Dinucleotide microsatellite primers designed for a critically endangered primate,the black lion tamarin (Leontopithecus chrysopygus)

Black lion tamarin (BLT) monkeys (Leontopithecus chrysopygus) have suffered a severe reduction in their natural range and are consequently critically endangered. Because allozyme data showed very low levels of variation, it was not clear if these monkeys had much genetic diversity. We designed microsatellite primers for BLTs, and from them we identified nine polymorphic loci, seven of which were tested on golden lion tamarins (GLTs) (Leontopithecus rosalia). All of the seven polymorphic loci and two other monomorphic BLT loci were polymorphic in GLTs. The microsatellite markers identified here are directly applicable to ongoing lion tamarin population and conservation genetics studies.     

Census and distribution of the golden lion tamarin (Leontopithecus rosalia)

During 1990–1992, a survey of the golden lion tamarin, Leontopithecus rosalia, was carried out throughout its known distribution area. Forest remnants were identified by visual interpretation of Landsat‐TM satellite images. Localities occupied by L. rosalia were first identified by interviews with local people. All forests more than 20 ha in size, and for which two or more interviews suggested the presence of the species, were surveyed using “play‐back” recordings of lion tamarin long calls. The total wild population of L. rosalia, including that of the Poço das Antas Biological Reserve, was estimated to be 562 individuals in 109 groups. The lion tamarins were generally found in four major areas of forest (six or more groups per forest, not including Poço das Antas), with a further 12 groups isolated in small forest patches. Currently the species' distribution is restricted to just four municipalities in the state of Rio de Janeiro: Silva Jardim, Cabo Frio, Saquarema, and Araruama. Although they are typically confined to lowland forest of <300 m altitude, L. rosalia was recorded at an altitude of 550 m in one locality. Average group size varied from 3.6 to 5.7 individuals, and densities from 0.39 groups/km² to 2.35 groups/km² (2.17 individuals/ km² to 8.53 individuals/km²). Six of the isolated groups found during the survey were successfully translocated to a forest of 2,400 ha. There is now also a significant population of reintroduced lion tamarins. Overall, however, the possibilities for further expansion of the wild population are severely limited. Am. J. Primatol. 59:29–44, 2003. © 2003 Wiley‐Liss, Inc.     

Informative microsatellites for genetic population studies of black-faced lion tamarins (Leontopithecus caissara)

Leontopithecus caissara is a critically endangered primate species from the Brazilian Atlantic Forest. Nineteen microsatellite loci, previously developed for congeneric species, were tested with 34 L. caissara individuals from Superagüi Island. Of the 19 loci, 17 (89.4%) produced robust alleles, nine (47.4%) of these proved to be polymorphic, with a total of 23 alleles and an average of 2.56 alleles per locus. Expected and observed heterozygosity averaged 0.483 and 0.561, respectively. The exclusion power for identifying the first parent of an arbitrary offspring was 0.315 over all loci. The results thus indicate both the usefulness and limitations of these nine microsatellite loci in the genetic analysis of L. caissara, as well as their potentiality for genetic investigation in other congeneric species.     

Microsatellite variation within and among recently fragmented populations of the golden lion tamarin (Leontopithecus rosalia)

Four variable microsatellite loci were used toexamine the genetic diversity and differentiation of golden lion tamarins (Leontopithecus rosalia) in four populations recently isolated by habitat fragmentation. Using Rst estimates of genetic differentiation, a considerable genetic divergence was detected among these populations, with an averagedifferentiation of 31%. Significant differences in allele number among these populations were found. However, the heterozygosity among these populations was not statistically different. These results suggestthat loss in allele diversity was faster than loss in heterozygosity. Conservation implications, particularly for golden lion tamarins, are then discussed. Loss of allelic diversity might be as serious a concern to endangered species as heterozygosity or inbreeding.     

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.     

Genetic structure of the threatened Phaedranassa schizantha (Amaryllidaceae)

Phaedranassa schizantha (Amaryllidaceae) is an endangered species endemic to Ecuador and two varieties have been described: P. schizantha var. schizantha and P. schizantha var. ignea. We assessed population genetic structure and demographic patterns in 11 populations across the range of the species using 13 microsatellite loci. Our data show that genetic diversity was generally lower in the southern part of the range and was especially low in populations closest to cities. We found significant population differentiation (F_ST = 0.14, D_EST = 0.34) and evidence of a genetic bottleneck. Genetic variation did not show isolation by distance. Instead, results suggest genetic barriers around two main cities. Bayesian analysis identified two genetic groups, neither of which represents either of the two varieties previously recognized. Coalescent analysis indicates a relatively recent colonization pattern between the two genetic groups (< 3000 generations). Conservation efforts need to be taken to facilitate genetic exchange between the groups, especially between locations that seem to be genetically isolated.     

Genetic divergence between Melipona quadrifasciata Lepeletier (Hymenoptera,Apidae) populations

Melipona quadrifasciata is a stingless bee widely found throughout the Brazilian territory, with two recognized subspecies, M. quadrifasciata anthidioides, that exhibits interrupted metasomal stripes, and M. quadrifasciata quadrifasciata, with continuous metasomal stripes. This study aimed to estimate the genetic variability of these subspecies. For this purpose, 127 colonies from 15 Brazilian localities were analyzed, using nine species-specific microsatellite primers. At these loci, the number of alleles ranged from three to 15 (mean: 7.2), and the observed heterozygosity (H_o) ranged from 0.03–0.21, while the expected heterozygosity (H_e) ranged from 0.23–0.47. The genetic distances among populations ranged from 0.03–0.45. The F_ST multilocus value (0.23) indicated that the populations sampled were structured, and the clustering analysis showed the formation of two subgroups and two more distant populations. The first group contained the subspecies M. quadrifasciata quadrifasciata, and the other, the subspecies M. quadrifasciata anthidioides and the two M. quadrifasciata populations with continuous metasomal stripes from northern Minas Gerais. These results confirmed that the yellow metasomal stripes alone are not a good means for correctly identifying the different subspecies of M. quadrifasciata.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

Population structure in the endangered Mauna Loa silversword, Argyroxiphium kauense (Asteraceae), and its bearing on reintroduction

Reintroduction of populations of endangered species is a challenging task, involving a number of environmental, demographic and genetic factors. Genetic parameters of interest include historical patterns of genetic structure and gene flow. Care must be taken during reintroduction to balance the contrasting risks of inbreeding and outbreeding depression. The Mauna Loa silversword, Argyroxiphium kauense, has experienced a severe decline in population size and distribution in the recent past. Currently, three populations with a total of fewer than 1000 individuals remain. We measured genetic variation within and among the remnant populations using seven microsatellite loci. We found significant genetic variation remaining within all populations, probably related to the recent nature of the population impact, the longevity of the plants, and their apparent self-incompatibility. We also found significant genetic differentiation among the populations, reinforcing previous observations of ecological and morphological differentiation. With respect to reintroduction, the results suggest that, in the absence of additional data to the contrary, inbreeding depression may not be a substantial risk as long as propagules for the founding of new populations are adequately sampled from within each source population before additional inbreeding takes place. The results further suggest that if mixing of propagules from different source populations is not required to increase within-population genetic variation in the reintroduced populations, it may best be avoided.     

Low genetic diversity and limited genetic structure across the range of the critically endangered Mexican howler monkey (Alouatta palliata mexicana)

Genetic diversity provides populations with the possibility to persist in ever-changing environments, where selective regimes change over time. Therefore, the long-term survival of a population may be affected by its level of genetic diversity. The Mexican howler monkey (Alouatta palliata mexicana) is a critically endangered primate restricted to southeast Mexico. Here, we evaluate the genetic diversity and population structure of this subspecies based on 83 individuals from 31 groups sampled across the distribution range of the subspecies, using 29 microsatellite loci. Our results revealed extremely low genetic diversity (H_O = 0.21, H_E = 0.29) compared to studies of other A. palliata populations and to other Alouatta species. Principal component analysis, a Bayesian clustering method, and analyses of molecular variance did not detect strong signatures of genetic differentiation among geographic populations of this subspecies. Although we detect small but significant F_ST values between populations, they can be explained by a pattern of isolation by distance. These results and the presence of unique alleles in different populations highlight the importance of implementing conservation efforts in multiple populations across the distribution range of A. p. mexicana to preserve its already low genetic diversity. This is especially important given current levels of population isolation due to the extreme habitat fragmentation across the distribution range of this primate.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Genetic structure, conservation genetics and evidence of speciation by range expansion in shy and white-capped albatrosses

Six variable microsatellite loci were used to examine genetic structuring in the closely related shy albatross (Thalassarche cauta) and white-capped albatross (T. steadi). First, levels of genetic differentiation between the species, and among three populations within each species, were analysed using amova, FST and RST. We found high levels of genetic structuring and detected many unshared alleles between the species, which provide strong evidence against any contemporary gene flow between them. Within each species, shy albatross populations were found to be genetically distinct whereas white-capped albatross populations were undifferentiated, which implies that dispersal events are much rarer in the former than in the latter. These results formed the basis for the recommendation that the three white-capped albatross populations (as a whole) and each shy albatross population be treated as separate units for conservation. Second, levels of genetic diversity and allelic patterns in shy and white-capped albatrosses were assessed for whether they support earlier mtDNA results suggesting that shy albatrosses arose through range expansion of white-capped albatrosses. All measures indicated lower genetic diversity within shy albatrosses than within white-capped albatrosses and upheld the hypothesis that shy albatrosses were founded by white-capped albatrosses.     

Genetic variation in populations of the endangered fish Ladigesocypris ghigii and its implications for conservation

1. The genetic variation of the endangered freshwater fish Ladigesocypris ghigii, endemic to the island of Rhodes (Greece), was investigated for nine populations, originating from seven different stream systems and a reservoir, both at the mtDNA and nuclear level, in order to suggest conservation actions. 2. Both restriction fragment length polymorphism analysis of five segments of mitochondrial DNA (ND‐5/6, COI and 12S‐16S rRNA) amplified by polymerase chain reaction, and random amplified polymorphic DNA analysis, revealed extremely low levels of intra‐population polymorphism. It is highly likely that the low intra‐population variability is the result of successive bottleneck events evident in shrinkage and expansion of the populations year after year, which may have led to a complete loss of several genotypes and haplotypes, and an increased degree of inbreeding. 3. Inter‐population genetic structuring was high, with fixation of haplotypes within six of the nine populations and fixation of alleles within populations originating from different waterbodies. It is probable that all haplotypes and/or alleles found were initially represented in all populations. However, because of the long time of isolation coupled with successive bottleneck and subsequent genetic drift, common mtDNA haplotypes and alleles among the populations may have become rare or extinct through stochastic lineage loss. 4. Although nucleotide divergence among haplotypes was very shallow, half of the haplotypes recorded (three of six), resulted from nucleotide changes on the 12S–16S rRNA segments, which are the most conserved part of the mitochondrial genome. This fact may indicate that the observed genetic variation did not necessarily result only from the retention of ancestral polymorphism, but may have arisen through mutation and complete lineage sorting over a relatively small number of generations, once the populations had become isolated from one another. 5. Our data suggest that two of the L. ghigii populations may be on independent evolutionary trajectories. Considering that each population appears so far well adapted within each site, all populations should be managed and conserved separately.     

Genetic diversity and gene flow within and between two different habitats of Primula merrilliana (Primulaceae), an endangered distylous forest herb in eastern China

Understanding whether and how different habitats shape population genetics is a fundamental question and a specific goal for evolutionary and conservation biology research. This study examined genetic diversity and gene flow within and between mountain and foothill habitats of Primula merrilliana, an endangered distylous forest herb in eastern China. Eleven population characteristics, including area, size and density variation, from the two habitats were also investigated. Mountain populations had significantly higher mean genetic diversity than foothill populations, which may be explained by stronger self‐incompatibility breeding system, more opportunity to use elevational shifts to track suitable sites under conditions of climate change and more heterogeneous environments in the former habitat, rather than by the differences of population size, gene flow and genetic drift intensity between them. Genetic analysis revealed that two distinct lineages, corresponding to the two habitats, diverged at China's ‘Last Glaciation’ (11 700–67 500 yr BP), suggesting this divergence was probably triggered by warmer climates during inter‐ (or post‐) glacial periods. Low unidirectional gene flow from mountain to foothill habitats, chiefly by seed dispersal, played a more important role in overall gene flow between habitats than within‐habitat gene flow. Within habitats, pollen contributes more substantially to gene flow than seed dispersal, especially in foothill habitats, possibly due to higher individual density and larger population sizes. These results have implications for the conservation in this and similar landscape areas and indicate the need to protect suitable habitats with wide elevational spans and sufficient size to permit ecological and elevational shifts in response to climatic changes. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 172–189.     

Genetic diversity, population structure, and conservation of Sophora moorcroftiana (Fabaceae), a shrub endemic to the Tibetan Plateau

Sophora moorcroftiana is a perennial leguminous low shrub endemic to the middle reaches of Yarlung Zangbo River in Tibet. It is an important species to fix sand dunes and to avoid the formation of shifting sands; therefore, its progressive over-exploitation may enhance land desertification. The levels and distribution of genetic variability of this species were evaluated from 10 natural populations at 24 loci encoding 13 enzymes, using allozyme analysis by starch gel electrophoresis. Data obtained revealed moderate levels of genetic variation within populations (Pp=27.5%, Ap=1.5, Hep=0.122) and a considerable divergence among populations (FST=0.199). Significant positive correlations (r2=0.49, p<0.05; r2=0.46, p<0.05) were found between elevation and both mean number of alleles per locus (A) and gene diversity (He) in the studied populations of S. moorcroftiana. Lower genetic diversity in lower elevation populations might be due to the negative effects of human pressures and habitat fragmentation, to adaptation to high altitudes as a consequence of a peripatric speciation process, or to directional gene flow along the river basin from the source populations located in the west at higher altitudes. The evaluation of the degree of threat has led to the inclusion of this species in the category of EN ("endangered"), and conservation strategies for this endemic species are discussed on the basis of these findings.     

Isolation and characterization of microsatellite markers for Sabatia campestris (Gentianaceae), an Illinois state‐endangered plant

Sabatia campestris is a regionally endangered prairie plant. Eight polymorphic loci have been generated for S. campestris (Gentianaceae) using non‐enriched and enriched libraries. Polymorphism was quantified from an isolated population from central Illinois yielding mean observed and expected heterozygosities of 0.723 and 0.801, respectively. The number of alleles per locus for this population ranged from three to 25 with a median of 15.5. These genetic markers will be used to determine population genetic structure of Illinois populations as well as to estimate fine‐scale gene flow rates.     

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

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