungary. Enzyme polymorphism was investigated at 10 loci (Aldox, Est, Got, Gpdh, Hk, Idh, Mdh, Me, Pgi and Pgm) in both species. High levels of polymorphism were detected in all samples. Gpdh proved to be diagnostic as there were no common alleles in the two species. At four further loci (Got, Hk, Mdh and Me), the two species had one common allele together with one or more differentiating alleles. We detected high F _IT values implying a high level of genetic variation. The positive F _IS values suggested a tendency of heterozygote deficiency in both species. The highly significant overall F _ST values indicated clear genetic differentiation among the local populations. Thus our results confirmed the taxonomic status of these two species. The dendrogram constructed on the basis of Nei's genetic distances and the results of the PCA analyses fully confirmed those obtained by F-statistics.     

Population genomics study for the conservation management of the endangered shrub Abeliophyllum distichum

Natural monuments are IUCN Category III protected areas that play an important role in biodiversity conservation as they provide species refuge and allow species migration. Despite their status, natural monuments are often confined to cultural and fragmented landscapes due to anthropogenic land-use demands. In this population genomic study, we surveyed 11 populations of the endemic shrub Abeliophyllum distichum Nakai (Oleaceae), including five natural monument habitats, covering its range-wide distribution in South Korea. Using 2,254 SNPs as markers, our results showed a mean expected heterozygosity (He) of 0.319, with populations in the central distribution showing significantly higher He than those at the periphery. There was no significant heterozygote deficiency and inbreeding among studied populations overall (F_{IS =} ?0.098), except for a single natural monument population (GS-NM147). Population structure and differentiation was moderate to high (F_ST?=?0.196), while recent gene flow between populations appeared weak, which can be attributed to the fragmented distribution and the outcrossing mating system of the heterostylous plant. Based on these findings, we provide suggestions for the population conservation and management of this endangered species.

     

Allozyme polymorphisms, outerossing rates, and hybridization of South AmericanNothofagus

Electrophoretically detectable genetic variation was used to describe the genetic structure of three South American species ofNothofagus: the widespreadN. betuloides andN. dombeyi, and the geographically restrictedN. nitida. Although the widespread species possess higher levels of genetic variation, the three species have more genetic variation within than among populations. These results are consistent with the theoretical expectations for woody, presumably highly outerossed species with wind-borne seeds.Estimates of outcrossing rates from progeny arrays yielded slightly higher average t-values forN. nitida (1.158) andN. dombeyi (range 0.873–1.045) than forN. betuloide (0.878). Hierarchical analysis of population structure revealed values of F_IS and FIT that were positive and significantly different from zero at most loci and for each species. The levels of inbreeding detected by F-statistics indicate some degree of self-fertilization and/or population substructuring into discrete family groups. Reduced seed vagility and regeneration of natural stands after disturbance by a few remnant individuals would probably generate the recruitment of related seedlings underneath parent trees.The analysis of a putative hybrid population betweenN. nitida andN. betuloides indicated that individuals clearly segregated intonitida-like orbetuloides- like individuals. The reduced outcrossing rate ofbetuloides-like individuals from the hybrid site (t=0.585) is interpreted in concert with low pollen availability and the increased probability of selfing and/or hybridization inNothofagus.     

BREEDING SYSTEM OF THE FERN DRYOPTERIS EXPANSA: EVIDENCE FOR MIXED MATING

The levels and distribution of genetic variation within and among populations of the homosporous fern Dryopteris expansa were assessed using enzyme electrophoresis. Twelve loci representing six enzymes were examined in 502 sporophytes from nine populations. Values of P, H, and mean number of alleles per locus are much lower in D. expansa compared to values for other fern species. Values of F varied from –0.014 to 0.745 with an average F of 0.335. There was also considerable population to population variation in F. In three populations the observed heterozygosity closely approximated that expected at Hardy-Weinberg equilibrium, whereas in the remaining populations observed heterozygosity was significantly lower than the expected heterozygosity. In D. expansa the major component of F_IT is F_IS; the relatively high F_ST value suggests a high degree of interpopulational differentiation. Electrophoretic data suggest that this species possesses a mixed mating system. This study, in conjunction with other investigations, indicates that just as in angiosperms, a variety of breeding systems operates in homosporous ferns, ranging from extreme inbreeding, through mixed mating to outcrossing.     

Patterns of allozyme diversity in several selected rare species in Korea and implications for conservation

Because some endemic plants appear to be adapted to a narrow setof environmental conditions with limited genetic diversity, an analysis ofpopulation genetic structure is necessary to fully evaluate the impact of rarityon genetic variation. Listed as endangered species in Korea, only fewpopulations of Abeliophyllum distichum, Leonticemicrorhyncha, Bupleurum euphorbioides, andBerchemia berchmiaefolia were found. A reduced level ofgenetic variation in B. berchemiaefolia is consistent withthe occurrence of a genetic bottleneck and inbreeding. Leonticemicrorhyncha differed dramatically from other taxa in its observed level of geneticvariation, probably due to its predominant selfing. The level ofallozyme variation maintained by A. distichum was high forendemic species. Compared to species with similar traits, A.distichum maintained a relatively higher genetic diversity, probably dueto floral heteromorphism and preferred outcrossing. Bupleurumeuphorbioides maintained a higher genetic diversity due to outcrossing,but at the individual locus, deficiency of heterozygosity prevailed. Probablyinbreeding between local neighborhoods was frequent because A.distichum and B. euphorbioides were pollinatedby small fly species which might be less effective as a pollen dispersal, andtheir visits were extremely scarce and controlled by the weather conditions.Since much of the species-to-species variation in genetic diversity is due tothe specific ecological and evolutionary history of a species, any managementplan developed should be based on historical changes in the population size anddistribution to better predict the amounts and patterns of genetic diversity.     

Genetic consequences of fragmentation in “arbor vitae,” eastern white cedar (Thuja occidentalis L.), toward the northern limit of its distribution range

We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive F_is values and heterozygote deficiency were observed in populations from the marginal (F_is = 0.244; P_HW = 0.0042) and discontinuous zones (F_is = 0.166; P_HW = 0.0042). However, populations from the continuous zone were in HW equilibrium (F_is = −0.007; P_HW = 0.3625). There were no significant latitudinal effects on gene diversity (H_s), allelic richness (AR), or population differentiation (F_st). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.     

The influence of self‐fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression

The genetic structure, selfing rate and inbreeding depression of the hermaphroditic freshwater snail Physa acuta were jointly analysed in a population near Montpellier, France. Allozymic markers revealed moderate gene diversity (0.138), and no heterozygote deficiency. The mean outcrossing rate, estimated by using progeny arrays, was 0.9, with substantial variation among families. This also suggests that the number of fathers among outcrossed offspring of a given mother is low. Inbreeding depression was estimated over more than one generation using 83 first‐laboratory‐generation (G₁) families. The main parameters measured were parental (G₁) fecundity, offspring (G₂) survival and fecundity. Size and growth were also monitored. Parental fecundity was analysed under several conditions (isolation, pair and quadruplet outcrossing). The self‐fertilization depression, including parental fecundity, offspring survival and fecundity, was about 0.9 at the population level. The genetic data obtained in the same population indicate a value of about 0.3 using Ritland’s (1990) technique, suggesting that the depression over the whole life‐cyle might be even higher than 0.9. Grouping affected neither fecundity nor self‐fertilization depression. Substantial variation in depression for survival was detected among individuals, from no survival in some selfed families to better survival than that of outbred families in others. The overall result (outbred population structure, high outcrossing rate and high self‐fertilization depression) is consistent with what is expected in large outcrossing populations in which inbreeding depression is maintained by mutation‐selection balance.     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Genetic structure of a rare European conifer, Serbian spruce (Picea omorika (Panč.) Purk.)

Genetic variation in 13 populations of a Balkan endemic, Serbian spruce (Picea omorika (Panč.) Purk.), was investigated using 16 isozyme loci. Serbian spruce is characterized by low levels of genetic variation (average proportion of polymorphic loci was 20.9% and average expected heterozygosity was 0.067). In most populations, a significant surplus of heterozygotes was observed, indicating a strong selection against inbreds. In the largest populations, fixation indices were positive, probably due to within-population differentiation and the associated Wahlund effect. Despite a strong overall differentiation (F_ST=0.261), no geographical trends in the genetic variation could be identified. Genetic drift caused by small effective population sizes and a strong fragmentation is the most plausible explanation for such variation patterns. A Bayesian analysis of population structure revealed the existence of two clusters, which are supposed to be possible remnants of ancient differentiation within a large range of the predecessor of Serbian spruce, Picea omoricoides Weber. Ex situ conservation measures, namely establishing seed orchards from trees of mixed origin, are proposed as a necessary complement to in situ protection of natural stands.     

Development of 15 polymorphic microsatellite loci from <Emphasis Type="Italic">Garrulax morrisonianus</Emphasis> (Timaliidae), an endemic avian species of Taiwan

Garrulax morrisonianus, an endemic avian species of Taiwan, inhabits evergreen forests at high elevations of 2,000–3,952 m. In this study, we developed 15 microsatellite primer pairs for genetic study. These markers were screened for 52 samples collected from wild populations of different geographical regions. The number of alleles ranged from 4 to 13. The expected (H _E) and observed (H _O) heterozygosities were 0.430–0.725 and 0.000–0.500, respectively. All loci were significantly deviated from Hardy-Weinberg equilibrium due to the heterozygote deficiency. The authors Tzen-Yuh Chiang, Sao-Cheng Lao, Ya-Fu Lee contributed to the study equally.     

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.     

Allozyme variations in Michelia formosana (Kanehira) Masamune(Magnoliaceae), and the inference of a glacial refugium in Taiwan

  Michelia formosana is distributed islandwide in Taiwan and also occurs in the Ryukyu Archipelago, Japan. Allozyme genetic variability in M. formosana (Magnoliaceae) was investigated using five polymorphic loci (Pgm-1, Idh-1, Mr-1, Skdh-1, and Ppo-3) from five enzyme systems. The average value of expected heterozygosity (H_e) describing within-population variation was 0.241. The overall F_is (0.0736) indicates a significant deficiency of heterozygotes at the population level. This positive value of F_is is mainly contributed from populations found at Pinglin, Chienshi, Wufeng, and the Ryukyus, all located at northern latitudes, and is also caused by locus Ppo-3. Among-population variation, F_st, accounted for 10.6% of the total heterozygosity and deviates significantly from zero. This populational differentiation agrees well with that of general tropical woody species outcrossed by animals. The population on Lanyu (Orchid Island) has some morphological differences from the plants native to Taiwan, but this was not reflected in the cluster analysis using the unweighted pair group method. Pgm-1d, however, is the diagnostic characteristic for the plants growing on Lanyu as it is entirely absent from all other populations of M. formosana. About 18% of the alleles of this study show clinal geographical variation and were found to be significantly related to latitudinal gradients throughout the species range. This, however, is not a linear relation but a curve with a peak form which is exactly the same as the relationship between expected heterozygosities of populations and latitude. This observation suggests that Nantou County, in central Taiwan could be a glacial refugium for genetic diversity of M. formosana. This inference is further supported by analysis of published studies on seven widely spread plant species in Taiwan. Location of this glacial refugium is probably related to the first emergence of the Central Mountain Ridge millions of years ago that naturally becamed the major window to receive genetic diversity from the Asian mainland. Received: 16 April 2000 / Accepted: 12 May 2000