Characterization of microsatellite loci in the endangered grand spider orchid Caladenia huegelii (Orchidaceae)

The orchid genus Caladenia is species rich with many threatened and endangered taxa. We report on the isolation and characterization of microsatellite loci in the rare Caladenia huegelii for molecular evaluation of this and the closely related C. thinicola as part of the development of conservation initiatives. Eight di‐ and trinucleotide loci were screened using 30 samples from each species. All loci were highly variable, with similar levels of heterozygosity and number of alleles across both species. These markers will be highly informative for population studies in both species.     

Genetic diversity and differentiation in the critically endangered orchid (Amitostigma hemipilioides): implications for conservation

Amitostigma hemipilioides is an endangered terrestrial orchid endemic to China. In the Southwestern China, the species is found restricted growing in the karst limestone or rock in the edge of farmland, moist hillsides or river. In the present study, the genetic diversity and differentiation was estimated within and among habitats, populations and groups of this species by ISSR markers. Using 13 polymorphic primers, an intermediate level of genetic diversity was found at the species level and population level with the percentage of polymorphic bands (P) of 64.7 and 50.9 %, Shannon index of diversity (I) of 0.3873 and 0.2949, respectively. The analysis of molecular variance (AMOVA) showed that the high level of population differentiation was presented with 45.63 % relative to the total genetic variation residing among eight populations. It was noteworthy that as much as 69.17 % of the total diversity was most likely attributed to the difference among the populations in fragmentation habitat conditions, while 16.32 % of the total diversity could be attributed to the difference among the populations in stable and favorable habitat conditions. The in situ conservation is a top strategy, thus the mycorrhizal fungi and pollinators are protected by protecting habitat and avoiding fragmentation which is helpful to the cycle of this endangered orchid species and recovery of its wild populations. In addition, to maintaining the germplasm bank of this species, the ex situ conservation by habitat simulation and reintroduction has been considered in the living collection at Kaili University ecological garden and Yuntai Mountain scenic spot using the plants collected in this study.     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Genetic diversity in natural populations of the endangered Neotropical orchid Telipogon peruvianus

Telipogon peruvianus is a highly restricted, sexually deceptive, Neotropical orchid species, endemic to the southern Peruvian Andes. It is only known from two localities, which are affected by anthropogenic disturbance. Here, we investigated whether the restricted distribution of T. peruvianus has led to low genetic diversity and inbreeding, thus threatening its survival. We isolated 10 novel microsatellite loci specific for T. peruvianus (and that also cross-amplified in related species) from two adjacent populations. We found that genetic diversity within populations was only moderately reduced, along with some evidence of inbreeding. We found low levels of genetic differentiation, suggesting connectivity by pollen/seed flow between the two populations. Effective population size was comparable to the real number of flowering individuals in the populations and we did not detect the signature of recent bottlenecks. Taken together, these results show that, despite increasing anthropogenic pressure, the two investigated populations of T. peruvianus still host valuable genetic diversity that should be preserved through appropriate conservation strategies.     

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 of Astragalus nitidiflorus, a critically endangered endemic of SE Spain,and implications for its conservation

Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population structure in five populations of Astragalus nitidiflorus, a critically endangered species endemic to southeast Spain. Eight primers amplified 78 bands with 40 (51.3%) being polymorphic. Statistical results indicated a low genetic diversity at the population and species level, with percentages of polymorphic bands (PPB) ranging from 28.2 to 37.2% (an average of 31.8%), and means of gene diversity (H_E) of 0.129 and 0.171 respectively. The Shannon’s index (SI) ranged from 0.160 to 0.214 at the population level and was 0.260 at the species level. A low level of genetic differentiation among populations was detected, based on the Shannon’s information index (0.297), the coefficient of genetic differentiation between populations (G_ST = 0.2418) and AMOVA analysis (Φ_ST = 0.255). The estimated gene flow (Nm) was 0.789. The high genetic connectivity found among populations of A. nitidiflorus is an evidence of a recent habitat fragmentation. In addition, a bottleneck event in the past has been revealed, with a subsequent reduction of population size and a loss of genetic variation. Based on these results, the conservation strategy of A. nitidiflorus was proposed.     

Contemporary breeding biology of critically endangered Regent Honeyeaters: implications for conservation

Identifying factors influencing the demographics of threatened species is essential for conservation, but a lack of comprehensive demographic data often impedes the effective conservation of rare and mobile species. We monitored breeding of critically endangered and semi‐nomadic Regent Honeyeaters Anthochaera phrygia (global population c. 100 pairs) over 3 years throughout their range. Overall nest success probability (0.317) was highly spatially variable and considerably lower than previous estimates for this (and many other honeyeater) species, as was productivity of successful nests (mean 1.58 juveniles fledged). Nest surveillance revealed high predation rates by a range of birds and arboreal mammals as the primary cause of nest failure. An estimated 12% of pairs either failed to establish a territory or their nests did not reach the egg stage. We also found a male bias to the adult sex ratio, with an estimated 1.18 males per female. Juvenile survival for the first 2 weeks after fledging was high (86%). Management interventions that aim to increase nest success in areas of low nest survival must be investigated to address an apparent decline in reproductive output and avoid extinction of the Regent Honeyeater. We show that temporal and spatial variation in the breeding success of rare and highly mobile species can be quantified with robust population monitoring using sampling regimens that account for their life histories. Understanding the causes of spatio‐temporal variation in breeding success can enhance conservation outcomes for such species through spatially and temporally targeted recovery actions.     

Floral and pollination biology of the critically endangered insular orchid Calanthe amamiana: Implications for in situ conservation

Calanthe amamiana is a terrestrial orchid that thrives on the shady forest floors of broad-leaved forests on Amami Island, central Ryukyus, Japan. Here, I report on the floral and pollination biology of this critically endangered orchid based on a 7-year study conducted in the species' natural habitats. As the nectarless flowers are probably self-compatible but are unable to autonomously self-pollinate, they need to attract pollen vectors by deceit for pollination. To advertise themselves, the flowering inflorescences, which comprise a few to dozens of flowers, use whitish floral colors that are comparatively conspicuous against the shaded background. The exclusive pollinator was the long-horned bee Eucera okinawae, which nests in soils of the forest floor, although bee abundance varied considerably among study years. Natural fruit-set ratios were generally low but showed interannual variation. The annual fruit-set ratios were positively correlated with annual bee abundance, thereby suggesting that under natural conditions, the orchid is pollinator-limited. Given that C. amamiana is dependent exclusively on E. okinawae for pollination and is also probably pollinator-limited, conservation plans for this orchid should take into consideration the welfare of long-horned bees. In particular, as nectariferous flowers were unavailable within broad-leaved forests, to satisfy the foraging requirements of long-horned bees, conservation managers should be cognizant of conservation values of flowering plants distributed in sun-lit sites near orchid habitats, and manage those in an appropriate manner.     

Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation

Eighteen populations of the endangered aromatic and medicinal plant Mentha cervina (Lamiaceae) were sampled across its natural range, in the western half of the Iberian Peninsula, and inter-simple sequence repeats (ISSRs) markers were used to assess genetic diversity and population structure. M. cervina populations exhibited a relatively low genetic diversity (percentage of polymorphic loci PPB = 14.2–58.3%, Nei's genetic diversity H_e = 0.135–0.205, Shannon's information index I = 0.08 − 0.33). However, the genetic diversity at species level was relatively high (PPB = 98.3%; H_e = 0.325; I = 0.23). The results of the analysis of molecular variance indicated very structured populations, with 50% of the variance within populations, 44% among populations and 6% between regions defined by hydrographic basins, in line with the gene differentiation coefficient (G_ST = 0.532). A Mantel test did not find significant correlation between genetic and geographic distance matrices (r = 0.064), indicating that isolation by distance is not shaping the present genetic structure. The levels and patterns of genetic diversity in M. cervina populations were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, low capacity of dispersion, small effective size and habitat fragmentation. The high genetic differentiation among populations indicates the necessity of conserving the maximum possible number of populations. The results also provide information to select sites for ex situ conservation. Optimal harvesting strategies, cultivation and tissue culture should also be developed as soon as possible to guarantee sustainable use of the species under study.     

Autogamous seed set in a critically endangered orchid in Japan: pollination studies for the conservation of Nervilia nipponica

The pollination biology of the critically endangered Nervilia nipponica was investigated at two sites on Shikoku Island, Japan, to shed light on the species' ecological strategy and to help prioritise management decisions for its conservation. Though the solitary flowers failed to attract pollinators, high rates of fruit set were observed in control plants at both sites. Comparable rates were obtained in bagged plants and following induced autogamous and xenogamous pollinations, indicating that the species is capable of outbreeding, but self-compatible and not pollinator-limited for fruit set under natural conditions; rates were significantly lower following emasculation. The number of seeds set per fruit and their fertility did not differ significantly between open-, self- and cross-pollinated treatments. Epifluorescence microscopy revealed that, in open-pollinated plants, pollen grains germinate within the anther on the first day of anthesis, and that fertilisation occurs within the following 96 hours. The position of the stigma, which overlaps the clinandrium, friable pollinia, and the absence of a rostellum ensure efficient, non-mechanical autopollination. Though this confers reproductive assurance, inbreeding depression may be limiting fitness at subsequent stages of the recruitment cycle and low genetic diversity is inferred.     

Reproductive biology of Cyrtopodium punctatum in situ: implications for conservation of an endangered Florida orchid

Cyrtopodium punctatum (Linnaeus) Lindley is an endangered epiphytic orchid restricted in the USA to southern Florida. This species has been extensively collected from the wild since the early 1900s, and today only a few plants remain in protected areas. As part of a conservation plan, a reproductive biology study was conducted to better understand the ecology of this species in Florida. Cyrtopodium punctatum relies on a deceit pollination system using aromatic compounds to attract pollinators. Nine aromatic compounds were identified as components of the fragrance of C. punctatum inflorescences, including two compounds that are known to be Euglossine bee attractants. However, this group of bees is not native to Florida. Of the four bee species observed to visit C. punctatum flowers in the present study, carpenter bees ( Xylocopa spp.) are likely to be the main pollinators. Pollination experiments demonstrated that C. puntatum is self-compatible, but requires a pollinator and thus does not exhibit spontaneous autogamy. In addition, the rates of fruit set were significantly higher for flowers that were outcrossed (xenogamy) than for those that were self-crossed. Thus, the species has evolved a degree of incompatibility. Examples of natural pollination and fruit set were observed during the present study (2007–2008), but the rates of reproduction were modest as a consequence of the low plant numbers and possible changes in insect densities as a result of anthropogenic influences.     

Genetic diversity and differentiation between the two remaining populations of the critically endangered Mediterranean monk seal

The Mediterranean monk seal Monachus monachus , is a critically-endangered species of which only two populations, separated by c . 4000 km, remain: the eastern Mediterranean (150–300 individuals) and the Atlantic/western Sahara populations (100–130 individuals). We measured current levels of nuclear genetic variation at 24 microsatellite loci in 12 seals from the eastern Mediterranean and 98 seals from the western Sahara population and assessed differences between them. In both populations, genetic variation was found to be low, with mean allelic richness for the loci polymorphic in the species of 2.09 and 1.96, respectively. For most loci, the observed allele frequency distributions in both populations were discontinuous and the size ranges similar. The eastern Mediterranean population had 14 private alleles and the western Sahara had 18, but with a much larger sample size. Highly significant differences in allele frequencies between the two populations were found for 14 out of 17 loci. F _ST between the two populations was 0.578 and the estimated number of migrants per generation was 0.046, both clearly indicating substantial genetic differentiation. From a conservation perspective, these results suggest that each population may act as a source for introducing additional genetic variation into the other population.     

Genetic diversity of the endangered species Rosa rugosa Thunb. in China and implications for conservation strategies

Rosa rugosa Thunb. is one of the dominant and important shrub species in estuary dunes and shingle beaches of northern China. However, its area of distribution, the number of populations, and the size of each population have decreased rapidly in the past two decades because of habitat degradation and loss. Random amplified polymorphic DNA markers were used to determine the genetic diversity of four remaining large natural populations of R. rugosa and to discuss an effective conservation strategy for this endangered species in China. High genetic variations were detected in R. rugosa populations in China. The mean percentage of polymorphic loci （P%） within four local populations was 57.99%, with the P% of the total population being 75.30%. Mean Shannon＇s information index （H0） was 0.2826, whereas total Ho was 0.3513. The genetic differentiation among populations was 0.1878, which indicates that most genetic diversity occurs within populations. Population Tumenjiang （TMJ） showed the highest genetic diversity （P% = 66.27%; H0 = 0.3117） and contained two exclusive bands. Population Changshandao （CSD） showed higher genetic diversity （P% =59.04%; H0 = 0.3065）. Populations TMJ and CSD contained 95.33% and 99.33%, respectively, of loci with moderate to high frequency （P〉0.05） of the total population. These results indicate that populations TMJ and CSD should be given priority for in situ conservation and regarded as seed or propagule sources for ex situ conservation. The results of the present study also suggest that R. rugosa in China has become endangered as a result of human actions rather than genetic depression of populations; thus, human interference should be absolutely forbidden in R. rugosa habitats.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):implications for its conservation

Omphalogramma souliei Franch. Is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. Souliei in NW Yunnan, China. The genetic diversity at the species level is low with P= 42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenog-amy predominated and autogamy played an assistant role in O. Souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae): implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

Genetic diversity analysis of the endangered slipper orchid Phragmipedium longifolium in Costa Rica

Phragmipedium longifolium is an endangered terrestrial orchid. In Costa Rica, these plants are found growing in small and isolated patches, some of them consisting of just four to six individuals. Information about the genetic variability within and among populations is very important for the conservation of this endangered species. A total of 160 samples were collected in six locations and analyzed with amplified fragment length polymorphism technique. The genetic diversity of P. longifolium in Costa Rica (Hw = 0.1711) is high and differentiation among sampled locations is moderate (φ_pt = 0.2013) in comparison with results of studies in some other terrestrial orchid species using the same technique. The percentage of polymorphic loci was, on average, 51.5. The analysis of molecular variance (AMOVA) indicated the main genetic variation was within sampled locations (80%), even though the variation among locations was also significant. In situ conservation is recommended because, in addition to protecting habitat and avoiding fragmentation, mycorrhizal fungi and pollinators are also protected. Close proximity between populations is required to maintain high genetic variability through a gene flow continuum. It is suggested that conservation of patches with higher genetic variability be prioritized as many are located in unprotected areas. A germplasm bank for ex situ conservation has been established in the living collection at Lankester Botanical Garden using the plants collected in this study. Finally, a search for new locations of this species is also suggested.     

Genetic diversity and population structure of endangered endemic Paeonia jishanensis in China and conservation implications

Paeonia jishanensis, one of the most important ancestral species of cultivated tree peonies, is an endangered ornamental and medicinal plant endemic to China. A total of 236 individuals of P. jishanensis from 10 extant populations were analysed using 21 EST-SSR markers to assess their genetic diversity and population structure. Moderate genetic diversity levels (H_E = 0.340) and high genetic differentiation among populations (F_ST = 0.335) were revealed. Combining the results of STRUCTURE, PCoA and neighbour-joining analyses, the 10 populations were divided into four genetic groups that were significantly related to their geographical origins, which was further supported by hierarchical AMOVA showing the highest variation of 17.9% among groups. The Mantel test showed a significant positive correlation between genetic distance and geographic distance among populations (r = 0.873, P < 0.0001). The genetic structure of P. jishanensis may be due to limited gene flow hindered by vicariance and its breeding system: facultative vegetative reproduction. This study carries significant implications for the conservation and utilization of this endangered species.     

Genetic diversity within and among populations of the endangered species Taxus fuana (Taxaceae) from Pakistan and implications for its conservation

The West Himalayan yew, Taxus fuana Nan Li & R.R. Mill (Taxaceae), is an endangered species endemic to the Western Himalayas. An investigation of the genetic diversity of wild populations of T. fuana in Pakistan was undertaken. The genetic diversity and genetic structure was quantified using random amplified polymorphic DNA (RAPD) variation in 219 individuals of the 10 populations. Of the 32 universal primers screened 16 produced highly reproducible, clear RAPD bands. Using these primers, 193 discernible DNA fragments were generated, of which 164 (84.97%) were polymorphic. The statistical results indicated that there was a relatively low genetic diversity within populations (with percentages of polymorphic bands, PPB, ranging from 29.53 to 50.26%, with an average of 38.34% and a Nei's genetic diversity index (H_E) of 0.1165), and a high genetic differentiation among populations (G_ST = 0.5842, Φ_ST = 0.5685) within these populations. The gene flow (N_m) was low with only 0.3558.