Evaluation of the extent of genetic variation in mahoganies (Meliaceae) using RAPD markers

Despite the economic importance of mahoganies (Meliaceae) little is known of the pattern of genetic variation within this family of tropical trees. We describe the application of a polymerase chain reaction (PCR)-based polymorphic DNA assay procedure random amplified polymorphic DNAs (RAPDs) to assess the extent of genetic variation between eight mahogany species from four genera. Pronounced genetic differentiation was found between the species and genera. There was a clear separation of Cedrela odorata from the other species, with 95% of the variable amplification products differing, whereas Lovoa trichilioides, Khaya spp. and Swietenia spp. were more closely grouped. These results are consistent with the current taxonomic viewpoint. A number of markers were found to be diagnostic for particular species, which could be of value in determining the status of putative hybrids. The application of RAPDs to the study of genetic variation in mahoganies is discussed in the context of developing genetic conservation and improvement strategies for these species.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Farmers Drive Genetic Diversity of Thai Purple Rice (Oryza sativa L.) Landraces

Purple or black rice (Oryza sativa L.) is a culturally important germplasm in Asia with a long history of cultivation in northern Thailand. Purple rice is identified by the color of the rice pericarp, which varies from purple to black with the accumulation of phenolic acids, flavonoids, and anthocyanins. In the present study, we assessed molecular variation within and between wetland purple rice landraces germplasm from northern and northeastern Thailand using 12 microsatellite loci. All purple rice varieties surveyed showed high levels of homozygosity within varieties and strong genetic differentiation among varieties, indicating the fixation of genetic differences among them. This pattern is consistent with purple rice farming practices in northern Thailand, where a small portion of harvested seed is selected and replanted based on farmers’ preferences. The reduced genetic diversity and high homozygosity observed for purple rice is also consistent with patterns expected for this inbreeding crop. Genetic differentiation among the varieties showed some degree of structuring based on their geographical origin. Taken together, these data highlight that the genetic diversity and structure of wetland purple rice landraces is shaped by farmer utilization and cultivation through local cultural practices, and that conservation should focus on ex situ conservation across its cultivation range, along with on-farm, in situ conservation based on farmers’ seed-saving practices. In situ conservation may prove especially valuable for preserving the genetic identity of local varieties and promote adaptation to local environments.

     

Low genetic diversity as revealed by SPAR methods possibly leads to extinction of two critically-endangered and endemic species of Mantisia

Mantisia spathulata Schult. and M. wengeri Fischer, two critically-endangered, endemic and rare species of the genus Mantisia (Zingiberaceae), have been rediscovered from Lunglei province of Mizoram, India, after two decades. For sustainable conservation and utilization of the Mantisia species, in vitro seed and clonal propagation methods have been developed earlier by our research group and plantlets have been reintroduced to their natural habitat for species recovery. To comprehend the plausible reasons for endemism and endangeredness of both the species at DNA level, they were analyzed to assess natural genetic variation using three different polymerase chain reaction (PCR) based DNA markers viz. random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and directed amplification of minisatellite DNA regions (DAMD), both individually and cumulatively, which are popularly regarded as single primer amplification reaction (SPAR) methods. A total of 107 primers belonging to three SPARs are used which collectively endow low genetic variation (15 and 20 %, respectively) in both M. spathulata and M. wengeri. The use and efficacy of SPAR methods to reveal the natural genetic variation in Mantisia species at intra-specific level has been recorded for the first time. To impede the extinction risk of these two species of genus Mantisia, large scale conservation strategies including in situ and ex situ conservation are recommended.     

Genetic diversity and similarity revealed via molecular analysis among and within an in situ population and ex situ accessions of chiltepín (Capsicum annuum var. glabriusculum)

Molecular analysis of genetic diversity amongand within phenotypically similar wild Capsicum annuum var. glabriusculum(chile) populations revealed geneticdifferences among accessions spread over abroad geographic range. These chiles areregionally known as chiltepíns and are a 50metric ton per year wild harvest for the spiceindustry, as well as a genetic resource forcrop improvement. Understanding geneticvariability in this species providesinformation related to conservation efforts. The objective of this research was to surveygenetic diversity among and within an insitu population and ex situ accessionsof chiltepíns. Random AmplifiedPolymorphic DNA (RAPD) molecular markers wereused to study the genetic structure of an in situ population found at the nothernmostrange of this species and ex situaccessions collected from Mexico and Guatemala. Novel genetic variation was found in both thein situ northern disjunct population, aswell as some ex situ accessions, thussupporting conservation of this species viaboth in situ and ex situ strategies The evidence presented here supports effortsto conserve outlier populations via insitu management practices.     

Rapid genetic differentiation between ex situ and their in situ source populations: an example of the endangered Silene otites (Caryophyllaceae)

Ex situ cultivation in botanic gardens could be one possibility to preserve plant species diversity and genetic variation. However, old ex situ populations are often sparsely documented. We were able to retrieve three different ex situ populations and their source in situ populations of the endangered plant species Silene otites after 20–36 years of isolation. Furthermore, three additional wild populations were included in the analysis. Population genetic diversity and differentiation were analysed using AFLP markers. Genetic variation in the ex situ populations was lower than the variation found in the in situ populations. Strong differentiation (F_ST = 0.21–0.36) between corresponding in situ and ex situ populations was observed. Bayesian clustering approach also showed a distinct genetic separation between in situ and ex situ populations. The high genetic differentiation and loss of genetic diversity during spatial and temporal isolation in the ex situ populations can be attributable to small population sizes and unconscious selection during cultivation. Therefore, adequate sampling prior to ex situ cultivation and large effective population sizes are important to preserve genetic diversity. Near‐natural cultivation allowing for generation overlap and interspecific competition without artificial selection is recommended as being best for the maintenance of the genetic constitution. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, ??, ??–??.     

Evolutionary history of two endemic Appalachian conifers revealed using microsatellite markers

Fraser fir (Abies fraseri [Pursh] Poir.) and intermediate fir (Abies balsamea [L.] Mill. var. phanerolepis Fern.) exist in small populations in the Appalachian highlands of the southeastern United States. We used ten nuclear microsatellite markers to quantify genetic variation within Fraser fir and intermediate fir, and to examine their evolutionary relationships with the widespread balsam fir (Abies balsamea [L.] Mill.). We found little genetic differentiation among these taxa, suggesting that Fraser fir might best be classified as a variety of balsam fir. The results further appear to reject the hypothesis that intermediate fir was of hybrid origin between two comparatively distantly related species. Low levels of genetic diversity suggest that intermediate fir and Fraser fir have undergone at least some genetic degradation since post-Pleistocene isolation. The results may prove important for in situ and ex situ gene conservation efforts for Fraser fir and intermediate fir, which are imperiled by an exotic insect and by global climate change.     

Conservation of threatened relict trees through living <Emphasis Type="Italic">ex situ</Emphasis> collections: lessons from the global survey of the genus <Emphasis Type="Italic">Zelkova</Emphasis> (Ulmaceae)

Maintaining living ex situ collections is one of the key conservation methods in botanic gardens worldwide. Despite of the existence of many other conservation approaches used nowadays, it offers for many endangered plants an important insurance policy for the future, especially for rare and threatened relict trees. The aim of this research was to investigate the global extent of living ex situ collections, to assess and discuss their viability and inform the development of conservation approaches that respond to latest global conservation challenges. We used as a model taxon the tree genus Zelkova (Ulmaceae). The genus includes six prominent Tertiary relict trees which survived the last glaciation in disjunct and isolated refugial regions. Our comprehensive worldwide survey shows that the majority of botanic institutions with Zelkova collections are in countries with a strong horticultural tradition and not in locations of their origin. More importantly, the acutely threatened Zelkova species are not the most represented in collections, and thus safeguarded through ex situ conservation. Less than 20% of the ex situ collections surveyed contain plant material of known wild provenance while the majority (90%) of collections are generally very small (1–10 trees). Botanic gardens and arboreta particularly in regions where iconic relict trees naturally occur should play a vital role in the conservation of these species. The coordination of conservation efforts between gardens has to be enhanced to prioritise action for the most threatened relict trees. Large scale genetic studies should be undertaken, ideally at genus level, in order to verify or clarify the provenance of ex situ collections of relict trees in cultivation. For the most threatened relict tree genera, well-coordinated specialist groups should be created.     

Maintenance of Phenotypic and Genotypic Diversity in Managed Populations of Stenocereus Stellatus (Cactaceae) by Indigenous Peoples in Central Mexico

The columnar cactus Stenocereus stellatus is used in Central Mexico for its edible fruits which are harvested in wild, managed in situ and cultivated populations. Management in situ of wild populations is conducted by selectively sparing and enhancing the abundance of plants with desirable phenotypes when fields are cleared for agricultural use. Cultivation of desirable phenotypes is carried out by vegetative propagation in homegardens. Effects of human management on morphological and genetic variation of S. stellatus were analyzed by comparing morphological diversity indices (MD, based on Simpson’s index) and expected (H_e) heterozygosity indices from allozyme analysis, in wild, managed in situ, and cultivated populations from La Mixteca and the Tehuacán Valley regions. Morphological diversity was similar among regions, but populations from the wetter La Mixteca region averaged higher genetic variation (H_e = 0.279) than populations from Tehuacán (H_e = 0.265). On average, populations manipulated by people had higher levels of variation (MD = 0.479 ± 0.012, H_e = 0.289 in cultivated populations; MD = 0.461 ± 0.014, H_e = 0.270 in managed in situ populations) than wild populations (MD = 0.408 ± 0.017, H_e = 0.253), which is apparently due to a continual introduction and replacement of plant materials in the manipulated populations. The results illustrate that human management may not only maintain but also increase both morphological and genetic diversity of manipulated plant populations in relation to that existing in the wild. Managed in situ and cultivated populations of S. stellatus are important reservoirs of variation, and are crucial for the general maintenance of diversity in wild populations. These populations may play a principal role in designing strategies for the conservation of variation of this cactus.     

Ex situ cultivation affects genetic structure and diversity in arable plants

Worldwide, botanical gardens cultivate around 80,000 taxa, corresponding to approximately one‐quarter of all vascular plants. Most cultivated taxa are, however, held in a small number of collections, and mostly only in small populations. Lack of genetic exchange and stochastic processes in small populations make them susceptible to detrimental genetic effects, which should be most severe in annual species, as sowing cycles are often short. In order to assess whether ex situ cultivation affects genetic diversity of annuals, five annual arable species with similar breeding systems were assessed with 42 in situ populations being compared to 20 ex situ populations using a random amplified polymorphic DNA (RAPD) analysis approach. Population sizes tended to be lower under ex situ cultivation and levels of genetic diversity also tended to be lower in four of the five species, with differences being significant in only two. Ex situ populations showed incomplete representation of alleles found in the wild. The duration of cultivation did not indicate any effect on genetic diversity. This implies that cultivation strategies resulted in different genetic structures in the garden populations. Although not unequivocally pronounced, differences nonetheless imply that conservation strategies in the involved gardens may need improvement. One option is cold storage of seeds, a practice that is not currently followed in the studied ex situ collections. This may reflect that the respective gardens focus on displaying living plant populations.     

Genetic spatial clustering: significant implications for conservation of wild soybean (Glycine soja: Fabaceae)

Knowledge of spatial patterns of genetic variation within populations of wild relative species has significant implications with respect to sampling strategies for ex situ and in situ conservation. To study spatial genetic structure of wild soybean (Glycine soja Sieb. et Zucc.) at the fine scale, three natural populations in northern China were analyzed using inter-simple sequence repeat (ISSR) fingerprints for estimating kinship coefficients. A regression analysis of kinship coefficients against spatial distances revealed that individuals occurring close together tended to be more genetically related. The Sp statistic further indicated a comparable spatial pattern among the three wild soybean populations with similar Sp values (mean = 0.0734, varied from 0.0645 to 0.0943) detected across the three populations. Genetic patches were on average ca. 20 m in size, and the effective neighborhood sizes varied between 10 and 15 m. The spatial genetic structure evident in the wild soybean populations may be attributed to the restricted seed dispersal and predominant inbreeding mating system of this species. The detection of family structure in the populations of wild soybean has a significant implication for the effective conservation of the important genetic resources.     

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.     

Life‐history traits and geographical divergence in wild rice (Oryza rufipogon) gene pool in Indochina Peninsula region

Indochina Peninsula is the primary centre of diversity of rice and lies partly in the centre of origin of cultivated rice (Oryza sativa) where the wild ancestor (Oryza rufipogon) is still abundant. The wild gene pool is potentially endangered by urbanisation and the expansion of agriculture, and by introgression hybridisation with locally cultivated rice varieties. To determine genetic diversity and structure of the wild rice of the region we genotyped nearly 1000 individuals using 20 microsatellite loci. We found ecological differentiation in 48 populations, distinguishable by their life‐history traits and the country of origin. Geographical divergence was suggested by isolation of the perennial Myanmar populations from those of Cambodia, Laos and Thailand. The annual types would be most likely to have lost genetic variation because of genetic drift and inbreeding. The growing of cultivated and wild rice together, however, gives ample opportunities for hybridisation, which already shows signs of genetic mixing, and will ultimately lead to replacement of the original wild rice gene pool. For conservation we suggest that wild rice should be conserved ex situ in order to prevent introgression from cultivated rice, along with in situ conservation in individual countries for the recurrent evolutionary process through local adaptation, but with sufficient isolation from cultivated rice fields to preserve genetic integrity of the wild populations.     

Adaptations to Captivity in the Butterfly Pieris brassicae (L.) and the Implications for Ex situ Conservation

Captive breeding has been suggested as a method of conserving many threatened vertebrates, and is increasingly being proposed as a valuable conservation strategy for invertebrates. Potential genetic problems associated with ex situ conservation are widely recognized, but a further issue has received less attention: the possibility that populations will undergo adaptation to the captive environment, rendering them less well adapted to survival in the wild. We investigated six traits related to dispersal and reproduction in a culture of the large white butterfly Pieris brassicae (L.), that had been captive for c. 100–150 generations, and in recently wild stock reared simultaneously in a common environment. Individuals in the captive culture were heavier, with smaller wings and lower wing aspect ratios. Females from the captive culture laid many more eggs in cage experiments, and had higher ovary mass at the time of peak egg production. These differences are consistent with adaptation to captive conditions. Over time, similar evolutionary changes may affect invertebrates reared in ex situ conservation programmes, decreasing the likelihood that these species can be re-established in the wild. Although the timescale over which most vertebrates are likely to adapt to captivity is longer, and the traits involved will be different, invertebrates like P. brassicae may also provide a model of potential problems in long-term ex situ conservation programmes for both invertebrates and vertebrates. We suggest that measures to reduce or slow adaptation to captivity should be introduced alongside measures to reduce deleterious genetic effects in captive populations.     

Comparison of genetic variation in populations of wild rice, Oryza rufipogon, plants and their soil seed banks

Wild rice, Oryza rufipogon, has endangered species conservation status and it is subject to in situ conservation in China. To understand the potential of the seed bank in species conservation and population restoration, this study compared the genetic diversity of O. rufipogon plants with that of its soil seed banks in two marshes. A total of 11 pairs of rice SSR primers were used and 9 were polymorphic. Allele frequencies of the seeds differed significantly from those of surface plants and varied between soil layers. Relatively more alleles and higher genetic diversity (H _e) were found in plant populations, relative to seed banks. The numbers of germinable seeds and the level of genetic variation in seed banks decreased with the increasing of soil depth, indicating a rapid seed loss. Genetic differentiation was detected between sites and between plant and seed populations, as well as among seeds of different soil strata. Rapid seed loss, partly dormancy loss, and nonrandom seed mortality are discussed as the possible contributors to the pattern of reduced genetic variation within seed banks, compared to plants. These could also be responsible for the considerable genetic differentiation between populations. The seed population held about 72% of the total genetic variation of O. rufipogon in each marsh, indicating the potential of seed banks for restoring population variabilities if the plant populations were lost.     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.     

Using genetic structure data and phylogenetic criteria in attributing prioritization scores for conservation of spontaneous Capparis spinosa L. populations from Tunisia

In Tunisia, the increasing pharmacological demand for spontaneous capers is actually leading to progressive forest overexploitation and genetic erosion of the species that is actually represented by scattered populations. Therefore, inventories with emphasis on the characterization, management and conservation of this medicinal plant are becoming a great need.In this work, genetic and phylogenetic criteria based on isozyme markers were used for the prioritization of spontaneous Capparis spinosa L. populations for conservation according to Vane-Wright and collaborators taxic method.Enzymatic loci varied in their ability to detect variation and population analyses indicated that most of the variation is partitioned between rather than within populations. Fst, gene flow values, PCA and cluster analysis also revealed significant differentiation between all populations.When combing phylogenetic and genetic criteria we were able to prioritize some of the populations for conservation. Therefore, the standardization and the summatory of all indices indicated that the populations Nebeur, Nahli and Dyr rank as a priority for conservation.So far, results of the present study appear to justify the in situ conservation strategy, where core areas completely free from perturbation would be defined for the populations with the highest priority for conservation, with the populations presenting unique alleles. This would guarantee the maintenance of most of the species’ genetic variation. The ex situ conservation management strategy would imply the setting up of specialized nurseries and national collections that are still missing in the country to counteract population fragmentation, demographic loss and genetic erosion threatening such spontaneous populations.     

ISSR Analysis of the Genetic Diversity of the Endangered Species Sinopodophyllum hexandrum （Royle） Ying from Western Sichuan Province, China

Slnopodophyllum hexandrum （Royle） Ying Is an Important medicinal and endangered species. Inter-simple sequence repeats （ISSR） analysis was conducted on seven natural populations from western Slchuan Province to Investigate the genetic diversity of S. hexandrum. Leaf samples of 140 Individuals were collected. Of the 139 discernible fragments generated by 12 selected primers （among 100 primers）, 54 appeared to be polymorphlc. The percentage of polymorphlc bands （PPB） was 38.85% at the species level, and PPB within a population ranged from 7.91% to 23.74%. Low levels of genetic variation （He = 0.092, Ho = 0.142） and high levels of genetic differentiation among the populations （Gst= 62.25%） was detected on the basis of results from POPGENE and analysis of molecular variance （AMOVA）, respectively. Furthermore, the limited gene flow （Nm = 0.361） may result from biological characteristics, such as self-pollination and short distance seed dispersal. Based on the genetic and ecological Information available for S. hexandrum, we propose some appropriate strategies for the conservation of the endangered medicinal species in this region, namely rescuing and conserving the core populations for in situ conservation and sampling and preserving more populations with fewer Individuals from each population for ex situ conservation.     

Genetic footprints of habitat fragmentation in the extant populations of Sinojackia (Styracaceae): implications for conservation

Sinojackia, a member of the family Styracaceae, is an endangered genus endemic to China. The number of populations and population size of Sinojackia have decreased sharply because of habitat fragmentation and destruction. We studied the genetic diversity of extant populations in two different cohorts (adult and seedling) using eight microsatellite markers to investigate the genetic footprints of habitat fragmentation in four recognized Sinojackia spp. and to develop appropriate conservation measures. Data on intrapopulational genetic diversity suggest that Sinojackia populations have maintained relatively high levels of genetic diversity and low levels of genetic differentiation despite severe fragmentation. The high genetic diversity may be explained by the outcrossing mating system and high longevity of Sinojackia spp. The amount of genetic variation is not associated with population size, which was also supported by bottleneck analysis. In the species studied, there was no significant difference in the genetic diversity between the two cohorts analysed. However, inbreeding increased from adult trees to seedling populations, suggesting that the higher proportion of biparental inbreeding in the recent generations of seedlings is the result of restricted current genetic flow caused by habitat fragmentation. Average seed set per population was not significantly correlated with either population size or genetic diversity. Conservation management should aim to monitor inbreeding and outbreeding depression carefully to ensure the in situ and ex situ conservation of Sinojackia spp. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ?? , ??–??.