Two sibling species sympatrically breeding: a new conservation concern for the critically endangered Balearic shearwater

Two Puffinus shearwater species are endemic to the Mediterranean region: the Yelkouan shearwater P.␣yelkouan breeding in the central and eastern, and the critically endangered Balearic shearwater P.␣mauretanicus in the western, exclusively breeding at the Balearic archipelago. All individuals caught in Minorca, a Balearic breeding site, showed phenotypic traits of Yelkouan shearwaters. Ten birds from that colony were sampled and genetically analysed. A fragment of 857 base pairs of the cytochrome b was sequenced and compared to published sequences of both species obtained from GenBank. A Neighbor-Joining (NJ) reconstruction distinguished two clades with high bootstrap support, showing that both species bred sympatrically in that colony. This result was unexpected since shearwaters do not usually form mixed colonies. New conservation concerns arise also from our study: at first, breeding population of Yelkouan shearwaters is even smaller than previously thought; additionally, other potential threats emerge, such as hybridization and ecological competition.     

Polymorphic microsatellite markers for the critically endangered Balearic shearwater, Puffinus mauretanicus

Ten novel polymorphic microsatellite loci were isolated and characterized from the Balearic shearwater (Puffinus mauretanicus), a critically endangered seabird. The developed loci revealed a relatively low number of alleles per locus, as well as low levels of polymorphism (H_O = 0.377 ± 0.241). One of the loci appeared to be W‐linked. All polymorphic loci were successfully amplified in its closely related species, the Yelkouan shearwater (Puffinus yelkouan). These microsatellite markers would be useful for assessing population structure in the Balearic shearwater and the possible hybridization process between both shearwaters species.     

Genetic and phenotypic differentiation between the critically endangered Balearic shearwater and neighboring colonies of its sibling species

Understanding the demographic and evolutionary processes within and between populations is essential for developing effective management strategies. Thus, for establishing good conservation policies both genetic and phenotypic studies are crucial. We carried out an integrated analysis of genetic and phenotypic characters of the critically endangered Balearic shearwater Puffinus mauretanicus (182 individuals) and compared them with those of 2 nearby colonies of Yelkouan shearwater P. yelkouan (40 individuals), a species for which hybridization has been hypothesized. The results of the microsatellite analyses were compared with previous mitochondrial DNA analyses. Genetic variability was low in the Balearic shearwater and high levels of inbreeding were revealed at local scale. Most dispersal in Balearic shearwaters was to neighboring sites, even though low levels of population structure were found. The admixture between the 2 species was much higher at nuclear than at mitochondrial level, but phenotypic characters would seem to indicate that a lower level of admixture exists. Individual nuclear DNA, mtDNA, and phenotype did not match at individual level, showing that migration alone cannot explain this phenomenon. We suggest that these 2 young shearwater species could have been involved in processes of divergence and admixing. However, due to the longer coalescence times in nuclear markers, incomplete lineage sorting cannot be ruled out.     

Subpopulation range estimation for conservation planning: a case study of the critically endangered Cross River gorilla

Measuring and characterizing the area utilized by a population or species is essential for assessment of conservation status and for effective allocation of habitat to ensure population persistence. Yet population-level range delineation is complicated by the variety of available techniques coupled with a lack of empirical methods to compare the relative value of these techniques. This study assesses the effect of model choice on resulting subpopulation range estimation for the critically endangered and patchily distributed Cross River gorilla, and evaluates the conservation conclusions that can be drawn from each model. Models considered range from basic traditional approaches (e.g. minimum convex polygon) to newer home range techniques such as local convex hull (LoCoH). Overlap analysis comparing sub-sampled to complete data sets are used to evaluate the robustness of various modeling techniques to data limitations. Likelihood cross validation criterion is employed to compare core range model performance. Results suggest that differing LoCoH models produce similar range estimates, are robust to data requirements, provide a good fit for core habitat estimation, and are best able to detect unused habitat within the subpopulation range. LoCoH methods may thus be useful for studies into habitat selection and factors limiting endangered species distributions. However, LoCoH models tend to over-fit data, and kernel methods may provide similar information about animal space use while supporting protection of larger swaths of critical habitat. Subpopulation range analyses for conservation/management planning should therefore explore multiple modeling techniques, and employ both qualitative and quantitative assessments to select the best models to inform decision making for species of conservation concern.     

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 in fragmented populations of the critically endangered spider orchid Caladenia huegelii: implications for conservation

The Orchidaceae is characterised by a diverse range of life histories, reproductive strategies and geographic distribution, reflected in a variety of patterns in the population genetic structure of different species. In this study, the genetic diversity and structure was assessed within and among remnant populations of the critically endangered sexually deceptive orchid, Caladenia huegelii. This species has experienced severe recent habitat loss in a landscape marked by ancient patterns of population fragmentation within the Southwest Australian Floristic Region, a global biodiversity hotspot. Using seven polymorphic microsatellite loci, high levels of within-population diversity (mean alleles/locus = 6.73; mean H _E = 0.690), weak genetic structuring among 13 remnant populations (F _ST = 0.047) and a consistent deficit of heterozygotes from Hardy–Weinberg expectation were found across all populations (mean F _IS = 0.22). Positive inbreeding coefficients are most likely due to Wahlund effects and/or inbreeding effects from highly correlated paternity and typically low fruit set. Indirect estimates of gene flow (Nm = 5.09 using F _ST; Nm = 3.12 using the private alleles method) among populations reflects a historical capacity for gene flow through long distance pollen dispersal by sexually deceived wasp pollinators and/or long range dispersal of dust-like orchid seed. However, current levels of gene flow may be impacted by habitat destruction, fragmentation and reduced population size. A genetically divergent population was identified, which should be a high priority for conservation managers. Very weak genetic differentiation indicates that the movement and mixing of seeds from different populations for reintroduction programs should result in minimal negative genetic effects.     

New distributional records and conservation implications for the critically endangered greater bamboo lemur Prolemur simus

To improve our knowledge of the distribution of the critically endangered greater bamboo lemur Prolemur simus, we surveyed 6 sites in eastern Madagascar. We found its characteristic feeding signs at 5 sites and made a direct sighting at one of these. One site represents a northern extension of 45 km of the known extant range of the species. Two sites are located in a forest corridor approximately halfway between the previously known southern and northern populations, therefore suggesting a broadly continuous distribution of the species within its range rather than the previously suspected distribution of two distinct populations separated by a distance of over 200 km. Our results illustrate the benefit of species-focussed surveys in determining the true distribution of endangered species, a realistic measure which is necessary in order to assess their current status and to prioritise long-term conservation interventions.     

Translocation as an ultimate conservation measure for the long-term survival of a critically endangered freshwater mussel

Hydrobiologia - Pseudunio auricularius (Spengler, 1793) is one of the most threatened unionid species worldwide. Translocation is considered one of the ultimate actions that can save this species...     

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.     

High genetic structure of the Cozumel Harvest mice,a critically endangered island endemic: conservation implications

We assessed the genetic structure and diversity of Reithrodontomys spectabilis, a critically endangered, endemic rodent from Cozumel Island, México. A total of 90 individuals were trapped from September 2001 to January 2005. Microsatellite data analysis revealed high genetic diversity values: a total of 113 alleles (average 12.5 per locus), H _o  = 0.78, H _e  = 0.80. These high values can be related to Cozumel’s size (478 km²) and extensive native vegetation cover, factors that could be promoting a suitable population size, high heterozygosity and the persistence of rare alleles in the species, as well as some long-term movement of individuals between sampling localities. A strong genetic structure was also observed, with at least four genetic groups, associated with a pattern of isolation by distance. We found a strong allelic and genetic differentiation shown between localities, with negligible recent gene flow and low inbreeding coefficients. The species life history and ecological characteristics—being nocturnal, semi-terrestrial, a good tree climber, having lunar phobia and significant edge effect—are likely affecting its genetic structure and differentiation. The high genetic diversity and population structure award R. spectabilis a significant conservation value. Our results can serve as a basis for future research and conservation of the species, particularly considering the problems the island is facing from habitat perturbation, urbanization and introduction of exotic species. In view of the structure and genetic variability observed, it is essential to establish and reinforce protected areas and management programs for the conservation of the endemic and endangered Cozumel Harvest mice.     

Novel micropropagation and weaning methods for the integrated conservation of a critically endangered tree species, Medusagyne oppositifolia

Medusagyne oppositifolia is a critically endangered monotypic species with only a few trees left in the wild. Due to invasion and habitat clearance, genetic diversity of this species has been reduced to an alarmingly low level. Long-term in vitro cultures of M. oppositifolia were slow-growing and eventually lost the ability to multiply and root. Because of the lack of new seed and vegetative materials, efforts were made to rejuvenate the existing cultures. This was attempted by a series of steps involving serial pruning of the shoot tips and growing on a modified MS medium containing silver nitrate and thidiazuron. Rapid cycling clonal propagules were produced this way from cultured nodes. Isolated shoots were rooted on Florialite?, and rooted plants were weaned in a transition stage using a Nutrient Film Technique (NFT) hydroponic system before being completely weaned under glasshouse conditions. The importance of screening cultures for multiplication and rooting efficiency during long-term maintenance is discussed in the context of stress metabolite production and its influence on growth in culture. Factors contributing to rejuvenation of old cultures, application of unconventional supporting systems for rooting and application of the hydroponic system as a weaning tool are also discussed.     

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.     

Population genetics and conservation priorities for the critically endangered island endemic Delphinium pentagynum subsp. formenteranum (Ranunculaceae)

Isozyme electrophoresis was used to evaluate levels of genetic diversity and population genetic structure of the critically endangered (CR) perennial larkspur, Delphinium pentagynum subsp. formenteranum (Ranunculaceae), endemic to the island of Formentera (Balearic Islands, Spain). There is only one known population for this taxon, containing only 480 individuals. Moderate values of diversity were detected (P = 40.7%, A = 1.6 and H _e = 0.180), within the range observed in other surveyed larkspurs, but higher than most island endemics. Moderate levels of inbreeding were detected, probably as a consequence of the population's genetic structuring (biparental inbreeding). Threats to this taxon are mainly anthropogenic (fires, grazing, pathway works, and building pressures), although given that only one population exists, stochastic risks cannot be ignored. Conservation of D. pentagynum subsp. formenteranum requires in situ strategies, such as implementing a monitoring program and establishing a botanical reserve, and ex situ strategies, such as collection and long-term storage of seeds.     

Managing polyploidy in ex situ conservation genetics: the case of the critically endangered Adriatic sturgeon (Acipenser naccarii)

While the current expansion of conservation genetics enables to address more efficiently the management of threatened species, alternative methods for genetic relatedness data analysis in polyploid species are necessary. Within this framework, we present a standardized and simple protocol specifically designed for polyploid species that can facilitate management of genetic diversity, as exemplified by the ex situ conservation program for the tetraploid Adriatic sturgeon Acipenser naccarii. A critically endangered endemic species of the Adriatic Sea tributaries, its persistence is strictly linked to the ex situ conservation of a single captive broodstock currently decimated to about 25 individuals, which represents the last remaining population of Adriatic sturgeon of certain wild origin. The genetic variability of three F1 broodstocks available as future breeders was estimated based on mitochondrial and microsatellite information and compared with the variability of the parental generation. Genetic data showed that the F1 stocks have only retained part of the genetic variation present in the original stock due to the few parent pairs used as founders. This prompts for the urgent improvement of the current F1 stocks by incorporating new founders that better represent the genetic diversity available. Following parental allocation based on band sharing values, we set up a user-friendly tool for selection of candidate breeders according to relatedness between all possible parent-pairs that secures the use of non-related individuals. The approach developed here could also be applied to other endangered tetraploid sturgeon species overexploited for caviar production, particularly in regions lacking proper infrastructure and/or expertise.     

MtDNA diversity of the critically endangered Mekong giant catfish (Pangasianodon gigas Chevey, 1913) and closely related species: implications for conservation

Catfishes of the family Pangasiidae are an important group that contributes significantly to the fisheries of the Mekong River basin. In recent times the populations of several catfish species have declined, thought to be due to overfishing and habitat changes brought about by anthropogenic influences. The Mekong giant catfish Pangasianodon gigas Chevey, 1913 is listed as Critically Endangered on the IUCN Red List. In the present study, we assessed the level of genetic diversity of nine catfish species using sequences of the large subunit of mitochondrial DNA (16S rRNA). Approximately 570 base pairs (bp) were sequenced from 672 individuals of nine species. In all species studied, haplotype diversity and nucleotide diversity ranged from 0.118±0.101 to 0.667±0.141 and from 0.0002±0.0003 to 0.0016±0.0013, respectively. Four haplotypes were detected among 16 samples from natural populations of the critically endangered Mekong giant catfish. The results, in spite of the limited sample size for some species investigated, indicated that the level of genetic variation observed in wild populations of the Mekong giant catfish (haplotype diversity=0.350±0.148, nucleotide diversity=0.0009±0.0008) is commensurate with that of some other related species. This finding indicates that (1) wild populations of the Mekong giant catfish might be more robust than currently thought or (2) present wild populations of this species carry a genetic signature of the historically larger population(s). Findings from this study also have important implications for conservation of the Mekong giant catfish, especially in designing and implementing artificial breeding programme for restocking purposes.     

Applying molecular genetic tools to the conservation and action plan for the critically endangered Far Eastern leopard (Panthera pardus orientalis)

A role for molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Because conservation genetic analyses provide essential insights on taxonomic status, recent evolutionary history and current health of endangered taxa, they are considered in nearly all conservation programs. Genetic analyses of the critically endangered Far Eastern, or Amur leopard, Panthera pardus orientalis, have been done recently to address all of these questions and develop strategies for survival of the leopard in the wild. The genetic status and implication for conservation management of the Far Eastern leopard subspecies are discussed.     

Back from the brink: potential for genetic rescue in a critically endangered tree

Rare plant species are vulnerable to genetic erosion and inbreeding associated with small population size and isolation due to increasing habitat fragmentation. The degree to which these problems undermine population viability remains debated. We explore genetic and reproductive processes in the critically endangered long-lived tropical tree Medusagyne oppositifolia, an endemic to the Seychelles with a naturally patchy distribution. This species is failing to recruit in three of its four populations. We evaluate whether recruitment failure is linked to genetic problems associated with fragmentation, and if genetic rescue can mitigate such problems. Medusagyne oppositifolia comprises 90 extant trees in four populations, with only the largest (78 trees) having successful recruitment. Using 10 microsatellite loci, we demonstrated that genetic diversity is high (H(E) : 0.48-0.63; H(O) : 0.56-0.78) in three populations, with only the smallest population having relatively low diversity (H(E) : 0.26 and H(O) : 0.30). All populations have unique alleles, high genetic differentiation, and significant within population structure. Pollen and seed dispersal distances were mostly less than 100 m. Individuals in small populations were more related than individuals in the large population, thus inbreeding might explain recruitment failure in small populations. Indeed, inter-population pollination crosses from the large donor population to a small recipient population resulted in higher reproductive success relative to within-population crosses. Our study highlights the importance of maintaining gene flow between populations even in species that have naturally patchy distributions. We demonstrate the potential for genetic and ecological rescue to support conservation of plant species with limited gene flow.     

Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation

Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value.     

Extremely low levels of genetic variation in the critically endangered monotypic fern genus Mankyua chejuense (Ophioglossaceae) from Korea: Implications for conservation

We investigated allozyme variation in 16 populations of the rare monotypic fern Mankyua chejuense and 11 populations of a closely related, widespread fern Botrychium ternatum. Genetic diversity was extremely low in the critically endangered M. chejuense (%P_s = 10%, A_s = 1.13, and H_es = 0.007) relative to that found in B. ternatum (%P_s = 22.7%, A_s = 1.23, and H_es = 0.026). We detected homozygous genotypes for alternative alleles at a few loci, which suggests occurrence of intragametophytic self-fertilization. High degrees of population differentiation were found in both M. chejuense (F_ST = 0.245) and B. ternatum (F_ST = 0.414).The extremely low levels of genetic diversity in M. chejuense would be attributable to inbreeding coupled with random genetic drift and rarity. In situ protection of habitats of standing populations and ex situ preservation of several populations are suggested for the development of conservation strategies for M. chejuense.