Relationship between population size and genetic diversity in endangered populations of the European bullhead (Cottus gobio): implications for conservation

This study investigated the relationship between the current size of endangered bullhead (Cottus gobio) populations and microsatellite genetic variability. Additionally, the microsatellite data were used to evaluate whether a genetic test for population bottlenecks was able to provide evidence of recent severe population declines. Finally, our results were used to develop conservation priorities and measures. Population size appears to be a crucial parameter in determining the amount of genetic diversity that can be preserved in bullheads, since a significant positive correlation was observed between both variables. Furthermore, in some populations we were able to detect genetic signatures of the documented decline in population size. We suggest that the most immediate goal for bullhead conservation should be to increase the size and the range of the populations, and in doing so minimise or even reverse further genetic erosion. Potential management actions like habitat quality improvement, reduction of river fragmentation and supplementation programmes (translocation, supportive breeding) are discussed.     

Genetic variation in the endangered Inner Mongolia endemic shrub Tetraena mongolica Maxim. (Zygophyllaceae)

Tetraena mongolica Maxim, is a critically endangered and endemic species of westem Inner Mongolia in China. Genetic variability within and among eight extant populations of this species was assessed using ISSR PCR (13 primers). We expected a low genetic diversity level, but our results revealed an intermediate level of intraspecific genetic diversity, probably resulting from this species being in a refuge during the last glaciation (at population level: P=48.1%, A_e=1.305, H_E=0.177 and H_pop=0.264; at species level: P=63.3%, A=1.368, H_T=0.213 and Hsp=0.324). A low level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (16.91%), Shannon's diversity index (18.83%) and AMOVA (15.2%). Populations shared high levels of genetic identity (I=0.9516±0.013). The extensive gene flow was a plausible reason for the low genetic differentiation.     

Genetic diversity and local population structure of fragmented populations of Trillium camschatcense (Trilliaceae)

Trillium camschatcense, a long-lived common woodland herb, has been experiencing intensive habitat fragmentation over the last century in eastern Hokkaido, Japan. We examined the genetic diversity and population genetic structure of 12 fragmented populations with different population sizes using allozyme electrophoresis. The percentage of polymorphic loci and mean number of alleles per locus were positively related to population size, probably due to the stochastic loss of rare alleles (frequency of q<0.1) in small populations. Populations with 350 flowering plants or fewer had lost almost all of their rare alleles. While the heterozygosity and inbreeding coefficient were not related to population size, some small populations showed relatively high inbreeding coefficients. In spite of the low genetic differentiation among overall populations (F_ST=0.130), local population structuring was recognized between the two geographically discontinuous population groups. Within groups, sufficient historical gene flow was inferred, whereas a low dispersal ability of this species and geographical separation could produce apparent differentiation between groups.     

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY (CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.     

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.     

Impact of introduced honey bees on native pollination interactions of the endemic Echium wildpretii (Boraginaceae) on Tenerife, Canary Islands

The aim of this study was to investigate effects of introduced honey bees (Apis mellifera) on native pollination interactions of Echium wildpretii ssp. wildpretii in the sub-alpine desert of Tenerife. We selected two study populations, one dominated by honey bees, while the other was visited by many native insects. During peak activity period of insects, nectar was nearly completely depleted in flowers of the first, but not the latter population. Thus, a high abundance of honey bees may have suppressed visitation by native animals due to exploitative competition. Honey bees stayed longer and visited more flowers on the same inflorescence than native bees, thus potentially promoting self-pollination of the plants. Level of seed set and viability was similar in the two study populations. However, we cannot rule out long-term changes in genetic population structure due to changes in gene-flow patterns caused by foraging behaviour of honey bees vs. native flower-visitors.     

Unexpected high levels of genetic variability and the population structure of an island endemic rodent (Oryzomys couesi cozumelae)

Oryzomys couesi cozumelae is an endemic, threatened rodent from Cozumel Island, Mexico. We estimated its genetic diversity and structure by analyzing microsatellite loci in 228 samples from 12 sampling sites widely distributed throughout the island. Unexpected high levels of genetic and allelic diversity were found: a total of 54 alleles, an average of 10.8 alleles per locus, and high heterozygosity values (mean H_O = 0.624, H_E = 0.690 and H_Nei = 0.689). These values are higher than those reported for small sized insular mammals, higher than that found in 37 individuals of the mainland O. couesi from southern Mexico (H_O = 0.578) that we analyzed for comparative purposes, and similar to those of other mainland small mammal populations. Despite factors that affect Cozumel’s biota, such as exotic predators and competitors, hurricanes, seasonal population fluctuations and anthropogenic activities, no evidence of genetic bottlenecks was found. A significant population structure was observed and a model of isolation-by-distance was supported. Our findings render O. c. cozumelae a high conservation value, not only for its high genetic diversity and structure, but because available data suggests that its population has declined significantly in recent years. Further habitat fragmentation and population isolation could result in a higher genetic structure and loss of genetic diversity. The protection of habitat, the maintenance of habitat connectivity and the removal of introduced competitors and predators are a conservation priority. Acknowledging that the genetic structure of populations has crucial conservation implications, the present genetic information should be taken into account in management plans for the conservation of O. c. cozumelae.     

Conserving marginal populations of the food plant (Impatiens noli-tangere) of an endangered moth (Eustroma reticulatum) in a changing climate

Impatiens noli-tangere is scarce in the UK and probably only native to the Lake District and Wales. It is the sole food plant for the endangered moth Eustroma reticulatum. Significant annual fluctuations in the size of I. noli-tangere populations endanger the continued presence of E. reticulatum in the UK. In this study, variation in population size was monitored across native populations of I. noli-tangere in the English Lake District and Wales. In 1998, there was a crash in the population size of all metapopulations in the Lake District but not of those found in Wales. A molecular survey of the genetic affinities of samples in 1999 from both regions and a reference population from Switzerland was performed using AFLP and ISSR analyses. The consensus UPGMA dendrogram and a PCO scatter plot revealed clear differentiation between the populations of I. noli-tangere in Wales and those in the Lake District. Most of the genetic variation in the UK (H_T=0.064) was partitioned between (G_ST=0.455) rather than within (H_S=0.034) regions, inferring little gene flow occurs between regions. There was similar bias towards differentiation between metapopulations in Wales, again consistent with low levels of interpopulation gene flow. This contrasts with far lower levels of differentiation in the Lake District which suggests modest rates of gene flow may occur between populations. It is concluded that in the event of local extinction of sites or populations, reintroductions should be restricted to samples collected from the same region. We then surveyed climatic variables to identify those most likely to cause local extinctions. Climatic correlates of population size were sought from two Lake District metapopulations situated close to a meteorological station. A combination of three climatic variables common to both sites explained 81-84% of the variation in plant number between 1990 and 2001. Projected trends for these climatic variables were used in a Monte Carlo simulation which suggested an increased risk of I. noli-tangere population crashes by 2050 at Coniston Water, but not at Derwentwater. Implications of these findings for practical conservation strategies are explored.     

Seasonal movements in the Sumatran orangutan (Pongo pygmaeus abelii) and consequences for conservation

Preservation of orangutan habitat can only be effective when their range requirements are taken into consideration. Here, the effect of the variation in fruit supply at different altitudes on the seasonal movements of orangutans is investigated. Transects were surveyed every 2 months from 1998 to 2000, at different altitudinal zones in the larger Ketambe area, Indonesia. The density of orangutan and sources of ripe fruit with soft pulp were determined along these transects. Both orangutan density and the availability of fruit varied considerably throughout the survey period at different altitudinal zones. The peaks in fruit production at different altitudes occurred in successive periods. For each altitudinal zone a significant correlation was found between orangutan density and the number of fruit sources, which shows that orangutans follow the production of fruit between these altitudinal zones within the boundaries of their home range. Orangutans with core areas in intermediate and highland areas depend seasonally on fruit production of lowland areas, and vice versa. Thus, conservation of large tracts of primary forest with a range of different altitudes is necessary to preserve the remaining orangutan population.     

Regional conservation priorities for a large predator: golden eagles (Aquila chrysaetos) in the Alpine range

We compared the density of golden eagles (Aquila chrysaetos) and their nest dispersion, productivity and diet in Alpine and pre-Alpine areas. The comparison was made at two spatial scales: (1) at the territory level within two contiguous populations, and (2) at the population level, using published data for 22 populations scattered across four countries (France, Switzerland, Germany and Italy). Availability of golden eagle's main prey species was higher in Alpine than in pre-Alpine regions. The potential foraging ranges of pairs of eagles in the pre-Alps had larger amounts of woodland and lower amounts of suitable foraging habitat than those of Alpine pairs. At both spatial scales, density and productivity were lower in the pre-Alps. Also, pre-Alpine populations were characterised by a lower percentage of main prey species in the diet, which was significantly correlated with productivity, and by a higher breadth of diet, which was negatively correlated with density. Overall, higher prey availability and habitat suitability in the Alps resulted in higher density and breeding success, in a manner compatible with theoretical metapopulation models. We suggest that priority be given to conservation of eagle populations in high quality Alpine habitat, and that conservation action be carried out by a mixture of site-protection measures and conservation of the wider environment.     

Plantations of Convallaria majalis L. as a threat to the natural stands of the species: Genetic variability of the cultivated plants and natural populations

The level of polymorphism, genetic variability and relatedness of Convallaria majalis-populations (species native in Poland, under partial legal protection) obtained from three Polish regions and from commercial producers (Polish and Dutch) were studied. In addition the differences between the cultivated plants and those occurring in natural stands were analyzed. Each region was represented by at least 20 populations among which half were collected in natural stands and half from cultivation sites (botanical gardens, private gardens and cemeteries), and compared with samples obtained directly from commercial producers. Seven primer pairs used for AFLP profiling amplified 466 scoreable DNA fragments that were used for multidimensional scaling and clustering. The above analyses make it possible to clearly distinguish among individuals and revealed groups of populations according to their geographic origin. Samples from populations collected in natural stands and cultivated in the same region did not differ from each other significantly.These results suggested that cultivated plants were probably obtained directly from the natural stand and the influence of plant cultures on natural populations was rather small.     

Microsatellite analysis of genetic population structure of the endangered cyprinid Anaecypris hispanica in Portugal: implications for conservation

The endangered fish species Anaecypris hispanica is restricted to eight disjunct populations in the Portuguese Guadiana drainage. The genetic structure of these populations was studied in order to determine levels of genetic variation within and among populations and suggest implications for conservation of the species. Based on five microsatellite loci, the null hypothesis of population homogeneity was tested. Tests for genetic differentiation revealed highly significant differences for pairwise comparisons between all populations, and substantial overall population subdivision (F_ST=0.112). All sampled populations contained unique alleles. Our findings indicate marked genetic structuring and emphasise limited dispersal ability. The high levels of genetic diversity detected within and among A. hispanica populations suggest, however, that the observed fragmentation and reduction in population size of some populations during the last two decades, has impacted little on levels of genetic variability. Data imply that most A. hispanica populations should be managed as distinct units and that each has a high conservation value containing unique genetic variation. It is argued that geographic patterns of genetic structuring indicate the existence of eight management units.     

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.     

Low levels of genetic variation among southern peripheral populations of the threatened herb, Leontice microrhyncha (Berberidaceae) in Korea

Levels of genetic variation and intrapopulation genetic structures of Leontice microrhyncha S. Moore (Berberidaceae) were assessed for six populations in South Korea, representing the southern most range of a species found in Northeast China and the Korean peninsula. Detected genetic diversity (H_es) was very low (0.024) and F_IS values showed large heterozygote deficiencies. The small percentage of polymorphic loci and numbers of alleles per locus suggest that L. microrhyncha has a history of severe or long-lasting population bottlenecks that have eroded genetic diversity. This study suggests that the Korean population appears to consist of two historically isolated and independently evolving populations. It seems likely that these groups have been isolated and unstable for a significant period of time. However, the effects of recent habitat fragmentation on the historically disjunct and fragmented population system found in L. microrhyncha were not those predicted from the lack of significant relationships between population-level patterns of genetic variation and population sizes. Most non-unique genotypes were shared by most individuals and the lower level of diversity, high levels of inbreeding and population differentiation as well as high rate of seed production indicated that this species is autogamous and self-compatible and probably largely selfing. Therefore, to preserve extant genetic variation, all populations must be protected across the small geographic range of the species to retain both allelic and genotypic diversity.     

Ex situ genetic conservation of endangered Vatica guangxiensis (Dipterocarpaceae) in China

RAPD polymorphisms were applied to check the efficiency of ex situ genetic conservation of endangered Vatica guangxiensis X. L. Mo. endemic to southwestern China. Low level of genetic variation was revealed in three remaining natural populations. Twenty random primers, each with 10 base pairs, generated 231 bands with 53.68% being polymorphic, and with an average of 32.46% being polymorphic in each natural population. Strong population differentiation was revealed by AMOVA (analysis of molecular variance) and Gst value was 0.3764. The population ML ex situ conserved in the Xishuangbanna Tropical Botanical Garden contained an intermediate genetic variation compared with natural populations, with 30.74% bands being polymorphic. Of the total 231 bands generated in V. guangxiensis, 204 bands were also detected in population ML, indicating that 88.31% of the total genetic variations of this species were conserved in ex situ population. If only the alleles with moderate to high frequency (P>0.05) were considered, 204 out of 209 bands (97.61%) occurred in ex situ population ML. RAPD analysis also detected one exclusive band in natural population NS, and five in natural population NP, three of these exclusive bands were generated in every samples of natural population (NP), and other three had moderate to high frequencies. While none of these exclusive bands were detected in ex situ conserved population ML. Our conclusions are that the ex situ conserved population ML contains representative genetic variation to maintain long-term survival and evolutionary process of V. guangxiensis, and that more extensive ex situ sampling in natural population NS and NP is needed to conserve more exclusive alleles in ex situ population. The tropical area in the Botanical Garden would play a more important role in the ex situ conservation of rare and endangered plants.     

Genetic diversity and differentiation in Ethiopian populations of Phytolacca dodecandra as revealed by AFLP and RAPD analyses

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analyses were performed on six populations (a total of 89 individuals) of Phytolacca dodecandra (endod) collected in Ethiopia. The populations were selected based on our previous investigation to represent two altitude groups: lowland/central-highland (1600–2500 m) and highland (2501–3000 m). A total of 197 AFLP and 68 RAPD markers were scored from 5 primer pairs and 12 random primers, respectively. The overall patterns obtained for AFLPs and RAPDs from diversity, cluster and principal component analyses were very comparable. However, the moderate correlation (r = 0.56) between AFLP and RAPD similarity matrices as well as the discrepancies in diversity estimates between the two techniques in some populations and in the lowland/central-highland plants could be due to differences in sensitivity of reaction conditions, bias in scoring of bands, number of markers used for analyses, and/or parts of the genome surveyed. For both AFLP and RAPD, the lowland/central-highland populations showed higher polymorphism and Shannon information measure (H) than the highlands. Cluster and principal component analyses performed for both marker types have also clearly demonstrated the differentiation of all the lowland/central highland plants from those of the highlands, in agreement with our previous conclusion. Markers scored from any of the five AFLP primer pairs were sufficient to clearly distinguish the two altitude groups; with RAPD, selection of about 8 informative markers produced by seven random primers was needed for the same purpose.     

Comparing population growth rates using weighted bootstrapping: Guiding the conservation management of Petrogale xanthopus xanthopus (yellow-footed rock-wallaby)

In this study we compare estimates of the long-term mean stochastic population growth rates, E[r] for Petrogale xanthopus xanthopus (yellow-footed rock-wallaby), a threatened Australian species. In fluctuating environments such as semi-arid areas, herbivore populations respond directly to changes in the biomass of food resources. Biomass is generally correlated with rainfall, so it is often useful to model annual population growth rates of herbivores directly with rainfall. Models of this nature are referred to as numerical response models. The factors that are thought to threaten this species include competition from introduced herbivores and predation from foxes. Annual aerial survey data collected from 1997 to 2004 over approximately 600 km of transect line were analyzed in seven zones within South Australia. Using the Ivlev numerical response model, the annual population growth rates were found to correlate best with the rain that fell in the seven-month period immediately prior to the surveys. Not surprisingly, positive growth rates were found to be associated with higher rainfalls in this period, while negative growth rates were associated with lower rainfalls. We also used weighted bootstrapping to calculate confidence intervals around our estimates of long-term mean stochastic population growth rates, E[r]. The findings suggest that the estimates of E[r] are positive in areas where there is fox and herbivore management. However, we found no evidence that this species will decline in the absence of these treatments.     

Genetic diversity within Aspergillus flavus strains isolated from peanut-cropped soils in Argentina

The genetic diversity of Aspergillus flavus populations isolated from the peanut-cropped soils in the peanut-growing region at Cordoba Province was evaluated by analysis of vegetative compatibility group (VCG). VCG_s were determined through complementation assays between nitrate-nonutilizing (NNO) mutants. Fifty-six VCG_s were identified from 100 isolates. Twenty-five VCG_s contained two or more isolates and 31 VCG_s contained only a single isolate. In general, there were significant differences among VCG_s in aflatoxin and CPA production. One VCG group included a single atoxigenic strain since it was neither aflatoxin nor cyclopiazonic acid producer. This isolate could be useful as a biological control agent, since it was unable to form a stable heterokaryon in the complementation test with the other isolates. Seven A. flavus isolated from soil were atypical because they simultaneously produced aflatoxins B, G and CPA.     

Reproductive biology and conservation implications of three endangered snapdragon species (Antirrhinum, Plantaginaceae)

About 32% of Antirrhinum species are considered to be endangered; however, no field studies have focused on their reproductive biology. In this work, several aspects of the reproductive biology (flowering phenology, floral biology, breeding system) and potential limits on seed quantity and quality (pollen limitation, inbreeding depression) were studied in natural populations of three endangered species of the genus (Antirrhinum charidemi, Antirrhinum subbaeticum, Antirrhinum valentinum). Results disclose that all three species need insect visitors for seed production since fruit set after autonomous self-pollination was lower than under hand cross-pollination. A. charidemi and A. valentinum were mainly self-incompatible, whereas A. subbaeticum was self-compatible but herkogamous. Supplementary pollination in open-pollinated flowers only increased fruit set and seed set relative to controls in a given population of A. valentinum. Preliminary data on inbreeding depression at early life-cycle stages of the self-compatible A. subbaeticum revealed that the cumulative level was low. Despite the three species being closely related and sharing many ecological characteristics, they show different mating systems, and different factors limit seed quantity and quality. Thus, caution should be taken when making a common conservation plan for a group of closely related taxa.     

Habitat selection and conservation of Sharpe's longclaw (Macronyx sharpei), a threatened Kenyan grassland endemic

Montane grassland is a severely threatened habitat in Kenya. Despite a high level of faunal endemism, it has received very little conservation attention. We investigated habitat selection in a threatened grassland endemic bird species, Sharpe's longclaw (Macronyx sharpei) (family Motacillidae), in order to understand its likely response to land-use changes. Between November 1995 and May 1996, we studied 41 territories of this species on the Kinangop Plateau in central Kenya. With an overall density of 0.4 birds ha⁻¹, longclaws lived in permanent groups of two to seven individuals. They were sedentary and territorial, with a mean home range size of 0.5 ha. The species avoided non-grassland areas entirely, and within grassland showed a strong preference for short grass with tussocks. Territory sizes and foraging ranges were smaller, and rates of pecking for food higher, in this grassland type than in open short grass or long grass. Land use changes, in particular conversion to cultivation or woodlots and ploughing up of grassland to remove tussocks, pose a serious and immediate threat to this species. Dairy farming is potentially compatible with grassland conservation, and conservation-friendly livestock rearing needs to be promoted through economic incentives, awareness-raising and technical advice.