Phenotypic Characterization of <i>Oryza nivara</i> (Sharma <i>et</i> Shastry) Collected from Different Ecological Niches of Sri Lanka

Information on the genetic diversity of wild rice species in Sri Lanka is relatively meagre, though it plays a key role in crop improvement programs of cultivated rice (Oryza sativa L.). The present study was carried out to identify the morphological variation pattern of the wild populations of O. nivara in Sri Lanka. Seven populations (P1 to P7) collected from different agro-ecological regions were characterized in a common garden based on nine morphological traits. The findings revealed a high level of phenotypic variation between populations when compared to within a population. The most variable traits were the flag leaf panicle neck length (FLPNL) and flag leaf angle (FLA), whereas the least variable trait was the flag leaf length (FLL). Box plots clearly illustrated the large differentiation of phenotypic traits in the entire distribution of wild rice populations. The cumulative values of the two principal components, i.e., FLPNL and FLA, explained 58.7% of the total variance. Populations from similar natural habitats clustered together. The P7 was adapted to intercept more sunlight by increasing flag leaf width (FLW) and FLA to compete with weeds and other shrubs. P2 and P5 were the most closely related populations representing approximately similar ecological conditions of the dry zone. The P3 population from the intermediate zone showed a vigorous plant growth with the highest plant height, culm girth and awn length (P < 0.05). Knowledge of such morphological diversity would facilitate designing conservation strategies and basic information for the proper utilization of wild resources in rice genetic improvement.     

Genetic structure and gene flow in an endangered perennial grass, <Emphasis Type="Italic">Arctophila fulva</Emphasis> var. <Emphasis Type="Italic">pendulina</Emphasis>

Arctophila fulva var. pendulina is a rare endemic perennial grass confined to seashore and riverbank meadows around the Bothnian Bay, the northernmost part of the Baltic Sea. The number of A. fulva populations has decreased during the last few decades in Finland and Sweden, and nowadays there are only eight populations left in the drainage area of the Bothnian Bay. We investigated the distribution of genetic variation within and between six subpopulations in the largest remaining population at Liminka Bay, Finland, using amplified fragment length polymorphism (AFLP) markers. Relatively high amounts of variation were found in the subpopulations, the mean Nei’s expected heterozygosity being typical (0.267) for an outcrossing species. Despite the fact that no seedlings or viable seeds of A. fulva have been found in the previous field studies, the observed high genotypic diversity suggested that sexual reproduction has played an important role at some time during the history of the studied A. fulva population. Analysis of population structure revealed a low level of genotypic differentiation (Φ_ST=0.046) between subpopulations, and also significant sub-structuring within subpopulations. Isolation-by-distance between subpopulations was present on scales larger than 1 km. The overall pattern of genetic variation within and between subpopulations suggest that the population has characters of both stepping-stone and metapopulation models. Because our results suggested that subpopulations are more or less ephemeral, the conservation and management effort in this species should be targeted to conservation of the required habitat of the species instead of extant subpopulations.     

Conservation implications of high genetic variation in two closely related and highly threatened species of Crambe (Brassicaceae) endemic to the island of Gran Canaria: C. tamadabensis and C. pritzelii

We used data from 12 allozyme loci for two endemic Brassicaceae from Gran Canaria (the endangered narrow endemic Crambe tamadabensis and its more widespread congener C. pritzelii) to assess whether their genetic diversity patterns reflect their phylogenetic closeness and contrasting population sizes and distribution areas, and to derive conservation implications. Genetic diversity values are high for both species and slightly higher in C. tamadabensis, despite its narrow distribution in north‐western Gran Canaria. At odds with the generally high interpopulation diversity levels reported in Canarian endemics, values of G_ST in C. tamadabensis and C. pritzelii are rather low (0.067 and 0.126, respectively). We construe that the higher genetic structure detected in C. pritzelii is mainly a result of unbalanced allele frequencies and low population sizes at the edges of its distribution. The overall high allozyme variation detected in C. tamadabensis and C. pritzelii is nevertheless compatible with an incipient but consistent genetic differentiation between the two species, modulated by recurrent bottlenecks caused by grazing and drift. Our data suggest that conservation efforts aimed at maintaining the existing genetic connectivity in each species and ex situ conservation of seeds are the best strategies to conserve their genetic diversity.     

Population structure and genetic diversity of wild <Emphasis Type="Italic">Helianthus</Emphasis> species from Mozambique

The production of sunflower suffered a major decline in Mozambique after its independence in 1975. Civil war, human activities and environmental damage subjected the species to an ecological stress contributing to reduce the number and size of wild populations. As this reduction is often related to a loss of genetic variation we estimated the genetic diversity within and among populations of wild Helianthus from five districts of Mozambique using RAPD markers. The 44 accessions studied grouped into four major clusters exhibiting structured variability with regard to geographic origin. A high level of genetic diversity (He = 0.350 and I = 0.527) was retained at the population level. The genetic variation among populations was high (59.7%), which is consistent with low gene flow (Nm = 0.338). The proportion of total genetic diversity residing among these populations should be kept in mind to devise different conservation strategies in order to preserve these populations. Currently wild Helianthus genetic resources present in Maputo and Sofala are on the edge of extinction mainly due to excessive urbanization. Therefore, conservation of what remains of this plant genetic diversity is essential for sustainable utilization and can be useful for breeding programs.     

Molecular phylogeography and population evolution analysis of <Emphasis Type="Italic">Citrus ichangensis</Emphasis> (Rutaceae)

Ichang papeda (Citrus ichangensis), a wild and endemic perennial plant in Rutaceae, is characterized by the existence of wild and natural populations in southwestern and middle-west China. We analyzed a total of 231 individuals across 16 natural populations using chloroplast SSR markers, nuclear SSR markers, and single-copy nuclear genes. Standard population genetic analyses as well as Bayesian and maximum likelihood models were used to clarify the genetic diversity, population differentiation, barriers to gene flow, bottleneck events, isolation by distance, history migration, demographic history among populations, and phylogeny evolution. The chloroplast and nuclear genome analyses revealed a low level of genetic diversity in C. ichangensis. Clear signals of recent bottlenecks and strong patterns of isolation by distance were detected among different subpopulations, indicating a low extent of historical gene flow for this species and that genetic drift would occur after population differentiation. Bayesian clustering analyses revealed a clear pattern of genetic structure, with one cluster spanning the potential refugia in Wuling Mountains and Ta-pa Mountains, and other two clusters covering a more limited distribution range. The demographic history also supported the scenario that two isolated clusters originated in parallel from the genetic diversity center. Taxonomically, Ichang papeda may be a member of subgenus Citrus. Owing to the complicated topography, the mountainous regions and the Yangtze River have provided long-term stable habitats for C. ichangensis and acted as main barriers for its expansion, which might facilitate the process of speciation. Statistical population models and genetic data indicated strong genetic structure in C. ichangensis, which might result from the restricted gene flow, genetic drift, and population bottlenecks.     

Genetic diversity,genetic drift and mixed mating system in small subpopulations of <Emphasis Type="Italic">Dyckia ibiramensis</Emphasis>, a rare endemic bromeliad from Southern Brazil

Dyckia ibiramensis is a naturally rare, endemic and threatened bromeliad which occurs naturally on 4 km of rocky river outcroppings in Southern Brazil. For this study, subpopulations of the species were characterized based on size and genetics, to compile information for in situ and ex situ conservation strategies. A census of the rosettes was undertaken for each subpopulation and seven allozyme polymorphic loci were used to estimate genetic diversity and structure of adults and offspring and assess the mating system. In general, the subpopulations were small and most of the rosettes were aggregated into clumps. The species showed a high genetic diversity ([^(H)]_e = 0.219 \hat{H}_{e} = 0.219 ) and significant fixation index ([^(f)] = 0.642, \hat{f} = 0.642, P ≤ 0.05). The estimate of differentiation among all adult subpopulations indicate pronounced genetic structure ([^(G)]^￠_ST = 0.674 \hat{G}^{\prime}_{ST} = 0.674 ). D. ibiramensis has a mixed mating system and multilocus outcrossing rates [^(t)]_m \hat{t}_{m} were variable between subpopulations. This study demonstrates the importance of in situ preservation of all subpopulations for the maintenance of species diversity. For effective ex situ conservation, it would be necessary to collect seeds from 52 to 99 seed-rosettes, depending on the target population.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.     

Genetic structure in a population of a tropical tree Ocotea tenera (Lauraceae): influence of avian seed dispersal

We studied the influence of avian seed dispersal on the structuring of genetic diversity in a population of a tropical tree, Ocotea tenera (Lauraceae). The seeds of O. tenera are principally dispersed by four, relatively specialized, fruit-eating bird species (emerald toucanets, keel-billed toucans, resplendent quetzals, and three-wattled bellbirds). We found high genetic diversity within the overall population and significant, nonrandom structuring of that diversity among subpopulations. Subpopulations contained members of several sibling groups, and most saplings within subpopulations were shown not to be the progeny of adult trees within the same subpopulation. Our data indicate that O. tenera subpopulations are founded with several seeds from few maternal families, and that this mode of establishment is an important determinant of population genetic architecture.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

Genetic structure of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens and its implications for the management of threatened island populations

The Red‐headed Wood Pigeon Columba janthina nitens is endemic to the Ogasawara Islands, an oceanic island chain located 1000 km south of the main islands of Japan. The subspecies is at high risk of extinction because of its small population size and restricted habitat range. We undertook genetic analyses of this pigeon using sequences of a portion of the mitochondrial control region and five microsatellite markers to estimate the genetic characteristics of two wild populations from the Bonin and Volcano Islands, as well as one captive breeding population. The genetic diversity of the wild individuals was exceptionally low in both the mitochondria (nucleotide diversity = 0.00105) and at the microsatellite (3.2 alleles per locus and H_E = 0.12) loci. Higher numbers of microsatellite genotypes were observed in the Volcano Islands population than in the Bonin Islands population, which may be because of the relatively low impact of human disturbance. The most common mitochondrial haplotypes and microsatellite alleles observed in the two wild populations were completely fixed in the captive population. Our results suggest that the genetic diversity of the captive population needs to be increased. However, introduction of a wild individual into a captive population can lead to a decreased genetic diversity in the wild population and therefore should be done with caution. The genetic differentiation between the Bonin and the Volcano island groups was low, and the populations of the two island groups should be regarded as a single evolutionarily significant unit. However, special consideration is required for habitat conservation in the Volcano Islands, which may be functioning as a sanctuary for the Red‐headed Wood Pigeon. For the long‐term conservation of threatened bird species that live on remote oceanic islands, determination of management units considering gene flow caused by their flying capacity and maintenance of genetically suitable wild and captive populations are essential.     

Conservation Strategy for African Medicinal Species: <i>In Vitro</i> Biotechnological Approach

The use of medicinal plants for different therapeutic values is well documented in African continent. African diverse biodiversity hotspots provide a wide range of endemic species, which ensures a potential medicinal value. The feasible conservation approach and sustainable harvesting for the medicinal species remains a huge challenge. However, conservation approach through different biotechnological tools such as micropropagation, somatic embryogenesis, synthetic seed production, hairy root culture, molecular markers based study and cryopreservation of endemic African medicinal species is much crucial. In this review, an attempt has been made to provide different in vitro biotechnological approaches for the conservation of African medicinal species. The present review will be helpful in further technology development and deciding the priorities at decision-making levels for in vitro conservation and sustainable use of African medicinal species.     

Population size,center–periphery,and seed dispersers’ effects on the genetic diversity and population structure of the Mediterranean relict shrub Cneorum tricoccon

The effect of population size on population genetic diversity and structure has rarely been studied jointly with other factors such as the position of a population within the species’ distribution range or the presence of mutualistic partners influencing dispersal. Understanding these determining factors for genetic variation is critical for conservation of relict plants that are generally suffering from genetic deterioration. Working with 16 populations of the vulnerable relict shrub Cneorum tricoccon throughout the majority of its western Mediterranean distribution range, and using nine polymorphic microsatellite markers, we examined the effects of periphery (peripheral vs. central), population size (large vs. small), and seed disperser (introduced carnivores vs. endemic lizards) on the genetic diversity and population structure of the species. Contrasting genetic variation (H_E: 0.04–0.476) was found across populations. Peripheral populations showed lower genetic diversity, but this was dependent on population size. Large peripheral populations showed high levels of genetic diversity, whereas small central populations were less diverse. Significant isolation by distance was detected, indicating that the effect of long‐distance gene flow is limited relative to that of genetic drift, probably due to high selfing rates (F_IS = 0.155–0.887), restricted pollen flow, and ineffective seed dispersal. Bayesian clustering also supported the strong population differentiation and highly fragmented structure. Contrary to expectations, the type of disperser showed no significant effect on either population genetic diversity or structure. Our results challenge the idea of an effect of periphery per se that can be mainly explained by population size, drawing attention to the need of integrative approaches considering different determinants of genetic variation. Furthermore, the very low genetic diversity observed in several small populations and the strong among‐population differentiation highlight the conservation value of large populations throughout the species’ range, particularly in light of climate change and direct human threats.     

Genetic diversity and conservation evaluation of a critically endangered endemic maple,Acer yangbiense,analyzed using microsatellite markers

The newly discovered endemic maple Acer yangbiense of China has only five individuals left in the wild, and thus has been classified as a plant species with extremely small populations (PSESP). PSESP species call for emergency protection procedures, such as ex-situ conservation and reintroduction. Our objectives were to examine the genetic diversity of A. yangbiense and to evaluate former conservation strategies from a genetic point of view. Our results suggested that A. yangbiense was not genetically depauperate, but its genetic loss at a species level was obvious. A parentage analysis indicated a high selfing-rate in A. yangbiense and suggested the existence of a previously unknown wild individual. Former conservation strategies did not include all genetic variations of the wild population, and gene diversity of the ex-situ conserved seedlings is lower than that of the wild population. From our findings, we make suggestions to guide the subsequent protection of this species.     

Population structure of an endangered frog (<Emphasis Type="Italic">Babina subaspera</Emphasis>) endemic to the Amami Islands: possible impacts of invasive predators on gene flow

The otton frog (Babina subaspera) is an endangered species endemic to the Amami Islands, Japan. High predation pressure from an introduced carnivore, the mongoose, has caused declines in the frog populations and created a large habitat gap around an urban area. To promote effective conservation, we investigated the genetic status of the species and examined the effect of the habitat gap on gene flow among populations. Using five polymorphic microsatellite loci and mitochondrial DNA sequences, we investigated genetic diversity, genetic structure and gene flow in B. subaspera populations on the islands of Amami-Oshima and Kakeroma-jima. The expected heterozygosity (H _E) within each locality was generally high (range: 0.67–0.85), indicating that B. subaspera maintains high genetic diversity. However, genetic differentiation was observed, and the two populations, TAG and KAR, showed little gene flow with other populations. The clustering and F _ST analyses also predicted that these two populations were clearly distinct. According to the mitochondrial DNA analysis, the observed genetic differentiation occurred relatively recently. Possible barriers such as mountain ridges, rivers or roads did not result in genetic separation of the populations. These data support the hypothesis that the habitat gap created by an introduced predator prevented the gene flow among B. subaspera populations. When developing conservation strategies for B. subaspera, focus should be directed to these two isolated populations; careful monitoring of population size and genetic diversity should be conducted along with the mongoose elimination project ensues.     

Comparative molecular studies on the genetic diversity of an ex situ garden collection and its source population of the critically endangered polish endemic plant <Emphasis Type="Italic">Cochlearia polonica</Emphasis> E. Fröhlich

Cochlearia polonica (Brassicaceae) is a narrow endemic plant extinct in the wild, known only from one transplanted population in southern Poland. The suitable habitats for this species are confined to shallow water courses and springs on sandy ground. During the second half of twentieth century the natural populations of C. polonica declined due to anthropogenic effects (primarily due to a major change of ground water levels). Consequently, 14 individuals were transplanted in the 1970 s into a secondary locality of similar habitat conditions of Centuria river. In the late 1980s five individuals from the transplanted population were used to establish an artificial ex situ site in the Botanical Garden of the Polish Academy of Sciences in Warsaw. In order to investigate the effect of 18 years of ex situ conservation, the level of genetic diversity and genetic structure of the ex situ conserved botanical garden population and its source population were sampled and analyzed using inter simple sequence repeat markers. The percentage values of polymorphic bands and Nei’s gene diversity indicated average genetic variation at species level, whereas at the population level it was relatively low, especially for the garden population. The results suggested differences in genetic composition of the analyzed populations and implied homogenization of the genetic structure of the ex situ conserved population. Ex situ conservation resulted in a decrease of the species’ genetic diversity, implying that the artificial population only partly represents the primary genetic variability found in the source population.     

Development of microsatellite markers for <Emphasis Type="Italic">Metrosideros boninensis</Emphasis> (Myrtaceae), an endangered endemic plant species from the Bonin Islands,Japan

Metrosideros boninensis is an endangered endemic plant species from the Bonin Islands. We isolated and characterized nine microsatellite loci in this species. The expected heterozygosities of these nine markers ranged from 0.127 to 0.768. These markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow, and planning for conservation of M. boninensis.     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

Genetic variation in <Emphasis Type="Italic">Delonix s.l.</Emphasis> (Leguminosae) in Madagascar revealed by AFLPs: fragmentation,conservation status and taxonomy

The distribution of genetic diversity has potential to inform conservation efforts but is rarely incorporated when conservation status is assigned to a species. These data can be beneficial to the conservation assessment process by providing information on subpopulations, gene flow and effective population sizes, thus achieving more successful assessments. In order to obtain a better understanding of the patterns of genetic variation and their relationship to conservation in the fragmented flora of Madagascar, this study assessed genetic diversity among and within Delonix s.l. (Leguminosae) using AFLP markers. The genetic diversity of eight species of Delonix s.l. (covering 79 sample sites and 254 individuals) showed a range of values (25–61% for polymorphic loci, and 0.076–0.192 Shannon’s Index). Results from an analysis of molecular variance (AMOVA) suggest that a majority of the genetic variance is attributed to variation within species (87%), which is also supported by a principle coordinate analysis of genetic distances between sites. The results were used to compare the genetic difference between species of different threat status and show that even closely related species with the same IUCN threat status differ in their genetic structure, probably arising from differences in life history traits, pollen and seed dispersal, and fragmentation. Species that are recently affected by habitat destruction and fragmentation are likely to be at high potential risk of genetic erosion contributing to their ongoing decline. Thus, genetic variation should be taken into consideration in conservation assessments, whenever possible, to provide accurate and targeted conservation recommendations in order to achieve more successful conservation outcomes.     

Loss of genetic diversity in an outbreeding species: small population effects in the African wild dog <Emphasis Type="Italic">(Lycaon pictus</Emphasis>)

Genetic studies on the endangered African wild dog (Lycaon pictus) have primarily focused on the few remaining large and viable populations. However, investigations on the many isolated small African wild dog populations might also be informative for species management because the majority of extant populations are small and may contain genetic variability that is important for population persistence and for species conservation. Small populations are at higher risk of extinction from stochastic and deterministic demographic processes than larger populations and this is often of more immediate conservation concern than loss of genetic diversity, particularly for species that exhibit out-breeding behaviour such as long distance dispersal which may maintain gene flow. However, the genetic advantages of out-breeding behaviour may be reduced if dispersal is compromised beyond reserve borders (edge effects), further weakening the integrity of small populations. Mitochondrial DNA and 11 microsatellite genetic markers were used to investigate population genetic structure in a small population of out-breeding African wild dogs in Zambia, which occupies an historical dispersal corridor for the species. Results indicated the Zambian population suffered from low allelic richness, and there was significant evidence of a recent population bottleneck. Concurrent ecological data suggests these results were due to habitat fragmentation and restricted dispersal which compromised natural out-breeding mechanisms. This study recommends conservation priorities and management units for the African wild dog that focus on conserving remaining levels of genetic diversity, which may also be applicable for a range of out-breeding species.     

Reintroduction of the endangered and endemic plant species Cochlearia bavarica—Implications from conservation genetics

Population reintroduction is a common practice in conservation, but often fails, also due to the effects of inbreeding or outbreeding depression. Cochlearia bavarica is a strongly endangered plant species endemic to Bavaria in Germany, constantly declining since the late 1980s. Therefore, population reintroduction is intended. In this study, we analyzed genetic diversity within and genetic differentiation between all 32 remnant populations of the species in Swabia and Upper Bavaria using amplified fragment length polymorphisms. Our aim was to increase reintroduction success by providing data to avoid negative effects of inbreeding and outbreeding and to preserve the natural genetic pattern of the species. Genetic diversity within populations was low but similar to other rare and endemic species and varied strongly between populations but did not depend on population size. Our analysis revealed a strong geographic pattern of genetic variation. Genetic differentiation was strongest between Swabia and Upper Bavaria and at the population level, whereas differentiation between subpopulations was comparatively low. Isolation by distance and genetic differentiation was stronger among populations from Upper Bavaria than from Swabia. From the results of our study, we derived recommendations for a successful reintroduction of the species. We suggest using rather genetically variable than large populations as reintroduction sources. Moreover, the exchange of plant material between Swabia and Upper Bavaria should be completely avoided. Within these regions, plant material from genetically similar populations should preferably be used for reintroduction, whereas the exchange among subpopulations seems to be possible without a negative impact on genetic variation due to natural gene flow.