Long-term population isolation in the endangered tropical tree species Caesalpinia echinata Lam. revealed by chloroplast microsatellites

Habitat fragmentation represents the single most serious threat to the survival of tropical ecosystems. In formulating strategies to counteract the detrimental effects of fragmentation, knowledge of the levels and patterns of genetic diversity within and between natural populations is vital to the establishment of any conservation programme. We utilized polymorphic chloroplast microsatellite markers to analyse genetic diversity in populations of the endangered tropical tree Caesalpinia echinata Lam. representing the entire extant range of the species. Levels of within-population diversity were low, with only two of seven populations studied displaying any variation. The vast majority of the genetic variation was partitioned between geographical regions (36%) and between populations within regions (55%). These levels of genetic structuring, coupled with a calculated pollen-to-seed flow ratio of approximately 6.7:1, suggest that there has been little gene flow between the three major geographical regions over an extended period. Thus, the current tripartite distribution of the species is more consistent with the existence of separate glacial refugia, rather than reflecting any anthropogenic effects.     

Small edge populations at risk: genetic diversity of the green lizard (<Emphasis Type="Italic">Lacerta viridis viridis</Emphasis>) in Germany and implications for conservation management

Edge and central populations can show great differences regarding their genetic variation and thereby also in their probability of extinction. This fact might be of great importance for the conservation strategies of endangered species. In this study we examine the level of microsatellite variability within three threatened edge populations of the green lizard subspecies Lacerta viridis viridis (Laur.) in Brandenburg (Germany) and compare the observed variation to other edge and central populations within the northern species range. We demonstrate that the northernmost edge populations contain less genetic variation in comparison to the central population. However, there were no observable significant differences to the other edge population included in this study. Surprisingly, we observed a high genetic differentiation in a small geographical range between the three endangered populations in Brandenburg, which can be explained by processes like fragmentation, isolation, genetic drift and small individual numbers within these populations. We also detected unique genetic variants (alleles), which only occurred in these populations, despite a low overall genetic variation. This study demonstrates the potential of fast evolving markers assessing the genetic status of endangered populations with a high resolution. It also illustrates the need for a comparative analysis of different regions within the species range, achieving a more exact interpretation of the genetic variation in endangered populations. This will aid future management decisions in the conservation of genetic diversity in threatened species.     

High RAPD but no cpDNA sequence variation in the endemic and endangered plant, Heptacodium miconioides Rehd. (Caprifoliaceae)

Heptacodium miconioides Rehd. is an endangered species endemic to China and has suffered rapid decrease of distribution range and population size. This species has been disappeared in central China where the modal specimen was collected. We analyzed the genetic variation of the remaining populations to reveal whether the genetic diversity also suffered decrease and to provide some suggestions for conservation. All the nine known remaining populations were sampled. Genetic variation was analyzed based on RAPD markers and two fragments of cpDNA sequence, intergenic spacers of petG-trnP and trnS-trnG. No variation was observed in the two fragments of cpDNA sequence. However, the species exhibited high level of RAPD variation compared to other threatened or rare plants. Measures of genetic diversity within populations were strongly related to the log of estimated population size, indicating that large populations usually have more genetic diversity than that of small ones. About 25% of the variation was partitioned among populations. Significant relationship was observed between differentiation and geographical distance, indicating a pattern of isolation-by-distance. Given for few populations remaining, all the populations should be protected and urgent efforts be paid on the small populations to avoid their local extinction.     

Genetic variation in the Afromontane tree Prunus africana,an endangered medicinal species

An understanding of the patterns of variation within and among populations of tropical trees is essential for devising optimum genetic management strategies for their conservation and sustainable utilization. Here, random amplified polymorphic DNA (RAPD) analysis was used to partition variation within and among 10 populations of the endangered Afromontane medicinal tree, Prunus africana, sampled from five countries across the geographical range of the species (Cameroon, Ethiopia, Kenya, Madagascar and Uganda). Analysis of molecular variance ( AMOVA ) employed 48 RAPD markers and revealed most variation among countries (66%, P < 0.001). However, variation among individuals within populations and among populations within Cameroon and Madagascar was also highly significant. Analysis of population product frequency data indicated Ugandan material to be more similar to populations from Cameroon than populations from Kenya and Ethiopia, while Malagash populations were most distinct. The implications of these findings for determining appropriate approaches for conservation of the species, particularly in Cameroon and Madagascar, are discussed.     

Promiscuity in populations of the cushion plant Saxifraga oppositifolia in the Swiss Alps as inferred from random amplified polymorphic DNA (RAPD)

Overviews on patterns of genetic variation within and among plant populations show that widespread, outcrossing species should have a high proportion of the total genetic variation within populations and a low proportion among populations, which results in little population differentiation. However, in Alpine areas, large–scale distribution barriers as well as small-scale habitat heterogeneity could lead to geographical and temporal isolation, respectively. We investigated the genetic variation of Saxifraga oppositifolia from 10 populations of the Alps in southeastern Switzerland using random amplified polymorphic DNA (RAPD). Based on the banding patterns of four RAPD primers, 84 polymorphic markers identified all 189 sampled individuals as being genetically different. The genetic variation was mainly found within populations (95%), whereas less than 5% was found among populations and among regions. Analyses of molecular variance ( AMOVA ) suggested that population differentiation was highly significant. However, grouping populations differently into regions did not appear to result in a clear correspondence of genetic and geographical relatedness. Genetic variation did not significantly differ between populations of two elevational levels. This coincides with results of former pollination experiments that revealed a breeding system of S. oppositifolia which remains the same irrespective of the elevation. We assume that the high outcrossing rate, rare clonal reproduction, and some long-distance dispersal even among topographically separated populations are the crucial determinants for the pattern of genetic variation found in the investigated area.     

Conservation genetics of endangeredBrasenia schreberi based on RAPD and AFLP markers

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θ^B) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (N_em), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoringB. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.     

Population genetic variation in rare and endangered Iliamna (Malvaceae) in Virginia

Random amplified polymorphic DNA (RAPD) markers were used as input for an analysis of molecular variance (AMOVA), homogeneity of molecular variance analysis (HOMOVA), and cluster analysis to describe the population genetic structure of Iliamna corei, a federally endangered plant located only in Virginia, and I. remota , a rare plant in Virginia, Indiana, and Illinois. The analysis was performed to help clarify the taxonomic relationship between the two closely related species. We analysed four clones in the only known population of I. corei , breeding stock derived from seeds originating from the population site, and three I. remota populations in Virginia. Eighty-five percent of screened primers revealed DNA polymorphisms in Iliamna. Ninety-nine informative markers were generated using seven primers. No significant statistical differences (at P = 0.05) in RAPD variation was found between species (24% of variance) using the AMOVA procedure. However, within species/among populations (31 % of the variance) and within populations (45% of the variance) there were significant differences (P < 0.002). An unweighted paired group method using arithmetic averages (UPGMA) cluster analysis showed the federally endangered I. corei population to be genetically distinct from the apparently recently introduced (in Virginia: ∼ 100 ybp) I. remota. The lack of significant differences from the AMOVA and the high number shared bands between I. corei and I. remota suggest that I. corei may be more appropriately classified as a subspecies of I. remota. Iliamna corei plants in the natural population were genetically similar to one another while the I. corei breeding stock plants and I. remota plants were genetically relatively diverse.     

High levels of genetic structuring as a result of population fragmentation in the tropical tree species <Emphasis Type="Italic">Caesalpinia echinata</Emphasis> Lam.

We have investigated levels of genetic diversity within and among seven remnant populations of Caesalpinia echinata Lam., an endangered species found as fragmented populations in three major areas around the coastal regions of Brazil. Using amplified fragment length polymorphism (AFLP) genetic markers, we detected levels of within-population genetic diversity ranging from 0.092 to 0.163, with the lowest values generally being found in the smallest populations. Estimates of between-population genetic differentiation were strongly correlated with geographical distance (r = 0.884, p < 0.001), which,along with a neighbour-joining phylogenetic analysis, strongly suggested high levels of genetic isolation by distance. Over half (62%) of the total genetic diversity was partitioned between populations, further highlighting the genetic distinctness of individual populations. Taken together, these results suggest that fragmentation has led to an increase in population differentiation between fragments of C. echinata. These formations will be of great value in the development of conservation plans for species exhibiting high levels of genetic differentiation due to fragmentation, such as indication of conservation unit size, which populations should be chosen as priority in conservation plans and which samples should be introduced in areas with a low number of individuals of brazilwood.     

Genetic Diversity and Population Structure of Yellow Camellia (Camellia nitidissima) in China as Revealed by RAPD and AFLP Markers

Camellia nitidissima, a rare plant but a useful genetic resource for commercial cultivation of ornamental camellias, is distributed in a narrow region of South China and North Vietnam. In this study, RAPD and AFLP markers were used to assess the genetic diversity and population structure of six natural populations of C. nitidissima from Guangxi in South China. Twenty RAPD primers amplified 183 bands, of which 143 bands were polymorphic, and 8 AFLP primer pairs produced 502 bands, of which 364 were polymorphic. Independent as well as combined analyses of the cluster analyses of the RAPD and AFLP fragments showed that the six populations could be classified into two major genetic groups corresponding to the Nanning and Fangcheng areas. The Mantel test revealed significant correlation between the genetic and geographic distances of C. nitidissima populations (r = 0.953, p = 0.036). AMOVA analysis allowed the partitioning of the genetic variation between groups (36.09%), among populations within groups (25.78%), and within populations (38.14%). An understanding of both the genetic diversity and the population structure of C. nitidissima in China can also provide insight into the conservation and management of this endangered species.     

Patterns of genetic variation in native grape phylloxera on two sympatric host species

Random amplified polymorphic DNA (RAPD) markers were used to examine population genetic structure in populations of native grape phylloxera. This research asked: (i) do RAPD markers distinguish two groups corresponding to the two host plant species; and (ii) do RAPD markers distinguish groups according to spatial location, independent of host plant association? Forty‐nine phylloxera clones were collected from five pairs of adjacent individuals of two sympatric grape species in five sites along a 145 km transect in Missouri, USA. A high level of polymorphism was observed, with some evidence for structuring between host plant species and no evidence for spatial structuring. An analysis of molecular variance (amova ) found that 6.52% of the variance in RAPD banding patterns was attributable to host species and 7.96% of the variance was attributable to spatial location. A cluster analysis did not result in two groups corresponding to the two hosts, or to five groups corresponding to the geographical sites sampled. A Mantel test showed a low correlation between genetic similarity and spatial location. Two of the 93 RAPD markers were nonrandomly associated between the hosts. It is suggested that there may be a small host‐mediated effect on genetic variation but stochastic dispersal and a highly heterogeneous environment may be the primary influences on the observed polymorphism.     

Genetic diversity and structure of natural populations of Plathymenia reticulata (Mimosoideae), a tropical tree from the Brazilian Cerrado

Plathymenia reticulata is a tropical tree native to the Brazilian Cerrado, one of the most important and endangered ecosystems in Brazil. This species presents high-quality wood and potential for recovery of degraded areas. Despite its importance, almost nothing is known about its genetic or ecological features. Random amplified polymorphic DNA (RAPD) markers were used to investigate the genetic diversity and structure of six natural populations of P. reticulata. DNAs from 117 adult individuals were amplified with 10 random primers and Shannon's index and amova were used to evaluate the levels of genetic diversity within and among populations. Through 72 markers, 70.8% of which were polymorphic, it was possible to obtain 117 unique RAPD phenotypes. The levels of genetic variability found in the six populations of P. reticulata were considerable and most of the genetic variation was found between individuals within populations, although pairwise PH(ST) values indicated significant divergence between populations. The among-population component accounted for, respectively, 12.3% and 16% of the genetic variation, according to amova and Shannon's index. These results were compared with other genetic studies on plant species and such a level of differentiation among populations corresponds to that which has usually been observed for outcrossing plants. The importance of maintenance of the P. reticulata populations and implications of the analysis of adult individuals, considering the longevity of this species and the relatively recent Cerrado fragmentation, are discussed.     

Random amplified polymorphic DNA (RAPD) variation among native little bluestem [Schizachyrium scoparium (Michx.) Nash] populations from sites of high and low fertility in forest and grassland biomes

Random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among four native populations of Schizachyrium scoparium . Genotypes were collected from high- and low-fertility sites in both New Jersey (forest biome) and in Oklahoma (grassland biome), USA, and propagated in the greenhouse. Four oligonucleotide primers, 10 bp in length, produced a total of 60 RAPD markers, with the minimum marker difference between any two individuals being 14 markers. Euclidean metric distances were calculated among all individuals, and the analysis of molecular variance ( AMOVA ) technique was used to apportion the total genetic variation among individuals within populations, populations within fertility levels, populations within biomes, fertility levels, and biomes. Even though most genetic variation resided within populations, statistically significant differences were detected between populations within each biome. Furthermore, genetic distances between high and low fertility levels within biomes were equal to or greater than biome distances. Therefore, in this wide-ranging and highly variable species, RAPD analysis suggests that local site differences in fertility and ecological history can promote genetic differentiation equal to or greater than geographical differentiation.     

Random amplified polymorphic DNA analysis of southern brown bandicoot (Isoodon obesulus) populations in Western Australia reveals genetic differentiation related to environmental variables

Random amplified polymorphic DNA (RAPD) markers were used to analyse genetic variation within and between populations of Isoodon obesulus in Western Australia. Genetically controlled geographical variation in body size associated with habitat type and rainfall exists in this species, raising the question of whether local conditions may influence gene flow in I. obesulus. The RAPD markers displayed substantial genetic variation, with all animals possessing unique RAPD phenotypes over 39 polymorphic bands produced by three primers. Significant geographical subdivision was apparent (PhiST = 0.208) with southwest locations being divergent from all others, despite there being no physical barriers to gene flow. The pattern of subdivision was unrelated to physical distance between the locations, but was related to both annual rainfall and habitat type. Therefore, the most reasonable explanation for this pattern of subdivision appears to be that gene flow is restricted by selection against migrants between local populations with substantially different habitat type or rainfall. Restriction of gene flow through selection against migrants is rarely investigated, and the results of this study suggest that the importance of this process in the formation of population structure may be underestimated.     

Random amplified polymorphic DNA and amplified fragment length polymorphism assessment of genetic variation in Nicaraguan populations of Pinus oocarpa

Pinus oocarpa is the most widely distributed pine species of Mexico and Central America. The natural populations of Nicaragua have been affected by extensive human activities. As a consequence, their size has been reduced, and there is a serious threat to the development of mature woodland. Knowledge of population structures and the genetic diversity of the species is required for the design of sustainable use and conservation strategies. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation among 10 populations from three geographical regions of Nicaragua. Both markers revealed high levels of diversity in these populations. G(ST) values and analyses of molecular variance (AMOVA) found that most variation was within populations but there is still a significant differentiation between populations indicating that the populations sampled cannot be considered a single panmictic unit. The partitions created by AMOVA also showed that there was little differentiation between populations of different regions, although cluster analyses based on RAPDs and AFLPs indicated a closer relationship among most of the populations from a same geographical region. Management of P. oocarpa in Nicaragua should be aimed to maintain the high degree of genetic variation within individual populations that is still observed even in some of these highly degraded populations.     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

Genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) germplasm from Ethiopia assessed by random amplified polymorphic DNA (RAPD)

The extent and distribution of genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) collected from five different geographical regions in Ethiopia were analyzed using random amplified polymorphic DNA (RAPD) markers for 93 individuals representing 11 populations. Nine decamer primers generated a total of 83 polymorphic bands with 8-12 bands per primer and a mean of 9 bands across the 93 individuals. The amount of genetic variation among the populations (H = 0.37) and among the geographical region (H = 0.44) was low to moderate, despite the high degree of polymorphic bands per primer. Similarly, the mean genetic distance (0.08) among populations as well as among regions of origin (0.04) of the population was found to be low. The low genetic variation may be due to the reduced population size of the wild sorghum in Ethiopia because of habitat change. Partitioning of the genetic variation into between and within the population as well as between and within the regions of origin revealed that 75% and 88% of the variation was found within the populations and within the regions, respectively. Cluster analysis of genetic distance estimates further confirmed low level of differentiation of wild sorghum populations both on population and regional bases. The implications of the results for genetic conservation purposes are discussed.     

Genetic diversity in natural and anthropogenic inland populations of salt-tolerant plants: random amplified polymorphic DNA analyses of Aster tripolium L. (Compositae) and Salicornia ramosissima Woods (Chenopodiaceae)

Eight populations of Aster tripolium (Compositae) and six of Salicornia ramosissima (Chenopodiaceae) from inland, naturally salt-contaminated habitats and anthropogenic salt-polluted sites in central Germany (Thuringia, Anhalt-Saxony) were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the patterns of genetic variation. In both species, the genetic diversity observed in the younger, anthropogenic sites caused by potash mines during the last century was found to be not significantly lower than in the older, naturally salt-contaminated habitats. Therefore, it is speculated that the loss of genetic diversity caused by founder effects on the anthropogenic habitats was balanced by successive colonization events, actual gene flow between populations, or the rapid growth of populations on the secondary habitats after colonization. Analyses of molecular variance (amova) of the RAPD markers, neighbour-joining clustering of populations based on Reynolds' co-ancestry distances, and Mantel tests indicate that: (i) anthropogenic habitats were colonized independently; (ii) genetic differentiation among populations of S. ramosissima is more pronounced than in A. tripolium, which is considered to be mainly due to biological differences between the two species; and (iii) the geographical pattern of genetic diversity was considerably modulated by historical events and/or population genetic effects.     

Genetic structure and diversity in Ramonda myconi (Gesneriaceae): effects of historical climate change on a preglacial relict species

The importance of the Mediterranean Basin as a long-term reservoir of biological diversity has been widely recognized, although much less effort has been devoted to understanding processes that allow species to persist in this area. Ramonda myconi (Gesneriaceae) is a Tertiary relict plant species restricted to the NE Iberian Peninsula. We used RAPD and chloroplast markers to assess the patterns of genetic structure in eight mountain regions covering almost the full species range, to identify the main historical processes that have shaped its current distribution and to infer the number and location of putative glacial refugia. While no cpDNA variation was detected, the species had relatively high levels of RAPD variation. Maximum levels of diversity were found within populations (71%), but there was also a significant differentiation between geographical regions (20%) and among populations within regions (9%). A spatial AMOVA identified three main groups of populations, corresponding to previously recognized centers of endemism and species richness. In addition, we found a marked geographical pattern of decreasing genetic diversity and increasing population differentiation from west to east. Our results support a complex phylogeographic scenario in the Iberian Peninsula of "refugia-within-refugia" and suggest that the higher diversity observed in western regions might be associated with prolonged and more stable climatic conditions in this area during the Quaternary.     

Genetic variation in Fitzroya cupressoides (alerce), a threatened South American conifer

Fitzroya cupressoides (alerce, Cupressaceae) is a large and exceptionally long-lived conifer, endemic to a restricted area of southern Chile and neighbouring areas of Argentina. As a result of its high economic value, the species has been severely exploited for timber, and remnant populations are fragmented and often highly disturbed. The species is thought to have undergone a major range contraction during the last glaciation. In order to assess the extent of genetic variation using DNA markers within and between populations of this species, samples were obtained from throughout the natural range and analysed for random amplified polymorphic DNA (RAPD) variation. Eight 10-mer and three 15-mer primers were used to produce a total of 54 polymorphic bands. Shannon's diversity estimates were calculated to provide an estimate of the degree of variation within each population. Values varied from 0.343 to 0.636 with only the lowest value differing significantly from the others (Spop = 0.547). This indicated that there is a significant degree of variation within each population, and did not provide evidence for genetic 'bottle-neck' effects within the species. A pairwise distance measure calculated from the RAPD data was used as an input for principal coordinate (PCO) and AMOVA analyses. The first three principal coordinates of RAPD distances described 8.3, 5.9 and 5.4% of the total variance, respectively, and a degree of clustering of samples according to their geographical origin was detectable. AMOVA analysis indicated that although most of the variation (85.6%) was found within populations, a significant proportion (P < 0.002) was attributable to differences between populations. An UPGMA dendrogram constructed using phi ST values derived from AMOVA produced a pattern broadly similar to that produced by the PCO, highlighting differences between three main groups of populations within Chile: those from the northern Coastal Range, the southern Coastal Range and Central Depression, and the Andes. Populations from Argentina also emerged as significantly different from those in Chile. These results are interpreted in the context of the postglacial history of the species, and their implications for the development of conservation strategies for Fitzroya are discussed.     

Genetic diversity of Annona crassiflora (Annonaceae) in northern Minas Gerais State

Genetic diversity analyses of tropical tree species are relevant to landscape management, plant genetic resource inventory, and biological conservation of threatened species. Annona crassiflora is an endangered fruit tree native to the Cerrado biome that is threatened by reduction of natural populations and fruit extraction. We examined the intra- and interpopulational genetic diversity of this species in the northern region of Minas Gerais State. Seventy-two individuals from four natural populations were genotyped using RAPD markers. We found moderate genetic diversity among populations, with Shannon's I index varying between 0.31 and 0.44, and Nei's genetic diversity (H(E)) for the population set equal to 0.31. AMOVA indicated a greater genetic variation within (77.38%) rather than among populations (22.62%), tending towards isolation by distance (Mantel's r = 0.914; P = 0.089). Nei's genetic identity estimates among populations revealed a hierarchical pattern of genetic similarity of form [(CA1, CA2), MC], [(GM)], corroborating the high genetic differentiation between spatially isolated populations.