High population differentiation and genetic variation in the endangered Mexican pine Pinus Rzedowskii (Pinaceae)

Pinus rzedowskii is an endangered pine species from Michoaca´n (central Me´xico), which has been previously reported from only three localities. Classified within the subgenus Strobus, it exhibits intermediate morphological characters between subgenera Strobus and Pinus. We analyzed genetic aspects that could shed light on the evolution and conservation of this species. The genetic structure of nine populations was examined using 14 isozyme loci. Pinus rzedowskii has a relatively high level of genetic variation with 46.8% of the loci assayed being polymorphic, a total of 35 alleles, and a mean heterozygosity per population of 0.219. We calculated Wright's F_ST statistic to estimate gene flow indirectly and to evaluate whether or not there was genetic structuring among populations. We found a marked differentiation among populations (F_ST = 0.175) and significant inbreeding (F_IS = 0.247). No pattern of isolation by distance was found. We also constructed a dendrogram based on a genetic distance matrix to obtain an overview of the possible historical relationships among populations. Finally, we found a convex relationship between the genetic distance among populations and the number of ancestral lineages, suggesting that demographically this species has not been at risk recently. Although endangered, with small and fragmented populations, P. rzedowskii shows higher levels of genetic variation than other conifer species with larger populations or similar conservation status.     

Habent Fata Sua: The Rise and Fall of Garden Plants

Background: Rare cactus in the Americas and other species worldwide are threatened species because of their high level of habitat specialisation, narrow distribution range and continuing population decline.

Aims: To identify management units (MUs) based on genetic variability and demographic structure in order to propose assertive conservation actions for Mammillaria crucigera and to provide a model case study for other species that are under similar threats.

Methods: We genotyped through eight microsatellite loci in 171 individuals and described demographic structures in six populations of this cactus based on plots of 1 m².

Results: Across populations with a mean density of 2.6 m^?2 and a total of ~500 individuals counted, 30% of the individuals were reproductive (diameter >2 cm). The total heterozygosity was low (H_O = 0.54), but the inbreeding coefficient (F_IS = 0.29) and the allele diversity (N_A = 20) were high. Four genetic groups were distinguished, although considering the demographic structure, we propose three MUs.

Conclusions: It is critical to maintain the genetic connectivity within and among MUs, which can only be achieved through cooperation between government authorities and local habitants to halt the degradation and further destruction of the remnant populations. Searching MUs allows the identification of critical areas for conservation issues for all species whose extant populations are in a fragmented landscape.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Genetic diversity and population structure in Elephantopus scaber (Asteraceae) from South China as revealed by ISSR markers

Abstract

We used inter-simple sequence repeat (ISSR) markers to investigate genetic variation in eight natural populations of Elephantopus scaber from South China, including Guangdong, Hainan and Hong Kong. Eleven primers produced 247 bands across all 184 individuals, of which 243 (98.4%) were polymorphic. The average genetic diversity at the species and population levels was estimated to be 0.283 and 0.103, respectively, using mean expected heterozygosity. The average gene differentiation (F _ST) among populations was 0.725. AMOVA analysis showed that the partition of molecular variation between and within populations was 72.5% and 27.5%, respectively. The effective number of migrants among populations based on the F _ST was relatively low (N _m = 0.095). Cluster analysis based on Nei's genetic distance and the neighbour-joining method revealed the genetic relationships among the populations of E. scaber. The Mantel test indicated that there was no significant correlation between population genetic and geographic distances. The results obtained from the AMOVA analysis, the cluster analysis, and the Mantel test all suggested that fragmented local environments and human disturbance might play important roles in shaping the population structure of E. scaber.     

Beyond F<Subscript>ST</Subscript>: Analysis of population genetic data for conservation

Both the ability to generate DNA data and the variety of analytical methods for conservation genetics are expanding at an ever-increasing pace. Analytical approaches are now possible that were unthinkable even five years ago due to limitations in computational power or the availability of DNA data, and this has vastly expanded the accuracy and types of information that may be gained from population genetic data. Here we provide a guide to recently developed methods for population genetic analysis, including identification of population structure, quantification of gene flow, and inference of demographic history. We cover both allele-frequency and sequence-based approaches, with a special focus on methods relevant to conservation genetic applications. Although classical population genetic approaches such as F _st (and its derivatives) have carried the field thus far, newer, more powerful, methods can infer much more from the data, rely on fewer assumptions, and are appropriate for conservation genetic management when precise estimates are needed.     

Genetic inference of epiphytic orchid colonization; it may only take one

Colonization of vacant habitat is a fundamental ecological process that affects the ability of species to persist and undergo range modifications in continually shifting landscapes. Thus, understanding factors that affect and limit colonization has important ecological and conservation implications. Epiphytic orchids are increasingly threatened by various factors, including anthropogenic habitat disturbance. As cleared areas (e.g. pastures) are recolonized by suitable host trees, the establishment and genetic composition of epiphytic orchid populations are likely a function of their colonization patterns. We used genetic analyses to infer the prevailing colonization pattern of the epiphytic orchid, Brassavola nodosa. Samples from three populations (i.e. individuals within a tree) from each of five pastures in the dry forest of Costa Rica were genotyped with neutral nuclear and chloroplast markers. Spatial autocorrelation and hierarchical genetic structure analyses were used to assess the relatedness of individuals within populations, among populations within pastures and among populations in different pastures. The results showed significant relatedness within populations (mean r = 0.166) and significant but lower relatedness among populations within a pasture (mean r = 0.058). Our data suggest that colonization of available habitats is by few individuals with subsequent population expansion resulting from in situ reproduction, and that individuals within a tree are not a random sample of the regional seed pool. Furthermore, populations within a pasture were likely colonized by seeds produced by founders of a neighbouring population within that pasture. These results have important ramifications for understanding conservation measures needed for this species and other epiphytic orchids.     

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

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

Genetic diversity,population genetic structure and demographic history of Przewalski’s gazelle (<Emphasis Type="Italic">Procapra przewalskii</Emphasis>): implications for conservation

The Przewalski’s gazelle (Procapra przewalskii) is one of the most endangered antelope species in the world. It is endemic to China and is a flagship species in the eastern part of the Qinghai–Tibet plateau. To establish effective conservation measures on this species, genetic information such as genetic structure is needed. However, there has not been a comprehensive genetic assessment on this gazelle using nuclear DNA markers yet. Here, we employed 13 microsatellite loci to investigate genetic diversity, population genetic structure and demographic history of Przewalski’s gazelle using noninvasive samples of 169 wild gazelles collected from nine populations. A total of 76 alleles were detected from the entire samples, mean allele number was 5.85, and overall H _O and H _E were 0.525 and 0.552, respectively. Structure and GENELAND analyses found six genetic groups in the nine populations. Between the inferred genetic groups, significant genetic differentiation and low migration rates were detected. Demographic analyses indicated that Przewalski’s gazelle experienced genetic bottleneck and severe population decline, with the ancestral effective population size reducing to less than one percent. Based on the results of this study, we provide several conservation recommendations for Przewalski’s gazelle, such as six management units, periodic monitoring and special conservation consideration on the Qiejitan population.     

Genetic structure and demographic history of the endangered tree species Dysoxylum malabaricum (Meliaceae) in Western Ghats,India: implications for conservation in a biodiversity hotspot

The impact of fragmentation by human activities on genetic diversity of forest trees is an important concern in forest conservation, especially in tropical forests. Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, one of the world's eight most important biodiversity hotspots. As D. malabaricum is under pressure of disturbance and fragmentation together with overharvesting, conservation efforts are required in this species. In this study, range‐wide genetic structure of twelve D. malabaricum populations was evaluated to assess the impact of human activities on genetic diversity and infer the species’ evolutionary history, using both nuclear and chloroplast (cp) DNA simple sequence repeats (SSR). As genetic diversity and population structure did not differ among seedling, juvenile and adult age classes, reproductive success among the old‐growth trees and long distance seed dispersal by hornbills were suggested to contribute to maintain genetic diversity. The fixation index (F_IS) was significantly correlated with latitude, with a higher level of inbreeding in the northern populations, possibly reflecting a more severe ecosystem disturbance in those populations. Both nuclear and cpSSRs revealed northern and southern genetic groups with some discordance of their distributions; however, they did not correlate with any of the two geographic gaps known as genetic barriers to animals. Approximate Bayesian computation‐based inference from nuclear SSRs suggested that population divergence occurred before the last glacial maximum. Finally we discussed the implications of these results, in particular the presence of a clear pattern of historical genetic subdivision, on conservation policies.     

Genetic variability and the effect of habitat fragmentation in spotted suslik Spermophilus suslicus populations from two different regions

Endangered species worldwide exist in remnant populations, often within fragmented landscapes. Although assessment of genetic diversity in fragmented habitats is very important for conservation purposes, it is usually impossible to evaluate the amount of diversity that has actually been lost. Here, we compared population structure and levels of genetic diversity within populations of spotted suslik Spermophilus suslicus, inhabiting two different parts of the species range characterized by different levels of habitat connectivity. We used microsatellites to analyze 10 critically endangered populations located at the western part of the range, where suslik habitat have been severely devastated due to agriculture industrialization. Their genetic composition was compared with four populations from the eastern part of the range where the species still occupies habitat with reasonable levels of connectivity. In the western region, we detected extreme population structure (F _ST = 0.20) and levels of genetic diversity (Allelic richness ranged from 1.45 to 3.07) characteristic for highly endangered populations. Alternatively, in the eastern region we found significantly higher allelic richness (from 5.09 to 5.81) and insignificant population structure (F _ST = 0.03). As we identified a strong correlation between genetic and geographic distance and a lack of private alleles in the western region, we conclude that extreme population structure and lower genetic diversity is due to recent habitat loss. Results from this study provide guidelines for conservation and management of this highly endangered species.     

Low Phylogeographic Structure in a Wide Spread Endangered Australian Frog <Emphasis Type="Italic">Litoria aurea</Emphasis> (Anura: Hylidae)

The green and golden bell frog (Litoria aurea) has a widespread distribution along the south-east coast of Australia. The species range, however, is highly fragmented and remaining populations are predominately isolated and restricted to the coastline. Previously, the range extended further inland and the species was considered common. Here we report a study designed to identify the phylogeographic and conservation genetic parameters of L. aurea. Mitochondrial DNA sequences were examined from 263 individuals sampled from 26 locations using both phylogenetic and population analyses. Despite a general consensus that amphibians are highly structured we found no phylogeographic divisions within the species, however, there was significant structure amongst extant populations (F _ST=0.385). Patterns of haplotype relatedness, high haplotypic diversity (mean h=0.547) relative to low nucleotide diversity (mean π=0.003) and mismatch distribution analysis supported a Pleistocene expansion hypothesis with continued restricted dispersal and gene flow. We conclude that the genetic structure of the species may permit ‘well managed’ intervention to mediate gene flow amongst isolated populations and provide some guidelines for the implementation of such conservation strategies.     

Allozyme diversity in the federally threatened golden paintbrush, <Emphasis Type="Italic">Castilleja levisecta</Emphasis> (Scrophulariaceae)

Castilleja levisecta (Scrophulariaceae), the golden paintbrush, is an insect-pollinated herbaceaous perennial found in the Pacific Northwest. Currently restricted to two island populations off British Columbia and nine populations (eight on islands) in Washington, C. levisecta is a rare species threatened with extinction. Allozymes were used to describe genetic diversity and structure in these eleven populations. Despite its threatened status and small geographic range, exceptionally high levels of genetic diversity are maintained within C. levisecta. All sixteen of the loci resolved were polymorphic within the species (P_s=100%), while the mean percentage of loci polymorphic within populations (P_p) was 65.7%. The mean number of alleles per polymorphic locus (AP_s) was 2.94 within the species and averaged 2.38 within populations (AP_p). Genetic diversity (H_es) was 0.285 for the species, whereas mean population genetic diversity (H_ep) was 0.213. Smaller populations had, on average, fewer observed alleles and less genetic diversity. A significant negative correlation (r = –0.72) was found between genetic identity and geographic distance, indicating reduced gene flow between distant populations. The most geographically isolated population was one of the larger populations, one of the most genetically diverse and the most genetically divergent. A wide range of pairwise population genetic identities (I = 0.771 – 0.992) was found, indicating considerable genetic divergence between some populations. Overall, 19% of the total genetic diversity was distributed among populations. Results of this survey indicate that genetic augmentation of existing populations is unnecessary. The high allelic diversity found for the species and within its populations holds promise for conservation and restoration efforts to save this rare and threatened plant species.     

Genetic population structure and management units of the endangered Tokyo bitterling, Tanakia tanago (Cyprinidae)

The Tokyo bitterling Tanakia tanago (Cyprinidae) was once found throughout the Kanto Plain, central Japan, but most of their habitats have been lost due to human activities such as urbanization and improvement of paddy fields. Subsequently, conservation efforts, including captive breeding and reintroduction, have been ongoing. However, the genetic relationships among populations of this species including captive and remnant wild populations have been uncertain and thus management units for this species have been unidentified. We examined the population differentiation among 12 populations, including four wild and eight captive populations, and their relative genetic diversities to assist in conservation management decisions. Phylogeographic analyses based on partial mitochondrial cytochrome b gene sequences and microsatellite polymorphisms revealed four geographically associated genetic groups in the populations. Northern Tochigi populations have diverged from other populations (0.77% of d _A ), likely stemming from allopatric fragmentation following a change in the route of the Naka River, which occurred during the middle of the Pleistocene epoch. Microsatellite analysis has revealed that the genetic diversity of each population is generally low, and that most of the populations have experienced genetic bottlenecks. For future in- and ex-situ conservation programs to succeed, the population structure and genetic variability of remnant populations need to be taken into consideration.     

Where do you come from,where do you go? Directional migration rates in landscape genetics

Identifying landscape elements that influence gene flow and migration in wild species is the current main topic of landscape genetics. Most landscape genetic studies infer gene flow and migration from genetic distances among populations or individuals and statistically relate these measurements to landscape composition and configuration. This approach assumes symmetrical gene flow between pairs of populations. Such an assumption, however, will often be violated, especially in source–sink systems. Source populations provide more emigrants than they receive immigrants, and sink populations get many immigrants, but release few emigrants. Source–sink dynamics cannot be explored using common landscape genetic approaches relying on genetic distances. In this issue of Molecular Ecology, Andreasen et al. ( 2012 ) apply an alternative approach allowing them to infer asymmetrical migration. They use a Bayesian assignment test among objectively defined populations of mountain lions (Puma concolor) in western USA to estimate recent and directional migration rates. The study shows that an area with a high amount of wildlife refuges and low hunting pressure harbours a source population for mountain lion dispersal, while areas with high hunting pressures form sink populations; a result helpful in making informed decisions in conservation management.     

Combining genetic and demographic data for prioritizing conservation actions: insights from a threatened fish species

Prioritizing and making efficient conservation plans for threatened populations requires information at both evolutionary and ecological timescales. Nevertheless, few studies integrate multidisciplinary approaches, mainly because of the difficulty for conservationists to assess simultaneously the evolutionary and ecological status of populations. Here, we sought to demonstrate how combining genetic and demographic analyses allows prioritizing and initiating conservation plans. To do so, we combined snapshot microsatellite data and a 30‐year‐long demographic survey on a threatened freshwater fish species (Parachondrostoma toxostoma) at the river basin scale. Our results revealed low levels of genetic diversity and weak effective population sizes (<63 individuals) in all populations. We further detected severe bottlenecks dating back to the last centuries (200–800 years ago), which may explain the differentiation of certain populations. The demographic survey revealed a general decrease in the spatial distribution and abundance of P. toxostoma over the last three decades. We conclude that demo‐genetic approaches are essential for (1) identifying populations for which both evolutionary and ecological extinction risks are high; and (2) proposing conservation plans targeted toward these at risk populations, and accounting for the evolutionary history of populations. We suggest that demo‐genetic approaches should be the norm in conservation practices.     

Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species

Captive breeding has become an important tool in species conservation programmes. Current management strategies for ex situ populations are based on theoretical models, which have mainly been tested in model species or assessed using studbook data. During recent years an increasing number of molecular genetic studies have been published on captive populations of several endangered species. However, a comprehensive analysis of these studies is still outstanding. Here, we present a review of the published literature on ex situ conservation genetics with a focus on molecular studies. We analysed 188 publications which either presented empirical studies using molecular markers (105), studbook analyses (26), theoretical work (38), or tested the genetic effects of management strategies using model species (19). The results show that inbreeding can be minimized by a thorough management of captive populations. There seems to be a minimum number of founders (15) and a minimum size of a captive population (100) necessary in order to minimize a loss of genetic diversity. Optimally, the founders should be unrelated and new founders should be integrated into the captive population successively. We recommend that genetic analyses should generally precede and accompany ex situ conservation projects in order to avoid inbreeding and outbreeding depression. Furthermore, many of the published studies do not provide all the relevant parameters (founder size, captive population size, H_o, H_e, inbreeding coefficients). We, therefore, propose that a general standard for the presentation of genetic studies should be established, which would allow integration of the data into a global database.     

Is the genetic diversity of small scattered forest tree populations at the southern limits of their range more prone to stochastic events? A wild cherry case study by microsatellite-based markers

Wild cherry (Prunus avium L.) is a widespread, partially asexual, noble hardwood European species characterized by a scattered distribution, small population sizes, and human exploitation for its valuable wood. These characteristics, especially at the southern limits of the species natural distribution where additional varying stresses may occur, render P. avium populations prone to potential stochastic, genetic, and demographic events. In this study, we used dominant inter simple sequence repeat (ISSR) and codominant simple sequence repeat (SSR) markers to infer the genetic structure of P. avium. Five populations from northern Greece were evaluated based on 46 ISSR and 11 SSR loci. Populations presented a relatively high level of genetic variation, with a mean genetic diversity of H _e?=?0.166 and H _e?=?0.740 regarding ISSR and SSR analysis, respectively. We observed moderate population differentiation for ISSR (G _ST?=?0.113) and SSR (F _ST?=?0.097) markers. AMOVA also detected significant differentiation among populations for ISSRs (?? _ST?=?0.338) and SRRs (?? _ST?=?0.162). According to linkage disequilibrium analysis, estimates of effective population size were generally sufficient for maintaining extant genetic variability and evolutionary potential. A possible bottleneck was detected for only one population. In general, it appears that despite the particular characteristics of the P. avium populations studied, genetic stochasticity events were not apparent. The studied populations, located at the rear edge of the species European distribution, reveal a wealth of genetic variation that is very valuable for the genetic conservation of local adaptive gene complexes, especially under contemporary climatic change scenarios.     

Considerations for monitoring population trends of colonial waterbirds using the effective number of breeders and census estimates

Detecting trends in population size fluctuations is a major focus in ecology, evolution, and conservation biology. Populations of colonial waterbirds have been monitored using demographic approaches to determine annual census size (N_a). We propose the addition of genetic estimates of the effective number of breeders (N_b) as indirect measures of the risk of loss of genetic diversity to improve the evaluation of demographics and increase the accuracy of trend estimates in breeding colonies. Here, we investigated which methods of the estimation of N_b are more precise under conditions of moderate genetic diversity, limited sample sizes and few microsatellite loci, as often occurs with natural populations. We used the wood stork as a model species and we offered a workflow that researchers can follow for monitoring bird breeding colonies. Our approach started with simulations using five estimators of N_b and the theoretical results were validated with empirical data collected from breeding colonies settled in the Brazilian Pantanal wetland. In parallel, we estimated census size using a corrected method based on counting active nests. Both in simulations and in natural populations, the approximate Bayesian computation (ABC) and sibship assignment (SA) methods yielded more precise estimates than the linkage disequilibrium, heterozygosity excess, and molecular coancestry methods. In particular, the ABC method performed best with few loci and small sample sizes, while the other estimators required larger sample sizes and at least 13 loci to not underestimate N_b. Moreover, according to our N_b/N_a estimates (values were often ≤0.1), the wood stork colonies evaluated could be facing the loss of genetic diversity. We demonstrate that the combination of genetic and census estimates is a useful approach for monitoring natural breeding bird populations. This methodology has been recommended for populations of rare species or with a known history of population decline to support conservation efforts.     

Conservation genetics of the endangered endemic Sambucus palmensis Link (Sambucaceae) from the Canary Islands

Five polymorphic microsatellites (simple sequence repeat; SSR) markers were used to estimate the levels of genetic variation within and among natural populations from different islands of the endangered endemic from the Canary Islands Sambucus palmensis Link (Sambucaceae). Genetic data were used to infer potential evolutionary processes that could have led to present genetic differentiation among islands. The levels of genetic variability of S. palmensis were considerably high; proportion of polymorphic loci (P = 100%), mean number of alleles per locus (A = 6.8), average expected heterozygosity (He = 0.499). In spite of its small population size and endemic character, 58 different multilocus genotypes were detected within the 165 individuals analyzed. All samples located in different islands always presented different multilocus genotypes. Principal Coordinates Analysis, genetic differentiation analysis (F _ST and G _ST ′) and Bayesian Cluster Analysis revealed significant genetic differences among populations located in different islands. However, this genetic differentiation was not recorded among Tenerife and La Gomera populations, possibly revealing the uncontrolled transfer of material between both islands. AMOVA analysis attributed 77% of the variance to differences within populations, whereas 8% was distributed between islands. The levels of genetic differentiation observed among populations, and the genetic diversity distribution within populations in S. palmensis, indicate that management should aim to conserve as many of the small populations as possible. Concentrating conservation efforts only on the few large populations would result in the likelihood of loss of genetic variability for the species.     

Population structure and genetic diversity of Rana dalmatina in the Iberian Peninsula

The increasing fragmentation of natural habitats may strongly affect patterns of dispersal and gene flow among populations, and thus alter evolutionary dynamics. We examined genetic variation at twelve microsatellite loci in the Agile frog (Rana dalmatina) from 22 breeding ponds in the Iberian Peninsula, the southwest limit of its range, where populations of this species are severely fragmented and are of conservation concern. We investigated genetic diversity, structure and gene flow within and among populations. Diversity as observed heterozygosities ranged from 0.257 to 0.586. The mean number of alleles was 3.6. Just one population showed a significant F _IS value. Four populations show evidence of recent bottlenecks. Strong pattern of structure was observed due to isolation by distance and to landscape structure. The average degree of genetic differentiation among populations was F _ST = 0.185. Three operational conservation units with metapopulation structure were identified. Additionally, there are some other isolated populations. The results reinforce the view that amphibian populations are highly structured even in small geographic areas. The knowledge of genetic structure pattern and gene flow is fundamental information for developing programmes for the preservation of R. dalmatina at the limits of its geographic distribution.