Current genetic isolation and fragmentation contrasts with historical connectivity in an alpine lizard (Cyclodomorphus praealtus) threatened by climate change

Assessing levels of genetic diversity, connectivity and historical demography for threatened species provides important information for conservation management. We used a combination of the mitochondrial ND4 gene and seven microsatellite markers to examine both historical and recent population genetic structure and demography of the threatened alpine she-oak skink, Cyclodomorphus praealtus. This species is restricted to the “sky islands” of the Australian alpine region. Based on mtDNA, the New South Wales and Victorian regions are reciprocally monophyletic and highly divergent, with among population variation of 0.9 and net sequence divergence of 4.28%, which suggests that they should be considered separate Evolutionary Significant Units for management purposes. The mtDNA data also indicate historical connectivity between the three Victorian populations. However, a model-based clustering analysis of microsatellite genotypes identified strong population structure in Victoria, with three distinct populations that have no current inter-population gene flow. This suggests that the Victorian populations are effectively isolated from each other, and is indicative of very low dispersal capacity and a high degree of habitat specialisation. This is reinforced by the substantially lower genetic diversity within the lowest elevation population compared to the other higher elevation populations. We found no genetic signature of major changes in effective population size. These data provide a baseline for assessing future impacts of climate change on the genetic structure of this alpine endemic species.     

Population genetic diversity of the endemic Sardinian newt Euproctus platycephalus: implications for conservation

The Sardinian mountain newt Euproctus platycephalus, endemic to the island of Sardinia, (Italy), is considered a rare and threatened species and is classed as critically endangered by IUCN. It inhabits streams, small lakes and pools on the main mountain systems of the island. Threats from climatic and anthropogenic factors have raised concerns for the long-term survival of newt populations on the island. MtDNA sequencing was used to investigate the genetic population structure and phylogeography of this endemic species. Patterns of genetic variation were assessed by sequencing the complete Dloop region and part of the 12SrRNA, from 74 individuals representing four different populations. Analyses of molecular variance suggest that populations are significantly differentiated, and the distribution of haplotypes across the island shows strong geographical structuring. However, phylogenetic analyses also suggest that the Sardinian population consists of two distinct mtDNA groups, which may reflect ancient isolation and expansion events. Population structure, evolutionary history of the species and implications for the conservation of newt populations are discussed.     

Hatchery introgression blurs ancient hybridization between brown trout (Salmo trutta) lineages as indicated by complementary allozymes and mtDNA markers

Populations living on the periphery of a species range are subjected to intense habitat pressures that stress their vulnerability to threats. South Iberian brown trout (Salmo trutta) populations are located on the southwestern boundary of the species range in Europe. This region has been described as a contact zone between Atlantic and Mediterranean populations in other fish species, and allozyme data detected natural hybridization between native Atlantic and Mediterranean brown trout in this area. Nevertheless, native pattern of populations relationships are threatened by hybridization with exogenous hatchery fish. Allozyme data and complete mtDNA control region sequences were used to analyse the ongoing processes of human mediated hybridization between native and hatchery brown trout in this region. Estimates of the persistence of hatchery genes detected a high level of introgression in some of the sampled locations (hatchery ancestry more than 25% in GF-1, GF-2 and GF-3), but results differed from both kinds of markers. The low intrapopulation diversity (H_s = 0.051) indicated that genetic drift could explain discrepancies between markers. At individual level, the complementary use of both markers detected more hatchery origin fish than those detected by the use of a single technique. Based on these results, the importance of considering a large number of genetic markers to evaluate introgression accurately is emphasized. The ability of the current management policies to the protection and conservation of marginal populations that retain complex and special evolutionary histories such as those studied is also questioned.     

Genetic differences between the two remaining wild populations of the endangered Indian rhinoceros (Rhinoceros unicornis)

The management of rare and endangered species in the wild and in captivity requires an understanding of the characterization of the genetic units within each species and their relationships to each other. The Indian rhinoceros (Rhinoceros unicornis) is an endangered species with a current population size of c. 2800 individuals. We analyzed 26 individuals of known origin kept in captivity and 21 wild ranging individuals of the two remnant large wild populations in Assam (India) and Nepal employing mitochondrial and microsatellite markers to determine whether the two geographically isolated populations show distinct patterns of genetic diversity, and whether the genetic diversity of the populations is influenced by past demographic bottlenecks. We identified 10 different mitochondrial D-loop haplotypes, of which 4 were specific to the Assam population (10 sequences examined) and 6 specific to the Nepal population (19 sequences). Similarly, the microsatellite analysis demonstrated a strong genetic differentiation between the Assam and Nepal populations and allowed to assign each individual to its origin with high confidence. Furthermore, our analyses revealed the occurrence of a bottleneck in the Assam population long before the reported bottleneck in 1908, and it revealed that the Nepal population is a recent (probably post-glacial) colonization. In summary, the extent of genetic divergence between the two remnant R. unicornis populations suggests separate conservation programs (even for captive individuals) as long as the persistence of the entire species is not severely threatened. The microsatellite markers can also be used to determine the origin of confiscated material such as horns.     

Two genetically distinct units of the Chinese sika deer (cervus nippon): analyses of mitochondrial DNA variation

Chinese sika deer, Cervus nippon, are currently threatened by habitat loss, fragmentation and human hunting, which has led to the extinction of three subspecies in the wild. The remaining subspecies subsist in the narrow regions of Jilin, Heilongjiang, Sichuan, Gansu, Jiangxi, Anhui, and Zhejiang provinces. In order to design effective conservation strategies for the Chinese sika deer, we have investigated genetic diversity, population structure and gene flow in the Chinese sika deer populations by analyzing ≈995 base pairs of the mitochondrial DNA (mtDNA) control region in 59 individuals sampled from the northeast of China, Sichuan, Jiangxi and Zhejiang. Chinese sika deer exhibited low mtDNA diversity and high gene flow among the four populations, and showed no strong geographical structure. The analyses of mtDNA variation among individuals of sika deer identified only two main phylogenetic groups even though three subspecies were sampled. These data singles out the Zhejiang population as being highly genetically distinct and worthy of separate conservation consideration. Therefore, it is recommended that a breeding program for the Zhejiang population be established.     

Phylogeographic study based on intraspecific sequence variation of chloroplast DNA for the conservation of genetic diversity in the Japanese endangered species Primula sieboldii

To gather information on intraspecific phylogeography for use in conservation programs for the endangered species Primula sieboldii in Japan, we analyzed sequence variation in five noncoding regions of chloroplast DNA. Twenty-two distinct haplotypes were recognized in total. The distribution of most haplotypes was geographically confined, but one haplotype was widely distributed throughout northern Japan, and several haplotypes were found in geographically distant regions. Three major clades were revealed by phylogenetic analysis of the haplotypes. Clade I was distributed in Kyushu and central Honshu, clade II in western Honshu and Hokkaido, and clade III from central Honshu to Hokkaido. According to analysis of molecular variance, 59.9% of the total cpDNA variation existed among regions, 32.5% among populations within regions, and 7.6% within populations. Therefore, if genetic conservation of the species is valued, transplanting of P. sieboldii among regions should be avoided. Multiple lineages often existed even in geographically narrow areas (e.g., within a 20-km range), so transplantation between adjacent populations in restoration activities should be carefully designed so as not to change the gene pool of local populations significantly. Also, the geographical distribution of cpDNA haplotypes may allow us to confirm the origin of plants collected for commercial purposes.     

Genetic variation and phylogenetic relationships among Indian Citrus taxa revealed by DAMD-PCR markers

Genetic variation and phylogenetic relationships among 50 wild and cultivated accessions of 19 Indian Citrus genotypes were examined through comparison of Directed Amplification of Minisatellite DNA (DAMD) markers and morphological characters. DAMD-PCR analysis with four primers resulted in amplification of a total of 45 bands, of which 35 (78 %) were polymorphic. Morphometric evaluation using 76 morphological characters showed high level of variability ranging from 0.18 to 1.00 (avg 0.39), whereas the Jaccard’s coefficient values of genetic similarity calculated from DAMD data ranged from 0.41 to 1.00 (avg 0.68), indicating moderate genetic divergence among the accessions studied. UPGMA dendrograms generated separately from morphometric and DAMD data segregated all the accessions of Citrus into four main clusters, each containing a true basic species and their probable hybrids. The grouping of individual accessions/genotypes under respective species or cultivars in DAMD dendrogram was based purely on their genetic relationships rather than geographical origin. There was no absolute congruence between the data and dendrograms generated from morphometric and DAMD analyses. The study demonstrates the resolving power of DAMD markers for discrimination of individual genotypes of Citrus under its respective species, hybrid or cultivar groups and inferring their genetic and phylogenetic relationships as well. This is the first report on application of DAMD markers in Citrus.     

Genetic structure, population dynamics, and conservation of Black caiman (Melanosuchus niger)

Microsatellite DNA polymorphisms were screened in seven populations of the largest Neotropical predator, the Black caiman Melanosuchus niger (n = 169), originating from Brazil, French Guiana and Ecuador. Eight loci were used, for a total of 62 alleles. The Ecuadorian population had the lowest number of alleles, heterozygosity and gene diversity; populations of the Guianas region exhibited intermediate diversities; highest values were recorded in the two populations of the Amazon and Rio Negro. During the last century Melanosuchus populations have been reduced to 1-10% of their initial levels because of hunting pressure, but no strong loss of genetic diversity was observed. Both the inter-locus g-test and the Pk distribution suggested no recent important recovery and/or expansion of current populations. On a global scale, the inter-population variation of alleles indicated strong differentiation (F_ST = 0.137).Populations were significantly isolated from each other, with rather limited gene flow; however, these gene flow levels are sufficiently high for recolonization processes to effectively act at regional scales. In French Guiana, genetic structuring is observed between populations of two geographically close but ecologically distinct habitats, an estuary and a swamp. Similar divergence is observed in Brazil between geographically proximate “black water” and “white water” populations. As a consequence, the conservation strategy of the Black caiman should include adequate ecosystem management, with strong attention to preservation of habitat integrity. Distribution of genetic diversity suggests that current populations originated from the central Amazonian region. Dispersal of the species may thus have been deeply influenced by major climatic changes during the Holocene/Pleistocene period, when the Amazonian hydrographic networks were altered. Major ecological changes such as glaciations, marine transgressions and a hypothesized presence of an Amazonian Lake could have resulted in extension of Black caiman habitats followed by isolation.     

Neutral DNA markers fail to detect genetic divergence in an ecologically important trait

The development of strategies for in situ, ex situ conservation and reforestation of the monkey puzzle tree (Araucaria araucana), a vulnerable tree endemic to southern South America, has led to an interest in the level and distribution of the genetic diversity of the species. Neutral DNA markers (RAPDs) and quantitative genetic techniques were used to characterise genetic heterogeneity within and among populations from throughout the natural range of the species. Both the level and pattern of genetic variation estimated using the different techniques were essentially uncorrelated. An important discrepancy was found with the neutral markers failing to detect an important quantitative genetic divergence across the Andean Range relating to drought tolerance. This study clearly demonstrates the potential problems associated with making recommendations for conserving the genetic resource of threatened species based solely on neutral marker studies. Alternative approaches are discussed, including a stronger focus on ecologically important traits and the potential use of surrogate measures of genetic variability.     

Combining genetic structure and ecological niche modeling to establish units of conservation: A case study of an imperiled salamander

Identification of conservation units below the species level has been difficult for researchers and conservation planners. Methods reliant solely on genetics to identify conservation units are widely used but have limited scope. Additionally, methods used to assess ecological distinctness are typically difficult to interpret, and hence not broadly applicable. Here, we attempt to reconcile these problems by defining conservation units utilizing both genetic and ecological methods. This study suggests a framework to evaluate discreteness and significance among populations for assessment of distinct population segments (DPSs). Specifically, we highlight a methodology that incorporates genetic analyses and niche-based distribution modeling to identify conservation units. As a case study, we sought to determine whether populations of an imperiled salamander (Notophthalmus perstriatus), appearing to exist in two regions separated by 125 km, exhibited genetic and ecological distinctness such that the regions demarcate separate conservation units. Using mtDNA (cyt-b), we found that haplotypes were shared between localities within each region but none were shared between regions. Niche-based distribution modeling revealed significant differences in the ecological setting between the two regions. In combination, the absence of evidence for recent genetic exchange and model-based support for differing ecological conditions utilized by newts between regions provides evidence that eastern and western populations are both distinct and significant. This study formalizes a method to assess DPS distinctness and significance providing general utility for this methodology as a conservation tool for many species.     

Natural history collections and the conservation of poorly known taxa: Ecological niche modeling in central African rainforest genets (Genetta spp.)

Natural history collections are crucial resources for conservation biology, particularly given the development of the ecological niche modeling (ENM) approach. We combined ENM with taxonomic investigations to address ecological, geographic, and phenotypic variation in the poorly known central African rainforest genets (Genetta cristata, Genetta servalina, Genetta victoriae) to provide new insights into their conservation status. Taxonomic identification was based on four discrete morphological characters. ENMs were developed using the Genetic Algorithm for Rule-Set Prediction (GARP), 10 environmental data layers and 310 georeferenced localities extracted from 667 museum specimens and 22 literature sources. Re-assessed taxonomic identifications allowed us to expand significantly the known range and ecological limits of the three genets. We suggested limited hybridization between G. cristata and G. servalina, in a zone of sympatry likely to cover central Cameroon, northwestern Gabon and Congo. ENM showed that almost all known occurrences were connected geographically by suitable ecological conditions, suggesting continuous potential distributions between supposedly isolated populations of the three genets. Our investigations indicated that G. cristata is distinct from G. servalina in morphology, geographic ranges and ecological niches, indicating species status for G. cristata, which, as such, should be the subject of appropriate conservation attention. Areas of predicted connectivity and actual zones of occurrence falling outside current forest cover should be surveyed to re-assess the status of the rainforest genets. Our study shows that combining ENM and taxonomic investigations can substantially improve data utilization from natural history collections, especially in the case of poorly known species.     

MtDNA genetic diversity and population history of a dwindling raptorial bird, the red kite (Milvus milvus)

The red kite (Milvus milvus) occurs in a relatively small area in the southwestern Palearctic region, with population strongholds in Central Europe. Following strong human persecutions at the beginning of the 20th century, populations have receded, particularly in peripheral areas and islands. In order to describe and compare levels of genetic diversity and phylogeographic patterns throughout its entire distribution in Europe, sequence variation of a 357 bps part of the mitochondrial DNA control region was assessed in eight populations and 105 individuals. Overall, results indicate that population declines have affected red kite mtDNA variation. We found low levels of genetic diversity (values of nucleotide diversity ranging from 0 in Majorca island to 0.0062 in Central Europe), with only 10 distinct haplotypes, separated by low levels of genetic divergence (mean sequence divergence = 0.75%). Highest haplotype and nucleotide diversities match with demographic expectations, and were found in Central European and Central Spanish samples, where present strongholds occur, and lowest values in the declining southern Spanish and insular samples. Φst estimates indicated moderate gene flow between populations. Phylogeographic patterns and mismatch distributions analyses suggest central European regions may have been colonized from southern glacial refugia (in the Italian or Iberian peninsulas). Interspecific phylogenetic comparisons and divergence date estimates indicated the genetic split between the red kite and its closely related species, the black kite (Milvus migrans), might be relatively recent. The low level of genetic variation found in the red kite mitochondrial control region, compared to the black kite, is likely the result of relatively recent divergence (associated with founder events), successive bottlenecks and small population sizes. As there are several ongoing projects aimed at reinforcing populations in countries such as the United Kingdom, Italy or Spain, our results may prove useful for the genetic management of the species.     

Home gardens: an assessment of their biodiversity and potential contribution to conservation of threatened species and crop wild relatives in Benin

Despite growing literature supporting the importance of home gardens (HG) as biodiversity hotspots, knowledge of patterns of their contribution to conservation of threatened species and crop wild relatives (CWR) across climate and culture in Africa is still limited. This investigation was conducted across three climatic zones to assess the floristic diversity of home gardens and the extent to which they contribute to conservation of threatened species and CWR. Overall, 240 home gardens were sampled and their floristic diversity assessed. The ecological importance of recorded species was determined per climatic zone using the importance value index (IVI). A cluster analysis was performed to group the species according to their IVI-values and a principal component analysis helped to identify the most important species. 285 species were inventoried throughout the study area. Home garden species’ diversity globally declined from the drier to the wetter zone but was highest in the transition zone. The average number of species found per HG was 10.1 and varied weakly across zones (9.07, Guineo-Congolean zone; 10.77, Sudano-Guinean zone; and 10.53, Sudanian zone). The most important home gardens species in the Sudanian, the Sudano-Guinean and the Guineo-Congolean zones were respectively: Abelmoschus esculentus (L.) Moench and Hibiscus asper Hook.f.; Solanum lycopersicum L. and Zea mays L.; Ipomoea aquatica Forssk. and Senna occidentalis (L.) Link. They were mainly vegetables and used as food and/or medicinal plant species. Twenty CWR and twelve threatened species were recorded and were also mainly used for food and medicinal purposes. Thorough research on socioeconomic factors supporting possession of HG and choice of managed species as well as indigenous management strategies of HG and dynamic of traditional knowledge related to HG may help to deeply assess home gardens’ effectiveness in biodiversity conservation.     

Genetic isolation and morphological divergence of Black Sea bottlenose dolphins

The Black Sea is a semi-enclosed body of water that differs from the adjacent Mediterranean Sea in terms of its biodiversity, oceanographical and ecological characteristics. There is growing international concern about pollution in the Black Sea and other anthropogenic threats to its fauna. The bottlenose dolphin (Tursiops truncatus) is one of three species of cetaceans living in the Azov-Black Sea basin. Despite considerable research on bottlenose dolphins elsewhere, the extent of human impacts on the Black Sea populations is unknown. Previous attempts to award special conservation status to Black Sea cetaceans have failed specifically because policy makers have viewed their ecological and evolutionary uniqueness as equivocal. This study assessed divergence between Black Sea, Mediterranean Sea and Atlantic Ocean bottlenose dolphins for 26 cranial measurements (n = 75 adult bottlenose dolphin skulls) and mitochondrial DNA (n = 99 individuals). Black Sea bottlenose dolphins are smaller than those in the Mediterranean, and possess a uniquely shaped skull. As in a previous study, we found the Black Sea population to be genetically distinct, with relatively low levels of mtDNA diversity. Population genetic models suggest that Black Sea bottlenose dolphins have so little gene flow with the Mediterranean due to historical isolation that they should be managed separately.     

Analysis of the genetic structure of red-legged partridge (Alectoris rufa, Galliformes) populations by means of mitochondrial DNA and RAPD markers: a study from central Italy

The strong hunting pressure on the red-legged partridge, Alectoris rufa, warranted its inclusion into the list of species of European conservation concern. During the last decades, restocking plans with farmed specimens have counterbalanced the hunting drawings from wild populations. Our concern was the study of A. rufa in the easternmost part of its range, the central Italy, to gain insights into the effects of this compensation practice on the genetic structure of its populations. Partridges from both a geographically isolated, long-time protected, wild population (Pianosa island, Tuscan Archipelago National Park) and two Tuscan farms (Bieri and Scarlino) were investigated. All the specimens were very similar in outward appearance, looking much like to A. rufa. Ninety-six sequences of both Cytochrome b and D-loop Control Region of mitochondrial DNA (mtDNA) were analysed to get evidence of ancestry at the population level, whereas, the Random Amplified Polymorphic DNA (RAPD) technique was employed to get fingerprinting at the individual level. Pianosa and Bieri populations showed both the A. rufa and Alectoris chukar mtDNA lineages, whereas the Scarlino one only the A. rufa-mtDNA line. However, a spread overall pattern of A. rufa × A. chukar hybridisation among specimens, whatever their mtDNA lineage could result to be, was disclosed by means of RAPD species-specific markers. This is the first genetically documented record of the A. rufa × A. chukar hybrids. The occurrence of the pure, native A. rufa genome in the easternmost part of the species’ geographical range may be guessed to be virtual.     

Minimum viable population and conservation status of the Atlantic Forest spiny rat Trinomys eliasi

A population viability analysis (PVA) was conducted to assess the minimum viable population (MVP) of the Atlantic Forest spiny rat Trinomys eliasi, a species threatened by habitat loss and restricted geographical distribution. Objectives were to suggest quasi-extinction thresholds, estimate minimum areas of suitable habitat (MASH) and MVPs, and compare results with the species’ current status. The computer package VORTEX was used. The model predicted sizes of 200 animals to achieve demographic stability, but buffering declines in genetic variability required populations of 2000 animals. Estimated MASHs were approximately 250 and 2500 ha for demographic and genetic stability, respectively. Mortality rate and mean litter size were the most sensitive parameters to changes in model assumptions. The protection of known populations and the search for extant populations are the first steps in conservation. T. eliasi's issue could help protecting the coastal shrubland ecosystem of Rio de Janeiro state. Observing IUCN's criteria for listing threatened species, it is suggested that T. eliasi should be ranked as vulnerable in red lists.     

Linking the gaps between conservation research and conservation management of rare dipterocarps: A case study of Shorea lumutensis

To conserve a rare plant, conservation programs must be guided by the biological attributes of the species. What constitutes the most critical biological information for plant conservation has been the issue of discussion for the last 30 years. Most scientists promote an approach that is either ecological or genetic in emphasis. Ecological and genetic processes will often interact synergistically to influence the population viability and to determine the persistence of populations in the long run. Consequently, conservation management of plant species, in addition to ecological information, requires a robust understanding of underlying genetic processes as well as the variation within and between populations. Conservation has a cost and the resources available for conservation programs are always limited. Therefore, conservation management strategies should not only be scientifically justified but also practical in terms of resource availability. Shorea lumutensis is a rare and endemic dipterocarp in Peninsular Malaysia. A comprehensive research activity was initiated to assess the population ecology and population genetics of S. lumutensis to elucidate specific ecological and genetic requirements and subsequently to set conservation strategies and priorities. This paper is apparently the first attempt at applying both the ecological and genetic approaches into conservation management of a rare dipterocarp. In addition, this paper also attempts to link the gaps between conservation research and conservation management in a realistic manner. It is our hope that this study will serve as a model for the study of other rare dipterocarps which should be given priority for conservation.     

Genetic diversity in wild Tunisian populations of Mentha pulegium L. (Lamiaceae)

The allozyme variation of 15 Tunisian wild populations of Mentha pulegium L. threatened by human activities (clearing, hard-grazing, ploughing, traditional uses) was surveyed by the analysis of 14 isozyme loci using horizontal gel starch electrophoresis. The species exhibited a high level of genetic variation within populations (the mean Ap = 2.20, P = 72%, Ho = 0.349 and He = 0.229), which indicates a predominately outcrossing mating system and the recruitment of new genotypes via dispersal seeds. The genetic structure analysis of the populations using F statistics indicates no inbreeding, and showed an excess of heterozygosity for few loci. The moderate differentiation of populations (F_ST = 0.110) and the low rate of gene flow between them (Nm = 2.02) might been caused by recent isolation of the populations through biotope disturbances. The value of Nei's genetic identity varied from 0.839 to 0.999 reflecting a relatively low genetic divergence between populations. Cluster analysis using UPGMA method and Nei's genetic identity values, showed that populations geographically close didn't always cluster together. However, populations within the same bioclimatic stage generally subclustered together indicating that differentiation between bioclimatic regions occurred.     

Extreme neutral genetic and morphological divergence supports classification of Adriatic three-spined stickleback (Gasterosteus aculeatus) populations as distinct conservation units

The fact that the intraspecific genetic differentiation in neutral genetic markers and genes coding for adaptive traits are not typically correlated has caused a great deal of conceptual and practical trouble in delimitation of conservation units. Although the importance of combining information on adaptive genetic divergence with information on historical and recent gene flow in the delimitation of conservation units has been recognized, integrated empirical studies to this end are still rare. We explored the evidence for the specific conservation status of two freshwater three-spined stickleback (Gasterosteus aculeatus) populations on the Adriatic side of the Balkan Peninsula by comparing their phenotypic and genetic characteristics to those of other representative European populations. Apart from focusing on the neutral genetic divergence in mitochondrial DNA sequences and microsatellite markers, we also compared the patterns of morphological differentiation (i.e. bony armour development) resulting from adaptation to freshwater environments. The Balkanic populations formed two distinct groups with regard to neutral genetic variation and had the least developed bony armour of all the examined populations. All morphometric analyses identified the two Balkanic populations as phenotypically – and hence most likely also ecologically – clearly distinct from other European three-spined stickleback populations. These results suggest that the two Balkanic populations (River Neretva and River Zeta) fulfil the most stringent criteria (i.e. lack of genetic and ecological exchangeability) to be classified as conservation units distinct from other European three-spined stickleback populations.