Highly reliable genetic identification of individual northern hairy-nosed wombats from single remotely collected hairs: a feasible censusing method

The highly endangered northern hairy-nosed wombat (Lasiorhinus krefftii) is extremely difficult to study in the wild, and its numbers correspondingly difficult to estimate. Disturbance to the animals caused by trapping and radio-tracking may not only constitute an excessive risk to the population's viability, but may also yield biased data. The results of a pilot study are presented, which clearly show noninvasive genotyping to be a highly feasible and reliable alternative censusing method for L. krefftii. The protocol can identify individual wombats from single hairs collected remotely at burrow entrances, using: (i) a panel of microsatellite markers giving individual-specific genotypes; and (ii) a Y-linked sexing marker in combination with a single-copy X-linked amplification control. Using just the eight most variable microsatellites (of 20 available), only one in 200 pairs of full-sibs are predicted to share the same genotype. From 12 wombat hair samples collected on tape suspended over burrow entrances, three known female, two known male and an unknown wombat of each sex were identified. The approach will allow censusing of individuals that evade capture, and will also reveal some otherwise problematic aspects of the behaviour of this elusive animal.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

The genetics of amphibian declines: population substructure and molecular differentiation in the yosemite toad, Bufo canorus (Anura, bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data

We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.     

Leaf litter inhabiting beetles as surrogates for establishing priorities for conservation of selected tropical montane cloud forests in Honduras, Central America (Coleoptera; Staphylinidae, Curculionidae)

Thirteen tropical montane cloud forests (TMCFs) in Honduras and adjacent El Salvador were evaluated for species diversity and endemism in leaf litter inhabiting Curculionidae (weevils) and Staphylinidae in June and August of 1994. Totals of 26,891 specimens representing 293 species of Curculionidae, and 7349 specimens representing 224 species of Staphylinidae, were collected. Regional endemism was high with 173 species (58.7% of total) of Curculionidae and 126 species (56.3% of total) of Staphylinidae, restricted to single sites. Measures of diversity (number of observed species [Sobs]; number of endemic species) and estimates of biodiversity (Chao 2, first and second order Jackknife and Bootstrap richness estimators) are given for each site for Curculionidae and Staphylinidae. Priority-areas analyses based on Sobs (Greedy), Sobs, Chao 2, number of endemic species, site complementarity and higher taxonomic diversity (Curculionidae only) are presented. Weak or lack of correlation was noted between site area and site diversity for either Curculionidae or Staphylinidae. The optimum sequence for site conservation was determined based on Sobs (Greedy). Four sites are identified as the highest conservation priorities; Parque Nacional Montecristo, P.N. La Muralla, P.N. Santa Barbara and P.N. Comayagua. Identification as high priority sites supports their designation as Parques Nacionales rather than the less diverse Reservas Biologias or Reservas Vidas Silvestres. While closely approximating performance of Sobs (Greedy), no one of Sobs, number of endemics, Chao 2 or site complementarity give results equivalent to the optimum sequence based on Sobs (Greedy) and the latter is concluded to be the best method for establishing conservation priorities in TMCF. Results of the analyses based on one of Curculionidae or Staphylinidae differ in the ordering of site priorities based on each of Sobs, Chao 2 or number of endemics at each site, with at best, weak positive correlations between results based on each taxon. Data from other taxa are necessary to determine if one of Curculionidae or Staphylinidae emerges as the better surrogate for general patterns of biodiversity in TMCF. Conservation strategies in Central America should emphasize the importance of TMCF particularly in view of high regional endemism. In formulating these strategies, consideration needs to be given to the preservation of many small regional preserves rather than fewer larger preserves. Data from inventories of other taxa should be included where available and all biological data should be integrated with social and cultural issues of regional importance.     

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

What do mammalogists want to save? Ten years of mammalian conservation biology

Recent upsurge of interest in biological diversity requests that resources for conservation be allocated to taxonomic groups and geographic areas of greater priority, independent of the attractiveness of individual species. The aim of this paper is to assess if contributed papers on four of the most important international conservation journals in the last ten years reflect our increasing concern for threatened mammals. Our results show that some orders receive disproportionally more attention than predicted by their absolute number and percentage of threatened species. The Nearctic and Palearctic Regions are far more studied than other like, for instance, the Oriental Region, which are badly neglected considering their higher rates of endangered and endemic mammals. Furthermore, it was found that among many orders most of the species covered in contributed papers are not presently considered threatened by IUCN. Our work highlights the need of assessing conservation priorities at least at continental level and of devoting more resources to research in tropical countries.     

Conservation of the Cedrus libani populations in Lebanon: history, current status and experimental application of somatic embryogenesis

Cedrus libani, the cedar of Lebanon, is a threatened conifer native to the Levant. Over 4000 years of exploitation have resulted in the fragmentation and degradation of the Lebanese cedar populations. Continued urban and agricultural development in Lebanon adds to the difficulty of effective conservation. Two protected areas have recently been established which contain two of the more important forests: a cedar dominated forest in the Shouf region and a mixed forest at Ehden. A number of other populations are protected by ministerial decrees, and there is a need for rigorous management of all the remaining populations. The application of in vitro techniques such as somatic embryogenesis may assist in the conservation of this species. We have produced somatic pro-embryos using immature zygotic tissue as explants cultured on half-strength MS medium containing an auxin and a cytokinin (10 M 2,4-D and 5 M BAP). The application of somatic embryogenesis to the Lebanese cedar would be in the propagation and preservation of selected genotypes, either those from old growth provenance for use in restoration, or those with desirable commercial or horticultural characteristics.     

The Central American squirrel monkey (Saimiri oerstedii): introduced hybrid or endemic species?

Although squirrel monkeys (Saimiri spp., Primates: Cebidae) are abundant and widespread in South America, the disjunct Central American species, Saimiri oerstedii, has been restricted to the Pacific wet lowlands of Costa Rica and Panama since the earliest historical records. This taxon is now endangered in Costa Rica and nearly extinct in Panama because of habitat loss, development, and the pet trade. Conservation efforts have been hampered because of the influential, but untested, speculation that S. oerstedii represents a hybrid species introduced by prehispanic Amerind traders from multiple localities in South America. Using nuclear and mitochondrial DNA sequence data to reconstruct the phylogenetic relationships among Saimiri from Central and South America, we reexamine the taxonomic status of squirrel monkeys from different geographic regions. The sequence data support P. Hershkovitz's (1984, Am. J. Primatol. 6: 257-281) taxonomy advocating four distinct species. Combining this information with evidence from the fossil record to date the divergence times among sister taxa, we test and reject the hypothesis that Central American squirrel monkeys are the result of human introduction.     

Greater male fitness of a rare invader (Spartina alterniflora, Poaceae) threatens a common native (Spartina foliosa) with hybridization

Hybridization with abundant invaders is a well-known threat to rare native species. Our study addresses mechanisms of hybridization between a rare invader, smooth cordgrass (Spartina alterniflora) and the common native California cordgrass (S. foliosa) in the salt marshes of San Francisco Bay. These species are wind-pollinated and flower in summer. The invader produced 21-fold the viable pollen of the native, and 28% of invader pollen germinated on native stigmas (1.5-fold the rate of the native's own pollen). Invader pollen increased the seed set of native plants almost eightfold over that produced with native pollen, while native pollen failed to increase seed set of the invader. This pollen swamping and superior siring ability by the invader could lead to serial genetic assimilation of a very large native population. Unlike California cordgrass, smooth cordgrass can grow into low intertidal habitats and cover open mud necessary to foraging shorebirds, marine life, navigation, and flood control in channels. To the extent that intertidal range of the hybrids is more similar to the invader than to the native parent, introgression will lead to habitat loss for shore birds and marine life as well to genetic pollution of native California cordgrass.