Patterns of chloroplast DNA polymorphism in the endangered polyploid Centaurea borjae (Asteraceae): implications for preserving genetic diversity.

A previous study with amplified fragment length polymorphism (AFLP) fingerprints found no evidence of genetic impoverishment in the endangered Centaurea borjae and recommended that four management units (MUs) should be designated. Nevertheless, the high ploidy (6x) of this narrow endemic plant suggested that these conclusions should be validated by independent evidence derived from non-nuclear markers. Here, the variable trnT-F region of the plastid genome was sequenced to obtain this new evidence and to provide an historical background for the current genetic structure. Plastid sequences revealed little genetic variation; calling into question the previous conclusion that C. borjae does not undergo genetic impoverishment. By contrast, the conclusion that gene flow must be low was reinforced by the strong genetic differentiation detected among populations using plastid sequences (global F_ST = 0.419). The spatial arrangement of haplotypes and diversity indicate that the populations currently located at the center of the species range are probable sites of long-persistence whereas the remaining sites may have derived from a latter colonization. From a conservation perspective, four populations contributed most to the allelic richness of the plastid genome of the species and should be given priority. Combined with previous AFLP results, these new data recommended that five, instead of four, MUs should be established. Altogether, our study highlights the benefits of combining markers with different modes of inheritance to design accurate conservation guidelines and to obtain clues on the evolutionary processes behind the present-day genetic structures.     

Selective recovery of founder genetic diversity in aquacultural broodstocks and captive, endangered fish populations

Hatchery broodstocks used for genetic conservation or aquaculture may represent their ancestral gene pools rather poorly. This is especially likely when the fish that found a broodstock are close relatives of each other. We re-analysed microsatellite data from a breeding experiment on red sea bream to demonstrate how lost genetic variation might be recovered when gene frequencies have been distorted by consanguineous founders in a hatchery. A minimal-kinship criterion based on a relatedness estimator was used to select subsets of breeders which represented the maximum number of founder lineages (i.e., carried the fewest identical copies of ancestral genes). UPGMA clustering of Nei's genetic distances grouped these selected subsets with the parental gene pool, rather than with the entire, highly drifted offspring generation. The selected subsets also captured much of the expected heterozygosity and allelic diversity of the parental gene pool. Independent pedigree data on the same fish showed that the selected subsets had more contributing parents and more founder equivalents than random subsets of the same size. The estimated mean coancestry was lower in the selected subsets, meaning that inbreeding in subsequent generations would be lower if they were used as breeders. The procedure appears suitable for reducing the genetic distortion due to consanguineous and over-represented founders of a hatchery gene pool.     

Patterns of ancestry and genetic diversity in reintroduced populations of the slimy sculpin: implications for conservation

Reintroductions are a common approach for preserving intraspecific biodiversity in fragmented landscapes. However, they may exacerbate the reduction in genetic diversity initially caused by population fragmentation because the effective population size of reintroduced populations is often smaller and reintroduced populations also tend to be more geographically isolated than native populations. Mixing genetically divergent sources for reintroduction purposes is a practice intended to increase genetic diversity. We documented the outcome of reintroductions from three mixed sources on the ancestral composition and genetic variation of a North American fish, the slimy sculpin (Cottus cognatus). We used microsatellite markers to evaluate allelic richness and heterozygosity in the reintroduced populations relative to computer simulated expectations. Sculpins in reintroduced populations exhibited higher levels of heterozygosity and allelic richness than any single source, but only slightly higher than the single most genetically diverse source population. Simulations intended to mimic an ideal scenario for maximizing genetic variation in the reintroduced populations also predicted increases, but they were only moderately greater than the most variable source population. We found that a single source contributed more than the other two sources at most reintroduction sites. We urge caution when choosing whether to mix source populations in reintroduction programs. Genetic characteristics of candidate source populations should be evaluated prior to reintroduction if feasible. When combined with knowledge of the degree of genetic distinction among sources, simulations may allow the genetic diversity benefits of mixing populations to be weighed against the risks of outbreeding depression in reintroduced and nearby populations.     

Phylogeography of the endangered Otago skink, Oligosoma otagense: population structure, hybridisation and genetic diversity in captive populations

Climatic cooling and substantial tectonic activity since the late Miocene have had a pronounced influence on the evolutionary history of the fauna of New Zealand's South Island. However, many species have recently experienced dramatic range reductions due to habitat fragmentation and the introduction of mammalian predators and competitors. These anthropogenic impacts have been particularly severe in the tussock grasslands of the Otago region. The Otago skink (Oligosoma otagense), endemic to the region, is one of the most critically endangered vertebrates in New Zealand. We use mitochondrial DNA sequence data to investigate the evolutionary history of the Otago skink, examine its population genetic structure, and assess the level of genetic diversity in the individuals in the captive breeding program. Our data indicate that the Otago skink diverged from its closest relatives in the Miocene, consistent with the commencement of tectonic uplift of the Southern Alps. However, there is evidence for past introgression with the scree skink (O. waimatense) in the northern Otago-southern Canterbury region. The remnant populations in eastern Otago and western Otago are estimated to have diverged in the mid-Pliocene, with no haplotypes shared between these two regions. This divergence accounts for 95% of the genetic diversity in the species. Within both regions there is strong genetic structure among populations, although shared haplotypes are generally evident between adjacent localities. Although substantial genetic diversity is present in the captive population, all individuals originate from the eastern region and the majority had haplotypes that were not evident in the intensively managed populations at Macraes Flat. Our data indicate that eastern and western populations should continue to be regarded as separate management units. Knowledge of the genetic diversity of the breeding stock will act to inform the captive management of the Otago skink and contribute to a key recovery action for the species.     

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.     

Strong population genetic structure and its implications for the conservation and management of the endangered Itasenpara bitterling

To infer the population genetic structure and genetic diversity of Itasenpara bitterling (Acheilognathus longipinnis), a cyprinid species endemic to Japan and distributed in only three specific regions, we investigated mitochondrial DNA variation. The distribution of the haplotypes among the three regions showed distinct geographic structure, and no common haplotypes were observed among regions. Analysis of molecular variance revealed a significant proportion of the genetic variance was partitioned among regions (93.1 %, P < 0.001), and pairwise estimates of D_A and Φ_ST between regions also revealed strong population structure. Given the strong genetic structure and low genetic diversity within regions, we strongly suggest that each region should be treated as a separate unit in any conservation program and any inter-regional translocations should be avoided.     

Strategies for maintaining genetic diversity in captive populations through reproductive technology

The maintenance of genetic diversity in captive populations is a primary goal of captive breeding plans, and it is becoming increasingly apparent that reproductive technology has much to offer captive breeding programs in attaining this goal. Reproductive technology can best assist captive breeding programs in this task by developing strategies that effectively increase the genetic contribution of new wild founders to a population as well as increase the reproductive life span of existing founders and their close descendents. This will act to reduce genetic drift and inbreeding effects in the population and thereby minimize the loss of genetic diversity. Considering only one aspect of reproductive technology, semen collection, this paper examines some of the genetic considerations that might be used for choosing which males in a population to collect semen from, assuming the goal of the captive breeding program is the preservation of genetic diversity. It is shown that semen collection and preservation, with future intent of artificial insemination, can make significant contributions to the maintenance of genetic diversity if careful consideration is given to the selection of donor males. Finally, the pedigree of the captive population of Asian lions (Panthera leo persica) is used to illustrate some of these genetic concepts that might be important in selecting males as semen donors.     

Rapid genetic deterioration in captive populations: Causes and conservation implications

Many species require captive breeding to ensuretheir survival. The eventual aim of suchprograms is usually to reintroduce the speciesinto the wild. Populations in captivitydeteriorate due to inbreeding depression, lossof genetic diversity, accumulation of newdeleterious mutations and genetic adaptationsto captivity that are deleterious in the wild.However, there is little evidence on themagnitude of these problems. We evaluatedchanges in reproductive fitness in populationsof Drosophila maintained under benigncaptive conditions for 50 generations witheffective population sizes of 500 (2replicates), 250 (3), 100 (4), 50 (6) and 25(8). At generation 50, fitness in the benigncaptive conditions was reduced in smallpopulations due to inbreeding depression andincreased in some of the large populations dueto modest genetic adaptation. When thepopulations were moved to `wild' conditions,all 23 populations showed a marked decline(64–86%percnt;) in reproductive fitness compared tocontrols. Reproductive fitness showed acurvilinear relationship with population size,the largest and smallest population sizetreatments being the worst. Genetic analysesindicated that inbreeding depression andgenetic adaptation were responsible for thegenetic deterioration in `wild' fitness.Consequently, genetic deterioration incaptivity is likely to be a major problem whenlong-term captive bred populations ofendangered species are returned to the wild. Aregime involving fragmentation of captivepopulations of endangered species is suggestedto minimize the problems.     

The structure of diversity within New World mitochondrial DNA haplogroups: implications for the prehistory of North America

The mitochondrial DNA haplogroups and hypervariable segment I (HVSI) sequences of 1,612 and 395 Native North Americans, respectively, were analyzed to identify major prehistoric population events in North America. Gene maps and spatial autocorrelation analyses suggest that populations with high frequencies of haplogroups A, B, and X experienced prehistoric population expansions in the North, Southwest, and Great Lakes region, respectively. Haplotype networks showing high levels of reticulation and high frequencies of nodal haplotypes support these results. The haplotype networks suggest the existence of additional founding lineages within haplogroups B and C; however, because of the hypervariability exhibited by the HVSI data set, similar haplotypes exhibited in Asia and America could be due to convergence rather than common ancestry. The hypervariability and reticulation preclude the use of estimates of genetic diversity within haplogroups to argue for the number of migrations to the Americas.     

Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974–1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Φ _ST = –0.00296–0.00284), increasing considerably at the height of stocking (1980s Φ _ST = 0.18932) and decreasing post-stocking (1990s/2002 Φ _ST = 0.04934–0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.     

Patterns of genetic variation within a captive population of Amur tigerPanthera tigris altaica

Eight founders and thirty-one descendants were sampled as the Founder group and the Offspring group respectively from a captive population of Amur tigerPanthera tigris altaica Temminck, 1844 for population genetic analysis with RAPD and ISSR markers. Integrated with demographic data during the initial recovery stage, results showed: (1) increasing the population size (N) and the effective population size (N _e) greatly retard lose of genetic variation induced mainly by genetic drift and selection; (2) recombination and admixture could cause the Offspring group (5.711%) and the Founder group (10.383%) to hold different linkage disequilibrium (LD); (3) further Ohta’s variance analysis indicated genetic drift (87.3%) and epistatic selection (12.7%) maintained LD in population, whereas GENEDROP analysis supported epistatic selection largely derived from artificial selection of managers; (4) both Tajima’s test and Fu’s test confirmed the statistic neutrality of genetic markers used, moreover the positive value of Tajima’sD (0.090) together with the result that π (25.286) was bigger than ϑ (24.898) revealed the Founder group was admixture population, while the negative Tajima’sD value (−0.053) together with the result that π (23.679) was less than ϑ (23.912) disclosed the Offspring group experienced selective sweep.     

Management implications for the reintroduction of the endangered Hawaiian state flower Hibiscus brackenridgei

Dry forests of Hawai'i are one of the most endangered forest types worldwide with 45% of all native tree and shrub species listed as federally threatened or endangered. Supplementation and reintroduction of endangered dry forest species are necessary to maintain viable population sizes. The goal of this research was to reintroduce Hibiscus brackenridgei mokuleianus, the state flower of Hawai'i and a federally endangered species, into its historical range on O'ahu. We determined the environmental variables contributing to plant survival and growth, assessed the effectiveness of a reintroduction program with minimal management, and evaluated the potential for habitat suitability models to assist in selection of optimal sites for reintroduction. Forty‐five individuals were grown from cuttings and transplanted in two regions: Ka'ena Point (2 sites, 10 individuals each site) and along Kealia Trail (5 sites, 5 individuals each site). Survival and height measurements were taken four times over the course of 25 months. There was 35% survival at Ka'ena Point and 76% survival at Kealia Trail and an average plant growth of 70.5 cm (±30.1 cm) and 121.6 cm (±61.7 cm), respectively. Ka'ena Point had significantly lower survival (p > 0.001) and growth rates (p > 0.001) than Kealia Trail. Plants survived best at sites with steep slopes and high soil moisture and grew the most in sites with steeper slopes, higher soil moisture, and lower soil bulk density. These findings provide insight into environmental variables which should be considered prior to planting as well as the intensity of site management.     

Effects of parental number and duration of the breeding period on the effective population size and genetic diversity of a captive population of the endangered Tokyo bitterling Tanakia tanago (Teleostei: Cyprinidae)

The maintenance of genetic diversity is one of the chief concerns in the captive breeding of endangered species. Using microsatellite and mtDNA markers, we examined the effects of two key variables (parental number and duration of breeding period) on effective population size (N_e) and genetic diversity of offspring in an experimental breeding program for the endangered Tokyo bitterling, Tanakia tanago. Average heterozygosity and number of alleles of offspring estimated from microsatellite data increased with parental number in a breeding aquarium, and exhibited higher values for a long breeding period treatment (9 weeks) compared with a short breeding period (3 weeks). Haplotype diversity in mtDNA of offspring decreased with the reduction in parental number, and this tendency was greater for the short breeding period treatment. Genetic estimates of N_e obtained with two single‐sample estimation methods were consistently higher for the long breeding period treatment with the same number of parental fish. Average N_e/N ratios were ranged from 0.5 to 1.4, and were high especially in the long breeding period with small and medium parental number treatments. Our results suggest that the spawning intervals of females and alternative mating behaviors of males influence the effective size and genetic diversity of offspring in bitterling. To maintain the genetic diversity of captive T. tanago, we recommend that captive breeding programs should be conducted for a sufficiently long period with an optimal level of parental density, as well as using an adequate number of parents. Zoo Biol 31:656‐668, 2012. © 2011 Wiley Periodicals, Inc.     

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.     

Patterns of spatial genetic structuring in the endangered limpet Patella ferruginea: implications for the conservation of a Mediterranean endemic

Patella ferruginea Gmelin, 1791 is an endangered marine gastropod endemic to the Western Mediterranean. Its range is restricted to the Sardinian-Corsican region (SCR), North Africa, a few scattered sites in Southern Spain, and Sicily. Inter-simple sequence repeat (ISSR) markers and three different mitochondrial DNA (mtDNA) regions, Cytochrome c Oxidase subunit I, 12S (small-subunit ribosomal RNA gene) and 16S (large-subunit ribosomal RNA gene), were used to investigate the presence of genetic population structuring. The mtDNA sequences showed very low levels of genetic differentiation. Conversely, ISSRs showed the presence of two main genetic groups, corresponding to Spain, North Africa and Sicily and the SCR. The SCR was further split into two subgroups. The ISSR results suggest that, on a regional scale, the genetic structure of P. ferruginea is mainly determined by the restriction of gene flow by dispersal barriers. On a more local scale human harvesting may play a crucial role in population structuring by increasing the effect of genetic drift.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.