Restoration genetics of the vernal pool endemic Lasthenia conjugens (Asteraceae)

Restoration of habitat for endangered species often involves translocation of seeds or individuals from source populations to an area targeted for revegetation. Long-term persistence of a species is dependent on the maintenance of sufficient genetic variation within and among populations. Thus, knowledge and maintenance of genetic variability within rare or endangered species is essential for developing effective conservation and restoration strategies. Genetic monitoring of both natural and restored populations can provide an assessment of restoration protocol success in establishing populations that maintain levels of genetic diversity similar to those in natural populations. California’s vernal pools are home to many endangered plants, thus conservation and restoration are large components of their management. Lasthenia conjugens (Asteraceae) is a federally endangered self-incompatible vernal pool annual with gravity- dispersed seeds. Using the molecular technique of intersimple sequence repeats (ISSRs), this study assessed levels and patterns of genetic variability present within natural and restored populations of L. conjugens. At Travis Air Force Base near Fairfield, California, a vernal pool restoration project is underway. Genetic success of the ecologically based seeding protocol was examined through genetic monitoring of natural and restored populations over a three-year period. Genetic diversity remained constant across the three sampled generations. Diversity was also widely distributed across all populations. We conclude that the protocol used to establish restored populations was successful in capturing similar levels and patterns of genetic diversity to those seen within natural pools. This study also demonstrates how genetic markers can be used to inform conservation and restoration decisions.     

Conservation genetics of an endemic and endangered epiphytic Laelia speciosa (Orchidaceae)

We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, P(p) = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity H(O) = 0.302, the average expected heterozygosity H(E) = 0.382). Moderate levels of inbreeding (?f = 0.216, 95% confidence interval = 0.029-0.381) were found. Low levels of genetic differentiation were observed among populations ((p) = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r(2) = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.     

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.     

Conservation genetics of the endangered endemic Hawaiian genus Brighamia (Campanulaceae)

The endemic Hawaiian genus Brighamia (Campanulaceae) comprises two federally endangered, morphologically similar species, B. insignis from Kaua`i and Ni`ihau and B. rockii from Moloka`i. To assist the design of conservation management programs for these taxa, isozyme analyses were performed to assess the levels of genetic diversity at the population and species levels, including comparisons within and among seven natural populations and one ex situ collection each of B. insignis and B. rockii. Our sampling (N = 80) represents ~41% of all known individuals in the wild. Isozyme analyses revealed levels of genetic variation comparable to those reported for other Hawaiian flowering plant taxa but low levels of genetic variation at the population and species levels when compared to flowering plants in general. Ex situ individuals (N = 61) were genetically representative of natural populations and hence may appropriately serve as stock for population augmentations. The two morphologically similar Brighamia species were highly distinct genetically. The combination of morphological and ecological similarity with allozymic dissimilarity observed in Brighamia is unique among the Hawaiian taxa studied to date.     

Conservation genetics of Amorpha georgiana (Fabaceae), an endangered legume of the Southeastern United States

Amorpha georgiana (Fabaceae) is an endangered legume species found in longleaf pine savannas in the Southeastern United States. Approximately 900 individuals and 14 populations remain, most of which are concentrated in North Carolina. Eleven microsatellite loci were used to explore genetic diversity, population structure and recent population bottlenecks using genotypic data from 132 individuals collected at ten different localities. Although A. georgiana is quite rare, it exhibited high levels of genetic diversity (17.7 alleles/locus; H _o = 0.65, H _E = 0.75). Most of the genetic variation was found within rather than between populations of this species. The single remaining Georgia population was well differentiated from populations of the Carolinas ( F _ST > 0.1), which had weaker structure among them ( F _ST < 0.1). Only a geographically disjunct population showed strong evidence of a recent population bottleneck, perhaps due to a recent founder event. Hybridization with A. herbacea was also detected. For conservation management plans, A. georgiana populations in each geographic region (North Carolina, South Carolina and Georgia) plus a disjunct population in North Carolina (Holly Shelter) should be treated as separate management units for which in situ conservation, including habitat restoration and use of prescribed burns, should ensure persistence of this species and preservation of its evolutionary potential.     

Conservation genetics of an endangered orchid in eastern Canada

Platanthera leucophaea, the Eastern Prairie Fringed-orchid, is a globally imperilled species native to the USA and Canada, with its Canadian distribution limited to the Province of Ontario. In Canada, where P. leucophaea is listed as endangered, approximately 40 % of populations have been extirpated in recent years, and many remaining populations have experienced substantial declines. In this study, we investigated whether reduced population sizes have led to low genetic diversity and inbreeding. We also investigated the extent to which hybridization with Platanthera psycodes may be threatening the genetic integrity of P. leucophaea populations. We found that overall, genetic diversity is low, and inbreeding is high. This is despite evidence of regular gene flow between proximate populations, although more distant populations show high levels of genetic differentiation. At sites where P. leucophaea is sympatric with P. psycodes, interspecific hybridization occurs in a bidirectional manner, i.e. with both parental species acting as either pollen donor or pollen recipient. Inbreeding and low genetic diversity in all populations, and hybridization in some populations, may pose future threats to P. leucophaea, and should be considered in the future by biodiversity managers.     

Seed germination traits of Trapella sinensis (Trapellaceae), an endangered aquatic plant in Japan: Conservation implications

We investigated the effects of cold stratification, temperature, light, and oxygen conditions on seed germination of Trapella sinensis Oliver, an endangered aquatic plant in Japan. Seeds had physiological dormancy, and final germination rate increased with an increasing period of cold stratification. Seeds of T. sinensis had an almost absolute requirement for aerobic conditions to germinate. Also, alternating temperatures significantly promoted germination regardless of light conditions, although final germination percentage was twice to four times higher in light under constant temperature conditions. Suitable sites for germination of T. sinensis appear to be shallow water with alternating temperatures and sufficient oxygen and sediment anoxia, caused by eutrophication of water bodies, may have prevented recruitment of T. sinensis from seed.     

Conservation genetics of Hebe speciosa (Plantaginaceae) an endangered New Zealand shrub

All extant populations of Hebe speciosa (Plantaginaceae), a threatened endemic New Zealand shrub, were investigated using the amplified fragment length polymorphism technique (AFLP). Genetic diversity indices varied significantly among geographical regions and were positively correlated with population size. Among-population genetic differentiation was high (mean pairwise Φ_ST = 0.47), implying complex historical relationships between disjunct populations and negligible contemporary gene flow. Southern populations exhibited extremely low genetic diversity relative to those found in Northland, suggesting that these populations may be more recent in origin. Patterns of genetic relationship among some populations indicate pre-European Māori dispersal and cultivation. The three northernmost populations were found to contain the majority of the species' remaining genetic diversity and thus, should be a focus for future conservation management. Some southern sites may also be culturally significant as evidence of Māori trade and cultivation of Hebe speciosa .  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 229–239.     

Conservation genetics of an endemic mountaintop salamander with an extremely limited range

Montane regions can promote allopatric speciation and harbor unique species with small ranges. The southern Appalachians are a biodiversity hotspot for salamanders, and several montane endemics occur in the region. Here, we present the first DNA sequence data for Plethodon sherando, a terrestrial salamander recently discovered in the Blue Ridge Mountains of Virginia. We sequenced two mitochondrial regions (cyt-b and CO1) from salamanders at reference sites near the center of P. sherando’s range and from two contact zones where P. sherando populations are replaced by Plethodon cinereus, the Northern Red-Backed salamander. We then used these sequence data to examine divergence and hybridization between the two taxa. We found P. sherando and P. cinereus morphotypes from contact zones to be reciprocally monophyletic and highly divergent (~17%). P. sherando exhibited very low sequence diversity (π = 0.0010) as compared to P. cinereus from the same locations (π = 0.0096). Salamander morphology in the contact zone was as distinct as morphology at reference sites, and discriminant function analysis based on morphology successfully classified 98% of salamanders to their mitochondrial lineage. Phylogenetic analysis of cyt-b sequences showed P. sherando to be sister to Plethodon serratus (the Southern Red-Backed salamander) rather than P. cinereus or any nearby mountaintop endemics. Our results suggest that P. sherando is a distinct lineage that is not subject to substantial introgression from P. cinereus and that may have a history of geographic isolation. Given its limited range (<80 km²), we believe P. sherando should merit a conservation status similar to that of other mountaintop salamanders in the region.     

Conservation genetics of the endangered Pampas deer (Ozotoceros bezoarticus)

The Pampas deer ( Ozotoceros bezoarticus L. 1758) is the most endangered neotropical cervid, and in the past occupied a wide range of open habitats including grassland, pampas, savanna, and cerrado (Brazil) from 5° to 41° S. To better understand the effect of habitat fragmentation on gene flow and genetic variation, and to uncover genetic units for conservation, we examined DNA sequences from the mitochondrial control region of 54 individuals from six localities distributed throughout the present geographical range of the Pampas deer. Our results suggest that the control region of the Pampas deer is one of the most polymorphic of any mammal. This remarkably high variability probably reflects large historic population sizes of millions of individuals in contrast to numbers of fewer than 80 000 today. Gene flow between populations is generally close to one migrant per generation and, with the exception of two populations from Argentina, all populations are significantly differentiated. The degree of gene flow was correlated with geographical distance between populations, a result consistent with limited dispersal being the primary determinant of genetic differentiation between populations. The molecular genetic results provide a mandate for habitat restoration and reintroduction of Pampas deer so that levels of genetic variation can be preserved and historic patterns of abundance can be reconstructed. However, the source of individuals for reintroduction generally should be from populations geographically closest to those now in danger of extinction.     

Phylogenetic relationship,biogeography, and conservation genetics of endangered Fraxinus chiisanensis (Oleaceae), endemic to South Korea

Endemic plants are important for understanding phylogenetic relationships, biogeographical history, and genetic variation because of their restricted distribution and their role in conserving biodiversity. Here, we investigated the phylogenetic relationships of the Korean endemic Fraxinus chiisanensis by reconstructing the molecular phylogeny of Fraxinus based on two nuclear DNA (nrITS and phantastica) and two chloroplast DNA (psbA-trnH and rpl32-trnL) regions. Within our fossil-calibrated phylogenetic framework, we also inferred the biogeographical history of F. chiisanensis. To provide a scientific basis for the conservation of F. chiisanensis, we determined the levels of genetic diversity and genetic differentiation in this species. Combining information from nuclear and chloroplast DNA sequence data, our molecular phylogenetic analyses identified F. chiisanensis as a genetically distinct unit from its sister group, Fraxinus platypoda from Japan. Our molecular dating analyses using nuclear and chloroplast DNA data sets show F. chiisanensis diverged from its sister F. platypoda in the Early or Middle Miocene and differentiated in the Late Miocene on the Korean Peninsula. Our results suggest that the divergence of F. chiisanensis was associated with the submergence of the East China Sea land bridge and enhanced monsoons in East Asia. When compared to F. platypoda, F. chiisanensis exhibits low genetic diversity within populations and high genetic differentiation among populations. These results help us to understand the evolutionary history of F. chiisanensis and to develop a conservation strategy for this species.     

Conservation of genetic diversity in British populations of the diploid endemic Coincya monensis ssp monensis (Isle of Man Cabbage): the risk of hybridisation with the tetraploid alien, Coincya monensis ssp cheiranthos

Coincya monensis is represented in the British flora by two, cytologically distinct subspecies. Coincya monensis ssp monensis is an endemic diploid with a coastal sand dune distribution that includes a number of isolated populations. Coincya monensis ssp cheiranthos is a tetraploid alien, well established in South Wales in early successional habitats. Both subspecies share similar life form traits, flowering times and pollinators. Cluster analysis and phylogenetic reconstruction based on sequences of the mitochondrial nad4 gene confirmed the distinction between alien and endemic taxa. Tetraploid populations carry more polymorphic RAPDs loci and their genetic diversity is partitioned more within than among populations. In contrast, C. monensis ssp monensis has a distinct population genetic structure. Analysis of the multilocus genetic data confirmed a structure of genetically isolated, endemic population clusters in Scotland, Arran, the Isle of Man and South Wales. Experimental hybridisation showed the two subspecies are interfertile. Multivariate analysis of RAPDs data resolved hybrids between alien and endemic clusters and hybrids contained a proportion of alien-specific polymorphic loci. Hybrids of alien maternal parentage contained the mitochondrial nad4 sequence characteristic of the alien subspecies. Since the alien subspecies can invade mobile sand dune communities from urban sites and compete for pollinators, there is a risk that alien and endemic populations will mix and introgress. Conservation of endemic genetic diversity in Britain will require protection for all C. monensis ssp monensis populations. Currently, the most disjunct endemic population in South Wales is most at risk from introgression.     

Population genetics of the threatened tree daisy Olearia gardneri (Asteraceae), conservation of a critically endangered species

All known populations of the nationally critical tree daisy, Olearia gardneri, were studied using AFLP markers. With fewer than 160 individual plants, Olearia gardneri is the third-rarest tree in New Zealand and a good model with which to study evolutionary process in fragmented endangered plants. Genetic variation was at similar levels to other long-lived tree species in New Zealand and also as in other studies to date had poor correspondence between genetic and geographic distance. Genetic factors such as inbreeding depression and the loss of genetic diversity might lower fitness and have substantial consequences for evolution and survival of rare threatened plants. Due to the decline of this species in recent times it is imperative that conservation measures are undertaken, including revegetation. Despite considerable emphasis on “eco-sourcing” in plant recovery programmes there is strong evidence that this may not be the best strategy for O. gardneri due to breeding system and population size considerations.     

Genetic structure among and within populations of the serpentine endemic Heteropappus hispidus ssp. leptocladus (Compositae)

Heteropappus hispidus ssp. leptocladus is an edaphic endemic taxon that is confined to serpentine and limestone-derived soils and is allopatrically distributed in three regions of western Japan. In this study, we attempted to detect genomic signatures of seven H. hispidus ssp. leptocladus populations along with eight other subspecies populations using eight nuclear microsatellite loci. The Mantel test supported an isolation by distance model across all H. hispidus populations, thus implying the possibility of parallel evolution for each subspecies. Results from AMOVA recognized relatively larger differentiations in geographic distribution compared to intraspecific taxonomy. Relationships indicated by neighbor-joining phylogenetic tree analysis and population structure generally did not reflect an intraspecific taxonomy. Populations from limestone-derived soil harbored a homogeneous genetic structure with neighboring populations from serpentine-derived soils. These results suggest that the edaphic ecotype may have derived allopatrically while a lack of edaphic constraint existed between serpentine and limestone soils.     

Population genetic structure of Monimopetalum chinense (Celastraceae), an endangered endemic species of eastern China

BACKGROUND AND AIMS: Monimopetalum chinense (Celastraceae) standing for the monotypic genus is endemic to eastern China. Its conservation status is vulnerable as most populations are small and isolated. Monimopetalum chinense is capable of reproducing both sexually and asexually. The aim of this study was to understand the genetic structure of M. chinense and to suggest conservation strategies. METHODS: One hundred and ninety individuals from ten populations sampled from the entire distribution area of M. chinense were investigated by using inter-simple sequence repeats (ISSR). KEY RESULTS: A total of 110 different ISSR bands were generated using ten primers. Low levels of genetic variation were revealed both at the species level (Isp=0.183) and at the population level (Ipop=0.083). High clonal diversity (D = 0.997) was found, and strong genetic differentiation among populations was detected (49.06 %). CONCLUSIONS: Small population size, possible inbreeding, limited gene flow due to short distances of seed dispersal, fragmentation of the once continuous range and subsequent genetic drift, may have contributed to shaping the population genetic structure of the species.     

Microsatellite loci from Taxus chinensis var. mairei (Taxaceae), an endangered and economically important tree species in China

Taxus chinensis var. mairei is an endangered and economically important tree species in China. Four polymorphic microsatellite markers were developed and characterized using the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) protocol. Polymorphism of each locus was quantified for five natural populations from Hubei Province, China. The number of alleles per locus varied from 6 to 24. The ranges of observed and expected heterozygosity were 0.493–1.000 and 0.440–0.845, respectively. These are the first microsatellites reported for the family of Taxaceae and will be helpful for the ongoing population and conservation genetics research of Taxus chinensis var. mairei.     

Micropropagation of Zeyheria montana Mart. (Bignoniaceae), an endangered endemic medicinal species from the Brazilian cerrado biome

Zeyheria montana Mart. has become endangered, primarily because of deforestation of its habitat, its use as a medicinal plant extract, and the strong endemism of the species. In this study, an efficient protocol was established for the micropropagation and conservation of Z. montana germplasm using isolated mature zygotic embryos as explants. Embryos germinated in vitro 4 d after isolation and inoculation on modified Murashige and Skoog (MS) medium containing 2.0 mg/l gibberellic acid (GA₃). The addition of GA₃ also improved the germination index and accelerated the process of germination. Nodal segments from seedlings were placed on modified ¼-strength MS medium containing 0.1 mg/l 6-benzyladenine and 0.5 mg/l GA₃. Nodal segments produced 7.3 shoots per explant within 60 d. Following transfer of shoots to a medium containing 1.5 mg/l indole-3-butyric acid, roots formed. All plantlets obtained were successfully acclimatized under greenhouse conditions, and approximately 68.5 acclimatized plants could be obtained per seed each year. This protocol provides a method to preserve this rare and endangered medicinal plant.     

Conservation genetics of the endangered Iberian steppe plant Ferula loscosii (Apiaceae)

Ferula loscosii (Lange) Willk (Apiaceae) is a threatened endemic species native to the Iberian Peninsula. The plant has a narrow and disjunct distribution in three regions, NE, C and SE Spain. Genetic variability within and among 11 populations from its natural distribution was assessed using allozymes. Intermediate levels of genetic diversity were detected in F. loscosii (P(99%) = 36.83; H(E) = 0.125; H(T) = 0.152). However, the highest genetic diversity (58%) corresponded to the threatened populations from SE and C Spain (H(T) = 0.169) rather than the more abundant and larger populations from NE Spain (Ebro valley) (H(T) = 0.122). Low to moderate levels of genetic structure were found among regional ranges (G(ST) = 0.134), and several statistical spatial correlation analyses corroborated substantial genetic differentiation among the three main regional ranges. However, no significant genetic differentiation was found among the NE Spain populations, except for a northernmost population that is geographically isolated. Outcrossing mating and other biological traits of the species could account for the maintenance of the present values of genetic diversity within populations. The existence of an ancestral late Tertiary wider distribution of the species in SE and C Spain, followed by the maintenance of different Quaternary refugia in these warmer areas, together with a more recent and rapid post-glacial expansion towards NE Spain, are arguments that could explain the low genetic variability and structure found in the Ebro valley and the higher levels of diversity in the southern Iberian populations.