Genetic analysis of populations of threatened snake species using RAPD markers

Snakes are a particularly threatened vertebrate taxon, with distributions of many species and populations becoming increasingly fragmented. At present, little is known about the degree of genetic differentiation that exists between isolated populations even though such information may be critical to their survival and conservation. As an example of how recently developed RAPD genetic markers can be used in conservation genetics, we present preliminary results from a study which used these DNA-based markers to assess population divergence in two threatened Canadian snakes, the black rat snake ( Elaphe o. obsoleta ) and the eastern massasauga rattlesnake ( Sistrurus c. catenatus ). We present information on the levels of variation and reliability of amplification for fragments generated from five primers. We then use a recently developed analytical technique to estimate levels of nucleotide diversity within populations and sequence divergence between populations. Our results show that intrapopulation levels of divergence as estimated by the methods of Clark & Lanigan ( Molecular Biology and Evolution 1993, 10 , 1096–1111) approximate those found for mtDNA in vertebrates and that diversity between snake populations is small and non-significant when tested using randomization procedures. Thus, our study provides an example of how RAPDs can be applied to conservation genetic studies of vertebrates and suggest that the snake populations we examined have only recently become isolated and maybe considered genetically equivalent from a conservation perspective, although this conclusion needs to be confirmed with other DNA-based markers.     

Genetic variation in Banksia saxicola (Proteaceae), a rare Australian plant with a markedly disjunct distribution

 Banksia saxicola A.S. George (Proteaceae) is a rare Australian endemic, found in only two locations in Victoria that are separated by approximately 500 kms: the Grampians and Wilson's Promontory National Parks. The organisation of genetic variation between and within populations at the two locations was assessed using the Amplified Fragment Length Polymorphism (AFLP) technique. Two populations were sampled in the Grampians National Park and one population was sampled at Wilson's Promontory. The three populations were genetically divergent from each other, in particular the Grampians and Wilson's Promontory populations, and this relates to the ancient geographic isolation of these two regions. The Wilson's Promontory population had lower genetic diversity than either Grampians populations, consistent with its smaller population size. The findings are important for strategies to conserve B. saxicola. Received October 3, 2000 Accepted January 3, 2001     

Genetic variation within and among populations of the threatened lichen Lobaria pulmonaria in Switzerland and implications for its conservation

The foliose epiphytic lichen Lobaria pulmonaria has suffered a significant decline in European lowlands during the last decades and therefore is considered as endangered throughout Europe. An assessment of the genetic variability is necessary to formulate biologically sound conservation recommendations for this species. We investigated the genetic diversity of the fungal symbiont of L. pulmonaria using 143 specimens sampled from six populations (two small, one medium, three large) in the lowland, the Jura Mountains, the pre-Alps and the Alps of Switzerland. Among all nuclear and mitochondrial regions sequenced for this study, variability was found only in the internal transcribed spacer (ITS I), with three polymorphic sites, and in the nuclear ribosomal large subunit (nrLSU), with four polymorphic sites. The variable sites in the nrLSU are all located within a putative spliceosomal intron. We sequenced these two regions for 81 specimens and detected six genotypes. Two genotypes were common, two were found only in the more diverse populations and two were found only in one population each. There was no correlation between population size and genetic diversity. The highest genetic diversity was found in populations where the fungal symbiont is reproducing sexually. Populations with low genetic diversity included only the two same common genotypes. Our study provides evidence suggesting that L. pulmonaria is self-incompatible and heterothallic. Based on our results we give populations with sexually reproducing individuals a higher rank in terms of conservation priority than strictly asexual populations. The remaining lowland populations are so small, that one single catastrophic event such as a windthrow might destroy the entire population. Hence we suggest augmenting such populations in size and genetic diversity using small thallus fragments or vegetative diaspores collected in other populations. As we did not detect any locally adapted genotypes, these transplants can be taken from any other genetically diverse population in Switzerland.     

Genetic variation in Costa Rican populations of the tropical timber species Cedrela odorata L., assessed using RAPDs

Cedrela odorata L. (Spanish cedar), an economically important timber species native to the American tropics, is the focus of increasing conservation concern due to high rates of deforestation within its native range. To assess the extent of the genetic diversity within and between populations of this species, samples were obtained from 10 widely dispersed populations within Costa Rica and analysed for random amplified polymorphic DNA (RAPD) variation. Fourteen 10-mer primers were used to generate 97 polymorphic RAPD bands. Presence/absence data for all bands were subjected to a pairwise genetic distance analysis, according to Jaccard's coefficient, then neighbour-joining cluster analysis was performed on these distances, as was an analysis of molecular variance ( AMOVA ), to assess levels of differentiation between populations and regions, and Shannon's Diversity Index was used to quantify levels of diversity within and between populations. Results indicated highly significant genetic differentiation ( P < 0.004, AMOVA ) between populations originating from the North Pacific and Atlantic/South Pacific regions of Costa Rica, with 35.3% of the total variation attributable to a difference between these areas. Little differentiation was recorded between populations from within the same region ( P = 0.757, AMOVA ), and 65.1% of the total variance was attributable to variation within populations. Estimated values for within-population diversity, calculated as H _pop/ H _sp by means of Shannon's Diversity Index, were found to vary greatly between primers, but the overall within-population component of genetic diversity was 0.45. Possible reasons for the high degree of intraspecific genetic variation within this species are discussed and the implications of these results for the conservation and use of its genetic resources are described.     

Genetic variation in low elevation Douglas-fir of British Columbia and its relevance to gene conservation

Patterns of variation were studied at 20 isozyme loci in 49 coastal, low-elevation Douglas-fir populations in SW British Columbia and NW Washington State. Several components of variation were estimated for each population including the number of alleles per locus N _a, number of alleles per polymorphic locus N _a(95), inbreeding F, heterozygosity H, and population divergence D. F was near zero indicating nearly complete outcrossing within populations. H was quite high (16%) and in aecord with previous studies of Douglas-fir. D values were low (equivalent to Wrights F _ST of 0.08) indicating levels of gene flow sufficient to largely homogenize populations. The parameters of diversity N _a, N _a(95), H, and D showed little intercorrelation across populations. A homogenous pattern of genetic relationship among populations was shown by the clustering of populations based on their inferred relationship, and by the principal components of the matrix of inferred genetic relationship. Because of the complex nature of gene diversity and the continuous nature of population differentiation in Douglas-fir, it is difficult with isozyme markers to identify specific populations of value for genetic conservation in this species.This paper is dedicated to J.C. Heaman on the occasion of his retirement.     

Microsatellite DNA variation in Bornean orangutans (Pongo pygmaeus)

Orangutans (Pongo pygmaeus) on the islands of Borneo and Sumatra are considered two separate subspecies. However, the genetic relationships between isolated populations on Borneo are not clear. This study determined the extent of variation within the Bornean subspecies of orangutan, using microsatellite DNA analysis. Blood samples were collected from 96 individuals of known origin from East, West and Central Kalimantan. Human microsatellite primer pairs located at human map position D2S141, D4S431, D 11S925, D16S420 and D17S791 were suitable for use in primates. D4S431 appeared monomorphic for all orangutans. In three cases (D2S141 East and West and D16S420 West), a highly significant excess of homozygous allele frequencies was detected, but with other primer pairs no significant difference in allele frequencies occurred. We conclude that the divergence between the different populations on Borneo is less than the variation within the populations. There was also evidence that inbreeding occurred within the populations.     

Genetic variation in a geographically restricted and two widespread species of South American Nothofagus

The amount and distribution of genetic variation is compared using starch gel electrophoresis among populations of the three evergreen species of South American Nothofagus : the geographically restricted Nothofagus nitida (Phil.) Krasser and the widespread Nothofagus betuloides (Mirb.) Oerst. and Nothofagus dombeyi (Mirb.) Oerst. Ten enzyme systems were resolved coding for fifteen putative genetic loci. N. betuloides and N. dombeyi were more genetically variable than N. nitida ; they had higher total number of alleles, mean number of alleles per locus, percent of polymorphic loci, mean expected heterozygosity, and mean total genetic diversity. These results are in agreement with the hypothesis that widespread species are often associated with historically large and more continuously distributed populations which in turn can maintain higher levels of genetic variation. Conversely, the genetic structure of N. nitida including its low genetic identity with the other two species and the presence of four unique alleles support the hypothesis that N. nitida has been isolated from the other two species for a considerable period of time. Genetic data in concert with the fossil record indicate that these taxa may be members of a pre-Pleistocene flora and consequently their genetic structures reflect a more ancient evolutionary history than previously hypothesized.     

Genetic variation in Holocene bowhead whales from Svalbard

Bowhead whales (Balaena mysticetus) are distributed in the Arctic in five putative stocks. All stocks have been heavily depleted due to centuries of exploitation. In the present study, nucleotide sequence variation of the mitochondrial control region was determined from bone remains of 99 bowhead whales. The bones, 14C dated from recent to more than 50,000 bp, were collected on Svalbard (Spitsbergen) and are expected to relate to ancestors of the today nearly extinct Spitsbergen stock. Fifty-eight haplotypes were found, a few being frequent but many only found in one individual. The most abundant haplotypes of the Spitsbergen stock are the same as those most abundant in the extant Bering-Chukchi-Beaufort (BCB) Seas stock of bowhead whales. Although F(ST) indicates a slight but statistically significant genetic differentiation between the Spitsbergen and the BCB stocks this was not considered informative due to the very high levels of genetic diversity of mitochondrial DNA haplotypes in both bowhead whale stocks. Other measures such as K(ST) also indicated very low genetic differentiation between the two populations. Nucleotide diversity and haplotype diversity showed only minor differences between the Spitsbergen and BCB stocks. The data suggest that the historic Spitsbergen stock--before the severe bottleneck caused by whaling--did not have substantially more genetic variation than the extant BCB stock. The similar haplotypes of the Holocene Svalbard samples and the current BCB stock indicate significant migration between these two stocks and question the current designation of five distinct stocks of bowhead whales in the Arctic.     

Genetic diversity is related to climatic variation and vulnerability in threatened bull trout

Understanding how climatic variation influences ecological and evolutionary processes is crucial for informed conservation decision‐making. Nevertheless, few studies have measured how climatic variation influences genetic diversity within populations or how genetic diversity is distributed across space relative to future climatic stress. Here, we tested whether patterns of genetic diversity (allelic richness) were related to climatic variation and habitat features in 130 bull trout (Salvelinus confluentus) populations from 24 watersheds (i.e., ~4–7th order river subbasins) across the Columbia River Basin, USA. We then determined whether bull trout genetic diversity was related to climate vulnerability at the watershed scale, which we quantified on the basis of exposure to future climatic conditions (projected scenarios for the 2040s) and existing habitat complexity. We found a strong gradient in genetic diversity in bull trout populations across the Columbia River Basin, where populations located in the most upstream headwater areas had the greatest genetic diversity. After accounting for spatial patterns with linear mixed models, allelic richness in bull trout populations was positively related to habitat patch size and complexity, and negatively related to maximum summer temperature and the frequency of winter flooding. These relationships strongly suggest that climatic variation influences evolutionary processes in this threatened species and that genetic diversity will likely decrease due to future climate change. Vulnerability at a watershed scale was negatively correlated with average genetic diversity (r = ?0.77; P < 0.001); watersheds containing populations with lower average genetic diversity generally had the lowest habitat complexity, warmest stream temperatures, and greatest frequency of winter flooding. Together, these findings have important conservation implications for bull trout and other imperiled species. Genetic diversity is already depressed where climatic vulnerability is highest; it will likely erode further in the very places where diversity may be most needed for future persistence.     

华南3个赤眼鳟群体Cytb基因的遗传变异

测定了珠江和韩江3个群体21尾赤眼鳟线粒体细胞色素b基因1 029bp序列片段,发现11个单倍型,14个变异位点。韩江群体单倍型多样度h(0.464)和核苷酸多样度π(0.000 97)较低,珠江水系左江和郁江群体较高(h=0.929-1,π=0.023 6-0.036 9)。在邻接树上不同地理来源的个体混杂,没有明显的谱系结构和地理聚群。Fst值和AMOVA分析亦显示珠江与韩江群体之间没有显著遗传分化。单倍型网络图呈星状结构,中性检测Tajima's D和Fu's Fs均为显著负值,核苷酸不对称分布分析呈单峰模式,说明华南赤眼鳟群体可能在晚更新世(164-66KaBP)曾经历过种群的快速扩张。     

Genetic variation in five Mediterranean populations of Juniperus phoenicea as revealed by inter-simple sequence repeat (ISSR) markers

BACKGROUND AND AIMS: The assessment of the genetic variability and the identification of isolated populations within a given species represent important information to plan conservation strategies on a genetic basis. In this work, the genetic variability in five natural populations of Juniperus phoenicea, three from Sardinia, one from Cyprus and the last one in the Maritime Alps was analysed by means of ISSRs, on the hypothesis that the latter could have been a refugial one during the last glaciation. METHODS: ISSRs were chosen because of their ability to detect variation without any prior sequence information. The use of three primers yielded 45 reproducible, polymorphic bands, which were utilized to estimate the basic parameters of genetic variability and diversity. KEY RESULTS: All of the populations analysed harboured an adequate amount of genetic variability, with H(S) = 0.1299. The proportion of genetic diversity between populations has been estimated by G(ST) = 0.12. The three Sardinian populations are separated, as tested by AMOVA, from the Cyprus and the continental ones. CONCLUSIONS: The results indicate that geographical isolation has represented a major barrier to gene flow in Juniperus phoenicea. This work represents a first step towards a full genetic characterization of a conifer from the Mediterranean, a world biodiversity hotspot confronted with climate change, and thus contributes towards the planning of genetics-informed conservation strategies.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

Genetic diversity among genotypes of Eryngium viviparum (Apiaceae): a plant threatened throughout its natural range

Eryngium viviparum (Apiaceae) is an endangered aquatic plant, listed as threatened in several European documents. The genotypes are distributed patchily in various wetlands in the north‐west of Spain and one is located in north‐west France. The study of the genetic diversity of a small population of a rare species is important for conservation and studies aimed at recovery programmes. Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity among five Spanish and one French genotype. This technique has contributed to the knowledge of the genetic diversity in E. viviparum, showing a greater genetic distance between the Spanish cluster formed by S1, S4 than the second cluster formed by S2, S3, S5 and the French genotype. Mantel testing did not show a significant correlation between genetic and geographical distances, but a significant correlation was found between altitude, habitat and genetic distance. The French genotype showed the highest level of polymorphism (28.16) and the highest percentage of exclusive markers (32%). One of these was isolated, purified, cloned and sequenced, revealing a high homology to a protein mainly expressed in roots. This could represent, for the F genotype, an adaptation to a specific habitat near the sea compared with the Spanish genotypes which grow inland. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 237–244.     

Phantom hitch-hikers mislead estimates of genetic variation in Antarctic mosses

Previous studies of Antarctic mosses employing RAPDs have reported extraordinarily high levels of genetic variation, even within a single clump of moss. This is unexpected given their extreme isolation in Antarctica and lack of sexual reproduction. We offer an alternative explanation: that unusually elevated levels of genetic variability are artefacts from contamination of a number of biota known to be naturally associated with Antarctic mosses. We utilized sequence variation of nrITS and RAPDs to further investigate the effect of naturally occurring contaminants on estimates of genetic variation of mosses. Our results indicate that these ``phantom hitch-hiker' contaminants hinder attempts to accurately and reliably estimate levels of genetic variation by non-specific PCR-based approaches. Furthermore, screening samples via amplification of nrITS failed to identify all contaminated samples, hence we caution against relying solely on ``quick' screening methods and suggest that suspect samples be carefully examined for contamination.     

Genetic diversity and morphological differentiation in Liatris helleri (Asteraceae), a threatened plant species

Liatris helleri (Asteraceae) is an insect-pollinated herbaceous perennial endemic to several high-elevation sites in the Blue Ridge Mountains of North Carolina. Allozymes were used to describe the genetic diversity and population structure in nine populations of this rare, federally listed species. Differences in leaf morphology were also examined for greenhouse-grown plants representing several populations. The proportion of the total genetic diversity found among populations, as indicated by the allozyme data, was 16%. Higher levels of population differentiation were found for differences in leaf shape; population of origin accounted for 37% of the variation in maximum leaf width, while families within populations accounted for 7%. In contrast to many endemic species, L. helleri maintains fairly high levels of genetic diversity. For the species, the percent polymorphic loci was 87.5, the average number of alleles at variable loci was 3.00 and the gene diversity was 0.276. Mean population values were percent polymorphic loci =58.4, mean number of alleles per polymorphic locus =2.59 and gene diversity =0.219. The estimated gene flow was low (Nm=0.85–1.32) and a relatively high correlation (r=0.55; p<0.005) was found between linear geographic and genetic distance. This suggests that the populations are partially isolated by distance, despite the limited range (<60 km) of the species. We recommend that population distinetiveness be maintained in restoration efforts.     

Genetic differentiation of Euterpe edulis Mart. populations estimated by AFLP analysis

Heart-of-palm (Euterpe edulis Mart.) is a wild palm with a wide distribution throughout the Atlantic Rainforest. Populations of E. edulis represent important renewable natural resources but are currently under threat from predatory exploitation. Furthermore, because the species is indigenous to the Atlantic Rainforest, which is located in the most economically developed and populated region of Brazil, social and economic pressures have devastated heart-of-palm forests. In order to estimate the partitioning of genetic variation of endangered E. edulis populations, 429 AFLP markers were used to analyse 150 plants representing 11 populations of the species distribution range. Analysis of the genetic structure of populations carried out using analysis of molecular variance (AMOVA) revealed moderate genetic variation within populations (57. 4%). Genetic differentiation between populations (FST = 0.426) was positively correlated with geographical distance. These results could be explained by the historical fragmentation of the Atlantic coastal region, together with the life cycle and mating system. The data obtained in this work should have important implications for conservation and future breeding programmes of E. edulis.     

Restoring the American Chestnut: Optimizing Founder Spacing to Promote Population Growth and Genetic Diversity Retention

Efforts are underway to return the American chestnut (Castanea dentata) to eastern forests of North America following its decline due to the introduction of the chestnut blight (Cryphonectria parasitica). Approaches include developing blight‐resistant chestnut lines through breeding programs and via genetic engineering. Reestablishment of resistant chestnut to eastern forests would produce one of the most extensive ecological restoration transformations ever attempted. However, this undertaking is costly and optimization of reintroduction methods is needed. We used the computer program NEWGARDEN to model whether some patterns of founder placement (regular vs. random spacing at differing densities) produce more rapidly expanding populations across a range of gene dispersal distance conditions (via both offspring and pollen). For a simulated introduction project employing 169 founders, placing founders randomly in a square of side 0.85 km produced higher rates of predicted population growth compared with larger or smaller squares under near gene dispersal conditions; this side distance was 1.0 km under far gene dispersal conditions. After 100 population bouts of mating and under near gene dispersal conditions, the trial with founder placement producing the greatest population expansion rate exhibited a 314% increase in census size compared with the founder pattern yielding the slowest expansion. Neither loss of alleles nor inbreeding or subdivision was significantly increased under the founder placement patterns yielding the most descendants. Exploring different numerical and geometrical founding scenarios using NEWGARDEN can provide first estimates of founding patterns or stand manipulations that will return the most descendants produced per founder planted in restoration projects.     

Genetic impoverishment of the last black grouse (Tetrao tetrix) population in the Netherlands: detectable only with a reference from the past

We have studied a small isolated population of black grouse (Tetrao tetrix) in the Netherlands to examine the impact of isolation and reduction in numbers on genetic diversity. We compared the genetic diversity in the last extant Dutch population with Dutch museum samples and three other black grouse populations (from England, Austria and Norway, respectively) representing isolated and continuous populations. We found significantly lower allelic richness, observed and expected heterozygosities in the present Dutch population compared to the continuous populations (Austria and Norway) and also to the historical Dutch population. However, using a bottleneck test on each population, signs of heterozygosity excess were only found in the likewise isolated English population despite that strong genetic drift was evident in the present Dutch population in comparison to the reference populations, as assessed both in pairwise F(ST)and STRUCTURE analyses. Simulating the effect of a population reduction on the Dutch population from 1948 onwards, using census data and with the Dutch museum samples as a model for the genetic diversity in the initial population, revealed that the loss in number of alleles and observed heterozygosity was according to genetic drift expectations and within the standard error range of the present Dutch population. Thus, the effect of the strong decline in the number of grouse on genetic diversity was only detectable when using a reference from the past. The lack of evidence for a population reduction in the present Dutch population by using the program bottleneck was attributed to a rapidly found new equilibrium as a consequence of a very small effective population size.     

Genetic variation in populations of the arctic perennial Pedicularis dasyantha (Scrophulariaceae), on Svalbard,Norway

Isozyme variability was examined in 13 geographically isolated populations of the endemic arctic hairy lousewort (Pedicularis dasyantha) in the Svalbard Archipelago, 80° N latitude, Norway. Of the 23 enzyme systems screened on five buffer systems 18 were interpretable. Of the 31 reliable loci, only 6-phosphogluconate dehydrogenase (6-Pgd), was polymorphic. However, no heterozygotes were detected. Frequencies for allele 1 among the populations varied from 1.00 in the north to 0.00 in the south and 0.53 in the central “overlap” region. At the species level the mean number of alleles per locus (A) was 1.03. Percentage of polymorphic loci (P) was 3%. Expected heterozygosity (H_e) was 0.016. At the population level the mean number of alleles per locus was 1.01, and 1.1% of the loci were polymorphic. H_e was 0.004. These values are low compared to endemic, widespread, selling, and outcrossed species. Flower color morphs were distinct. They varied within and among the 13 populations. The frequency of color morphs coincided with allele frequencies of 6-PGD: allele 1 was found in dark purple morphs, and allele 2 was found in light morphs. This species shows more isozyme genetic variability than the five other species reported in the genus but generally less variation than other species with limited regional distributions. Low-level genetic variation in this diploid species may be a result of colonization events coupled with genetic drift, founder effects, and strong natural selection. Additional factors include the self-compatible reproductive system and the long-lived perennial habit.