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.     

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.     

AFLP fingerprinting of Anchusa (Boraginaceae) in the Corso-Sardinian system: Genetic diversity, population differentiation and conservation priorities in an insular endemic group threatened with extinction

Although several plants endemic to Corsica and Sardinia are included in various redlists, no attempts have been made to analyse their genetic diversity with molecular techniques. Genus Anchusa occurs with seven taxa in either mountain or coastal habitats of the two islands, but the very restricted range and low population size pose these endemisms in a very precarious conservation status. Highly variable markers (AFLP) were therefore used to analyse the patterns and levels of genetic diversity in a sample of 11 populations from the entire range of the group.Results indicate the separation between a mountain genic pool including Anchusa formosa, Anchusa capellii and Anchusa montelinasana, and four groups of coastal accessions. In spite of small size, mountain taxa show low interpopulation differentiation (F_st = 0.02) and relatively high intrapopulation genetic variation (0.365), while coastal accessions showed on average a stronger differentiation (mean F_st = 0.20) and a lower diversity (0.281), possibly due to higher rates of inbreeding. The particularly low levels of variation found in A. sardoa, A. littorea and A. crispa ssp. maritima from the Coghinas bay are likely due to a historical decrease of populations and to bottleneck events caused by loss of habitat and natural stochastic factors on sand dune ecosystems. While habitat maintenance and regulation of grazing by domestic herbivores should be sufficient to ensure the persistence of the mountain endemics, additional actions of in situ and ex situ conservation are needed for the critically endangered coastal species A. sardoa and A. littorea. A. crispa showed a relatively high variation, especially on Corsica. No correlation between population size and genetic variation was found in the latter species, highlighting the importance of the small patches for its conservation. Also, the genetic separation between subspecies crispa and maritima stresses the need of keeping them distinct in redlists and conservation actions on Sardinia.     

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.     

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.     

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.     

Relationship between reservoir size and genetic differentiation of the stream caddisfly Stenopsyche marmorata

Reservoirs have the potential to block the gene flow of stream macroinvertebrates along a channel, which takes place via larval drift and adult flight, resulting in genetic differentiation above and below the reservoirs. Gene flow across the reservoirs may be strongly obstructed if the area of standing water is larger. Using random amplified polymorphic DNA markers, we investigated the genetic structure of Stenopsyche marmorata (Stenopsychidae: Trichoptera) populations found above and below the reservoirs, and the reference stream of six reservoirs with small to large water surface area ranging from 0.12 km² to 6.00 km². As a result, the two largest reservoirs with a standing water area larger than 3.27 km² showed significant differences in pairwise θ between fragmented and reference streams, whereas the other four reservoirs with a standing water area smaller than 1.64 km² showed insignificant differences. The genetic differentiations in the two largest reservoirs did not result in the reduction of genetic diversities in the fragments. Based on the significant correlation between relative population size and mean expected heterozygosity, we concluded that local genetic diversities were constrained in smaller populations due to the effect of genetic drift.     

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.     

Island differences in population size structure and catch per unit effort and their conservation implications for Komodo dragons

Species inhabiting archipelagos are often characterised by high levels of interpopulation divergence (e.g. size related traits). This divergence may, in turn, influence their life-history. To facilitate better management and conservation of the Komodo dragon (Varanus komodoensis), an island endemic, we identified demographic differences between two island populations in Komodo National Park, Indonesia. Comparison of data collected from dragon populations inhabiting Rinca Island and the much smaller Gili Motang Island indicated that between 1994 and 2004, the Komodo dragon population on Gili Motang significantly decreased its: (1) mean body mass, (2) body condition and (3) relative abundance. These results suggest that the numerically small Gili Motang population was oscillating downwards; in contrast, the Rinca Island population had been relatively stable. More importantly these results emphasize the necessity for managers of this priority conservation species to understand further the inherent functional differences among dragon populations to develop island specific management units. Current management practices (e.g. monitoring) instigated by Komodo National Park management ignore small island dragon populations and thus run the risk of being unable to detect adverse effects for populations that are potentially most prone to decline.     

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.     

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.     

Estimating population size in the subdesert mesite (Monias benschi): new methods and implications for conservation

The subdesert mesite, a terrestrial non-passerine bird endemic to the Mikea Forest of southwest Madagascar, is currently classified as globally threatened (category: Vulnerable). However, accurate assessment of threat in accordance with the IUCN Red List criteria (A, B and C) requires data on effective population size, area of habitat occupied and rate of decline, none of which is available for this species. Here we present the first empirical estimates of its population size using five complementary methods, three incorporating data on territory size and two using data gathered during call-playback surveys conducted throughout its entire global range. Estimates vary from 98,000 to 152,000 individuals, with the most reliable possibly being that generated by distance sampling (115,000). This figure is more than an order of magnitude greater than the only published estimate of <10,000 individuals. By analysing data on forest cover change, we estimate the population of the subdesert mesite to have declined by, at most, 10% in three generations. Although the rate of deforestation in 1994-1999 is double that calculated for 1962-1994, it is deemed unlikely that the population will decline by 20% over the next three generations. As such the species fails to meet criterion A. Although the subdesert mesite's range and area of occupancy are small, they are not fragmented and do not comprise fewer than 10 locations. Consequently, this species does not meet criterion B. Further, the species fails to meet criterion C, for which a maximum of 10,000 mature adults is required. According to IUCN (2000 Red List of Threatened Species) this species should therefore be downlisted in status. We discuss why it still warrants conservation attention and suggest the need for modifications to the criteria thresholds in relation to basic information about the ecology and taxonomic distinctness of species.     

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in tunisian soils

A collection of 299 isolates of rhizobia nodulating Medicago truncatula was isolated from 10 Tunisian soils and was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA gene. Results showed that 227 and 72 isolates were assigned, respectively, to Sinorhizobium meliloti and Sinorhizobium medicae. In 9 out of 10 soils S. meliloti was detected, whereas S. medicae was recovered from only 5 out of 10 soils. The cross-nodulation of three populations of M. truncatula grown on Bulla Regia soil, which contained naturally the two Sinorhizobium species, showed that M. truncatula population collected from Amra site was selective to S. meliloti at least in soil conditions. Forty-eight isolates of each Sinorhizobium species trapped by M. truncatula populations collected from Bulla Regia, Soliman and Rhayet sites on Bulla Regia soil were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and showed a clear distinction between the two Sinorhizobium species and a higher diversity for S. meliloti.     

Behavioural and morphological variation between captive populations of red junglefowl (Gallus gallus) - possible implications for conservation

The escalating threats to ecosystems worldwide have lead to a need for efficient methods to breed animals in captivity and to prepare captive-born animals for release back to the wild. However, life in captivity may lead to modifications in the animal’s behaviour mainly by genetic changes, including behavioural adaptations such as reduced predator responses. Such modifications may seriously affect survival after a reintroduction. The present study was a first screening of behavioural and morphological variation between different captive populations in standardized test situations using red junglefowl as a model species. The birds were tested in three different test situations in order to measure anti-predatory behaviour, social behaviour and exploratory behaviour. The results of this study clearly show that there are behavioural differences between the captive populations which potentially can be crucial for the animals in a reintroduction situation. However, the extent to which these differences are due to genetic changes caused by small breeding populations or adaptations to the different captive environments is not yet known, although morphological differences found suggest that genetic variation may cause some of the behavioural differences as well. The differences found imply that life in captivity can affect an animal’s behaviour and even though the red junglefowl is merely used as a model here, this suggests that these aspects may be important to consider also in other species where reintroduction is a more central motive for keeping the animals in captivity.     

Assessing habitat use of the endangered marine mollusc Patella ferruginea (Gastropoda, Patellidae) in northern Africa: preliminary results and implications for conservation

The limpet Patella ferruginea Gmelin, 1791, endemic to the Mediterranean, is the most endangered marine species on the list of the European Council Directive 92/43/EEC on the Conservation of Natural Habitat of Wild Fauna and Flora (1992). Its Mediterranean range has progressively contracted to a few restricted areas and the species is now threatened with extinction. Seventeen stations were sampled along the littoral zone on the coast of Ceuta, North Africa, for quantifying environmental factors (water movement, siltation and suspended solids), and 70 transects were selected for P. ferruginea sampling. The study revealed the presence of a well-established population of P. ferruginea with values of density and size of 0.67 individuals m⁻¹ ±0.96 and 48.94 mm±11.61 respectively (mean±standard error of mean). The biggest densities of P. ferruginea were found inside the harbour of Ceuta. The limpets were more abundant on artificial harbour stones than on natural rocky shores, and the areas under the highest human pressure were characterised by the lowest densities and the smallest specimens probably due mainly to the predation for food and fishing. Human pressure is probably the main contributing factor to the currently endangered status of P. ferruginea. Programmes of environmental education to avoid its collection for fishing, food or for fun as decorative objects should be conducted, and further experimental studies dealing with the reproductive biology of this species are needed to properly assess the future programmes of conservation.     

Pathogen exposure in endangered island fox (Urocyon littoralis) populations: Implications for conservation management

Island fox (Urocyon littoralis) populations on four California Channel Islands have declined severely since 1994. Canine distemper (CDV) was suspected to be responsible for the decline of the Santa Catalina Island fox, so knowledge of infectious disease exposure in the remaining island fox populations was urgently needed. This study reviewed previous pathogen exposure in island foxes and investigated the current threat by conducting a serologic survey of foxes on all islands and sympatric feral cats on three islands from 2001 to 2003 for antibodies against canid pathogens. Before the decline, foxes had evidence of exposure to CDV, canine adenovirus (CAV), canine parvovirus (CPV), and Toxoplasma, with exposure to these five pathogens differing greatly by island. Exposure to canine coronavirus (CCV), canine herpesvirus (CHV), and Leptospira was rare. In 2001-2003, wild-born foxes had evidence of exposure to CDV (5.2-32.8%) on 5 of 6 islands, CPV (28-100%) and CAV (4.7-100%) on five islands, and Toxoplasma gondii (2.3-15.4%) on four islands. Exposure to CCV, CHV and Leptospira was less common. Sharing of infectious agents between sympatric foxes and feral cats appeared minimal, but CDV exposure was detected in two cats on Santa Catalina Island. Domestic dogs have historically been present on the islands, but it is not known if canine diseases can be maintained in fox populations without the continual presence of dogs. Targeted vaccination programs against the most virulent pathogens and continued intensive disease surveillance may help protect the critically small remaining fox populations from disease outbreaks that could threaten the success of ongoing conservation efforts.     

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.     

Population genetic structure and the conservation of isolated populations of Acacia raddiana in the Negev Desert

There is much concern over the high mortality of many populations of Acacia raddiana, a keystone tree species in the Negev desert of Israel. We used random amplified polymorphic DNA (RAPD) to assess patterns of genetic variation within and among 12 populations of A. raddiana from the Arava (Syrian-African Rift) valley and western Negev. A high level of genetic polymorphism was recorded within populations. An analysis of molecular variance (AMOVA) showed that about 59.4% of total genetic variance occurred among populations, which is considerably greater population differentiation than that recorded for other outbreeding species. Cluster and principal coordinates analyses and AMOVA indicate that the western Negev and Arava valley populations are highly differentiated. We suggest that there may have been two invasions of A. raddiana into Israel: one across the northern Sinai/Gaza Strip area into the western Negev, with some plants reaching the Dead Sea and a second invasion across the southern part of the Sinai peninsula, or even from Saudi Arabia, up to the Arava valley. From the conservation point of view, each population should be conserved separately because they are genetically highly differentiated and loss of any one population would lead to a dramatic loss of genetic variation. The mixing of genetically distinct populations may give rise to outbreeding depression (particularly because of GXE interactions). An obvious first step to the maintenance of this species' genetic diversity is the separate management of the western Negev and Arava valley populations because of their different evolutionary histories.