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.     

Isolation and characterization of 15 polymorphic microsatellite markers for the fig-pollinating wasp,Blastophaga nipponica (Hymenoptera: Agaonidae)

We developed microsatellite markers for the fig-pollinating wasp Blastophaga nipponica Grandi using a dual-suppression-PCR technique. Twenty-one candidates of microsatellite loci were obtained, of which 15 yielded scorable patterns. The degree of polymorphism for the 15 loci was further characterized using summary statistics describing the genetic variation in 60 individuals from three natural populations in Japan. All 15 loci were polymorphic and yielded 2–27 alleles per locus. Overall observed heterozygosity (H _O) and expected heterozygosity (H _E) were 0.465 and 0.631, respectively. As expected, based on the inbreeding tendency of this species, the mean inbreeding coefficient (F _IS) was high (= 0.255). These markers will contribute to studies on the population structure of this species.     

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.     

Genetic Structure of Blastocerus dichotomus Populations in the Paraná River Basin (Brazil) Based on Protein Variability

The population structure of 147 marsh deer (Blastocerus dichotomus) from three areas in the Paraná River basin, Brazil, was studied by observing protein polymorphism at 17 loci. Six loci were polymorphic and 11 monomorphic. The proportion of polymorphic loci (P) was 35.29% and the average heterozygosity (H) was 6.31%. Wright's F_ST indicated that only 4.9% of the total variation in allelic frequencies was due to genetic differences between the three groups. The high value of F_IS (0.246) indicated inbreeding in the marsh deer. Genetic distance values (D = 0.014–0.051) showed little divergence between the three areas. We suggest that probable mechanisms accounting for the genetic structure are female phylopatry and polygyny and also that inbreeding has resulted from decreasing areas of wetland leading to isolation, overhunting, and diseases transmitted by cattle.     

Limited genetic differentiation in Labeo rohita (Hamilton 1822) populations as revealed by microsatellite markers

Labeo rohita, popularly known as rohu is a widely cultured species in the whole Indian subcontinent. Knowledge of the genetic diversity of this species is important to support management and conservation programs which will subsequently help in sustainable production of this species. DNA markers, mostly microsatellite markers are excellent tool to evaluate genetic variation of populations. Genetic variation of three wild and one farm population was assessed using eleven microsatellite loci. In analyzing 192 samples, the number of alleles ranged from 4 to 23; observed heterozygosity 0.500 to 0.870 and expected heterozygosity from 0.389 to 0.878. Exact test for Hardy Weinberg disequilibrium revealed that each riverine sample had at least one locus not in equilibrium except one river. Negative inbreeding coefficients (F_IS) were observed across populations indicating very high level of genetic diversity but little genetic differentiation among populations.     

Breeding system and population genetics of the vulnerable plant Dillwynia tenuifolia (Fabaceae)

Dillwynia tenuifolia Sieber ex DC. is a vulnerable species endemic to the Sydney region in Australia. This investigation focused on the breeding system and genetic structuring of the species in locations encompassing its entire geographical range. A field pollination experiment showed that D. tenuifolia produced fruit from both self‐ and cross‐pollination events, and thus has a facultative breeding system. Fruiting success from self‐ pollination was approximately half that observed from cross‐pollination. The genetic structure of D. tenuifolia populations was investigated by using allozyme electrophoresis. A survey of 16 loci revealed a high level of genetic variation with approximately two‐thirds of the loci being polymorphic and a mean of 1.84 alleles per loci. The genotype frequencies deviated from the Hardy–Weinberg equilibrium, with the observed level of heterozygosity significantly less than expected. The inbreeding coefficient was 0.31. There was very little divergence between populations (F_ST = 0.04) but the fixation indices were high within populations. Spatial autocorrelation, using Moran’s I, showed that neighbouring plants were closely related (I > 0.4). The genetic neighbourhood and effective population size in the species were estimated at one location as 1.1 ha and 10⁴ plants, respectively.     

GENETIC CONSEQUENCES OF OUTCROSSING IN THE CLEISTOGAMOUS ANNUAL,IMPATIENS CAPENSIS. I. POPULATION-GENETIC STRUCTURE

We examined the genetic structure of 11 populations of Impatiens capensis, a cleistogamous annual herb, using starch gel electrophoresis. We sampled both cleistogamous (CL) and chasmogamous (CH) progeny (if present) from maternal parents in each population to infer maternal genotypes and to estimate the extent and pattern of inbreeding within and among populations. Only eight of 31 loci were polymorphic, with one to six (mean = 3.1) loci varying within each population. Mean heterozygosity per individual is quite low (mean = 3.9%) and comparable to highly self-fertilized species. Gene flow is low, and genetic distances do not parallel geographical distances, suggesting a population structure similar to Wright's Island model with drift among the populations. Fixation indexes within populations (f? or F_IS) span the largest range yet reported for a plant species (0.26 to 0.94, mean = 0.57). Further inbreeding results from population substructuring , resulting in a total average inbreeding coefficient (F? or F_IT) of 0.77. Despite these high overall levels of inbreeding, chasmogamy significantly reduces fixation, which may account for the observed greater fitness of CH progeny.     

Interspecific Genetic Variability of the Oyster Mushroom Pleurotus ostreatusAs Revealed by Allozyme Gene Analysis

Allozyme variation in natural populations of basidiomycete fungus Pleurotus ostreatus (88 individuals) from three regions of central Russia was studied. The species was shown to have 92.86% of polymorphic allozyme loci and expected heterozygosity H _e = 0.49. The mean number of alleles per locus was 3.5. The genetic differences among populations were supported by F-statistics (F _ST = 0.750). The low level of inbreeding (F _IS = 0.018) suggests that the P. ostreatus populations are panmictic, and the main reproduction mode involves basidiospores dispersing at long distances. Using cluster analysis, geographically isolated populations and intersterile groups were differentiated within the complex P. ostreatus species.     

INBREEDING AND THE RATE OF SELF-FERTILIZATION IN A GRAPE FERN,BOTRYCHIUM DISSECTUM

Botrychium dissectum is a homosporous fern with bisexual, subterranean gametophytes. Because of these features, B. dissectum would be suspected of displaying a very high frequency of self-fertilization. Sporophytes collected from three populations of this species were assayed for heterozygosity by determining the electrophoretic mobility patterns displayed by two polymorphic enzymes. Extreme deviations from Hardy-Weinberg expectations were observed in each population and analyzed by means of F-statistics. The average inbreeding coefficient was found to be 0.951. A population genetic model is derived that demonstrates that the rate of intragametophytic self-fertilization in homosporous ferns is equal to the inbreeding coefficient calculated from deviations from Hardy-Weinberg expectations. It is therefore concluded that B. dissectum outcrosses about 5% of the time.     

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).     

Analysis of the relationship between allozyme heterozygosity and fitness in the rare Gentiana pneumonanthe L.

Especially for rare species occurring in small populations, which are prone to loss of genetic variation and inbreeding, detailed knowledge of the relationship between heterozygosity and fitness is generally lacking. After reporting on allozyme variation and fitness in relation to population size in the rare plant Gentiana pneumonanthe, we present a more detailed analysis of the association between heterozygosity and individual fitness. The aim of this study was to test whether increased fitness of more heterozygous individuals is explained best by the ‘inbreeding’ hypothesis or by the ‘overdominance’ hypothesis. Individual fitness was measured during 8 months of growth in the greenhouse as the performance for six life-history parameters. PCA reduced these parameters to four main Fitness Components. Individual heterozygosity was scored for seven polymorphic allozyme loci. For some of these loci (e.g. Aat3, Pgm1 and 6Pgdh2) heterozygotes showed a significantly higher relative fitness than homozygotes. To test the inbreeding model, regression analyses were performed between each Fitness Component and the number of heterozygous loci per individual. Multiple regressions with the adaptive distance of five loci as independent variables were used to test the overdominance model. Only the inbreeding model was a statistically significant explanation for the relationship between heterozygosity and fitness in G. pneumonanthe. The number of heterozygous loci was significantly negatively correlated with the coefficients of variation of three of the six initially measured fitness parameters. This suggests a lower developmental stability among more homozygous plants and may explain the higher phenotypic variation in small populations of the species observed earlier. The importance of the results for conservation biology is discussed.     

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     

Isolation and characterization of six microsatellite loci in the drywood termite Cryptotermes secundus (Kalotermitidae)

The Kalotermitidae is a family of lower termites and its species are ideal to study the evolution and maintenance of eusociality within the Isoptera. Co‐dominant genetic markers are an essential tool to elucidate the putatively complex colony and population structure. Six polymorphic microsatellite loci were identified in the drywood termite Cryptotermes secundus (Kalotermitidae). To test the variability of the markers, 18 colonies (n = 18) from Darwin (Australia) were assayed. We found two to eight alleles per locus with the level of observed heterozygosity at each locus ranging from 0.00 to 0.53. The significantly positive F_IT value suggests that some kind of inbreeding does occur in this population.     

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.     

Population genetic structure of the noxious weed Amaranthus retroflexus in Central Europe

Genetic variation was assessed in a range of populations of Amaranthus retroflexus using isoenzyme analysis. Population genetic diversity was measured by evaluating patterns of variation at six putatively neutral isoenzyme loci (comprising 24 putative alleles) within and among 20 populations of A. retroflexus collected in different habitats: ruderal habitats, cereal fields and hop gardens. Amaranthus retroflexus is a noxious weed of North American origin that infests various crops. Overall, A. retroflexus displayed moderate levels of genetic diversity in comparison with other herbaceous plants. The percentage of polymorphic loci was 50.0%, with mean values of 2.01, 0.142 and 0.227 for the average number of alleles per polymorphic locus (A), observed heterozygosity (H_o) and expected heterozygosity (H_e), respectively. A discrepancy between observed and expected heterozygosity and significant differences from H-W expectation indicate that there is an excess of homozygotes in many populations. As a result, there is strong evidence of inbreeding within populations (F_IS = 0.382) and significant population differentiation (F_ST = 0.270). Even though the species is partly autogamous, inbreeding does not lead to strong inbreeding depression resulting from self-pollination, as inbreeding has no effect on the success of the species in today's countryside. Moreover, allele frequencies detected in agricultural habitats (i.e., cereal fields and hop gardens) differed from those detected in populations collected from ruderal habitats, which is probably caused by systematic application of herbicides in agricultural ecosystems.     

Genetic structure in a swarming brown long-eared bat (<Emphasis Type="Italic">Plecotus auritus</Emphasis>) population: evidence for mating at swarming sites

Plecotus auritus, a small, gleaning bat species, lives in small, isolated summer colonies in which both males and females show a high degree of natal philopatry. Despite this, colonies have high gene diversities and low inbreeding coefficients. It has been suggested that inbreeding is avoided because mating occurs during autumnal and spring swarming at hibernation sites. We tested this hypothesis by comparing microsatellite profiles, based on eight loci, of bats from six summer colonies and two swarming sites they were known to visit from radiotelemetry studies. We found high gene diversities (H _s = 0.77) at both swarming sites and summer colonies which were not statistically different. There was no detectable isolation by distance and F_ST was low (0.001). Together, these results suggest high gene flow between sites. Despite this, there was small but significant genetic differentiation amongst summer colonies and between summer colonies and the primary swarming site. We suggest that swarming is important for gene flow and for maintaining genetic diversity in this highly philopatric species and discuss possible reasons for the genetic differentiation observed. The identification and protection of swarming sites should be a major conservation priority for this and other temperate bat species.     

Heterosis at Allozyme Loci under Inbreeding and Crossbreeding in PINUS ATTENUATA

The dependence of heterosis at isozyme loci on inbreeding and crossbreeding was studied in 10-yr-old trees of knobcone pine (Pinus attenuata Lemm.). Heterozygosity was determined at 24 polymorphic isozyme loci and related to the rate of vegetative growth and cone production. The inbreds, created by selfpollination, had 46% of the heterozygosity of their mothers; the crossbreds, created by interpopulation crossing, had 155% of the heterozygosity of their mothers. Within the crossbreds, heterozygosity was positively correlated with trunk growth, but negatively correlated with cone production. Results in the crossbreds, however, were strongly influenced by a few individuals that showed unusually slow growth, high reproduction and low heterozygosity. Without those individuals, there was no relationship of heterozygosity to either growth or reproduction.—Within the inbreds, heterozygosity was positively correlated with both trunk growth and cone production. Each locus that was heterozygous in the mothers was calculated to mark about 3% of the genome for identity by descent in the inbred progeny; the total proportion of the genome marked was between 10 and 11%. Using these estimates to relate heterozygosity to the inbreeding coefficient (F) gave estimates of inbreeding depression per unit of F that fell within the range of published values for conifers. The strength of heterosis found among the inbreds suggests that single-locus or multilocus overdominance should be exceedingly difficult to detect in natural populations of predominantly outcrossing species.     

Genetic divergence between populations of two closely related troglobitic beetle species (Speonomus: Bathysciinae,Coleoptera)

Genetic variability and divergence at 13 enzyme loci were studied in two species of troglobitic beetles (Speonomus zophosinus andhydrophilus 12 populations) collected from the Pyrenees mountains. These allopatric species exhibit a high degree of specialization to underground life and a very small geographical range. Four diagnostic loci arePgi, Est-2, Pac-1, Phi. Within each species polymorphic loci exhibited marked spatial variation of allele frequencies, sometimes over short distances (5–10 km). The levels of genetic variability are comparable to those observed in non-cave invertebrates (frequency of polymorphic loci: 0.31; average expected heterozygosity: 0.112±0.015). Moreover, a single species showed variability in the average level of heterozygosity (0.06 to 0.172). Patterns of genetic differentiation among species were also studied; mean genetic distance (D: 0.263±0.010) between the twoSpeonomus species was of the same order of magnitude as most data reported in the literature between species. A significant heterozygote deficiency occurs in local populations and for all loci. This deficit does not vary from locus to locus. Several hypotheses were examined in an attempt to account for these observations in connection with the species biology. This deficit probably is due to the breeding structure of the population (inbreeding and assortative mating) coupled with limited dispersal ability.     

Genetic diversity and conservation of Ipomoea microdactyla (Convolvulaceae): an endemic vine from the Bahamas,Cuba, and southeastern Florida

Ipomoea microdactyla Griseb. (Convolvulaceae) is restricted to the Bahamian archipelago, Cuba, and southeastern Florida. The species is listed as a state endangered species in Florida, where it is mostly restricted to the hyperfragmented pine rockland of Miami‐Dade County. Using seven DNA microsatellite loci, we assessed levels of genetic diversity for 12 populations of this species from Andros Island in the Bahamas (six sites), Cuba (one site), and Florida (five sites). We found significantly greater mean numbers of alleles, and higher mean values for both observed and expected heterozygosity in populations from the continuous forest on Andros than those from the habitat fragments in Florida. It is unknown if these patterns of genetic diversity in the Florida populations are the result of habitat fragmentation or founder effects. The population from Cuba exhibited relatively high levels of genetic variation, suggesting that this island is a major center of diversity and dispersal for this species. It appears that hybrid introgression for I. carolina alleles within I. microdactyla individuals occurred at a single site on Andros Island. Overall, the mean inbreeding coefficient value was 0.089, suggesting low levels of inbreeding. The highest inbreeding coefficient values were mostly recorded in Florida. Two groups were revealed, one containing the populations from Florida, and the second one encompassing those from the Bahamas and Cuba. Our results highlight the negative genetic consequences of habitat fragmentation and support initiatives recently established to establish corridors to connect the remnants of the pine forest of the Miami‐Dade County.     

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.