Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.     

分子生态学研究与运行多样性保护

分子生态学的发展揭开了生物多样性保护研究的新篇章,分子技术的应用克服了传统生态学法中的一些难题,如野外调查周期长,分辨率有限,实验条件不易控制等,应用各种分子标记（如：RFLP,VNTR,RAPD,DNA测序等）可以分析种群地理格局和异质种群动态,确定种群间的基因流,研究瓶颈效应对种群的影响以及确定个体间的亲缘关系等等,所有这些研究都是指导物种保护和淑危种群的恢复所必要的,种或品系特异性的分子标记技术能够解决形态分类中的模糊现象,确定基于遗传物质的谱系关系,还可以用来分析近缘种间杂交问题,这些问题的解决有助于确定物种优先保护顺序,选择保护地工,近年来引起重视的主要组织人性复合体（MHC）NDA异分析可能会在研究种群对疾病的易感性第一系列种群特异性问题方面非常有用,随着分子技术的不断发展,会有更多的保护生物学问题得到解决,尤其是结合野外调查统计数据应用多个分子标记对目标种群进行研究,所得到的结果会更精确,更有说服力。     

How global extinctions impact regional biodiversity in mammals

Phylogenetic diversity (PD) represents the evolutionary history of a species assemblage and is a valuable measure of biodiversity because it captures not only species richness but potentially also genetic and functional diversity. Preserving PD could be critical for maintaining the functional integrity of the world's ecosystems, and species extinction will have a large impact on ecosystems in areas where the ecosystem cost per species extinction is high. Here, we show that impacts from global extinctions are linked to spatial location. Using a phylogeny of all mammals, we compare regional losses of PD against a model of random extinction. At regional scales, losses differ dramatically: several biodiversity hotspots in southern Asia and Amazonia will lose an unexpectedly large proportion of PD. Global analyses may therefore underestimate the impacts of extinction on ecosystem processes and function because they occur at finer spatial scales within the context of natural biogeography.     

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next‐generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.     

Identification of 'extinct' freshwater mussel species using DNA barcoding

Freshwater mollusks are highly imperiled, with 70% of the North American species extinct, endangered, or at risk of extinction. Impoundments and other human impacts on the Coosa River of Alabama, Georgia and Tennessee of the southeastern USA alone are believed to have caused 50 mollusk species extinctions, but uncertainty over boundaries among several putatively closely related species makes this number preliminary. Our examination of freshwater mussels collected during an extensive survey of the upper-drainage basin, DNA barcoding and molecular phylogenetic analyses confirm the rediscovery of four morphospecies in the genus Pleurobema (Unionidae) previously thought to be extinct from the upper Coosa basin. A fifth 'extinct' form was found in an adjoining basin. Molecular data show that the Coosa morphologies represent at least three species-level taxa: Pleurobema decisum, P. hanleyianum and P. stabile. Endemism is higher than currently recognized, both at the species level and for multispecies clades. Prompt conservation efforts may preserve some of these taxa and their ecosystem.     

Isolation and characterization of microsatellite loci from the endangered New Zealand takahe (Gruiformes; Rallidae; Porphyrio hochstetteri)

Nineteen polymorphic microsatellite loci were characterized from the endangered takahe (Porphyrio hochstetteri). Like many of New Zealand's other native avian species, levels of polymorphism were low, with variation detected at only 19 of 110 (17.3%) loci, and most polymorphic loci (78.9%) were diallelic (mean number of alleles = 2.3). Despite these low levels of variation, the microsatellites developed here will be useful for parentage assignment for confirming pedigrees, and investigating relationships between genetic variation, pedigree‐based inbreeding and reproductive success in this highly endangered species.     

Promotion by phloroglucinol of micropropagation of Minuartia valentina, an endangered and endemic Spanish plant

Tissue culture techniques for the propagation and conservation of endemic or threatened plants can be used to complement the methods usually applied in ex situ conservation. Thus, Minuartia valentina (Caryophyllaceae), an endangered plant species endemic to the Valencia Community (Eastern Spain), was successfully regenerated through shoot proliferation from wild plants growing in their natural area. Nodal segments, 10~mm long, were cut from rametes of adult material, sterilised and established in vitro. Equally successful shoot multiplication was achieved on Murashige and Skoog (MS) medium with 80 mg l-1 phloroglucinol in combination with either 1 mg l-1 6-benzylaminopurine or 1 mg l-1 kinetin. Excised shoots were rooted in MS medium supplemented with an auxin (indole acetic acid, indole-3-butyric acid, or napththalene acetic acid). Shoots rooted well (96–100%) within three weeks in all auxin treatments. However, the use of napththalene acetic acid was discarded because this auxin delayed root differentiation, and induced adventitious root malformation. Rooted plantlets were transferred to pots and 85% of them acclimatized successfully four weeks after transfer to greenhouse conditions, where they exhibited normal morphology and growth.     

Distribution status and habitat characteristics of the endangered freshwater fish,Microphysogobio rapidus (Cyprinidae)

To investigate distribution, habitat characteristics, and current conservation status of the endangered endemic species, rapid small gudgeon Microphysogobio rapidus (Cyprinidae), we surveyed a total of 79 sites from the historic records (20 sites) plus additional sites (59 sites) with good habitat conditions, analyzed their sites, and compared them with historic recorded sites to reveal the factors of extinction threats and causes. We found only eight out of 79 sites in the Nam River areas. The habitats were greatly reduced and restricted compared with the historic sites, which mainly cause from habitat modification, such as various types of river renovations at the main stream and tributary streams of the Nakdong River. The present habitats are higher water temperature and more number of fish species than the absent ones, but conductivity, total nitrogen, and number of weir are lower. In addition, the present sites are lower low velocity at pool and higher mean substrate at pool. From this study, we suggest that maintaining good water quality and preventing anthropogenic impacts greatly aid conservation of the M. rapidus in South Korea.     

Changes in functional biodiversity in an invaded freshwater ecosystem: the Moselle River

The spread of non-indigenous species and the decline of autochthonous ones are leading to a homogenization of freshwater fauna in terms of systematic units, but the functional consequences are poorly documented. We studied the peculiar case of the lower, French section of the Moselle River where 20 invertebrate species have been introduced since 1854, with a rate increasing exponentially with time. Dredge sampling performed in 1994, 1996, 2000 and 2001 at four sampling stations allowed for an evaluation of faunal changes in terms of composition, structure and function. During this period, no structural changes were recorded in spite of multiple, new and successful introductions. The evaluation of functional modifications was based on a typology of taxa exhibiting homogeneous biological/ecological traits. Functional diversity, measured as the diversity of taxa distribution among functional groupings, revealed a significant increase between 1994/1996 and 2000/2001 because those species that were over-represented during the former period reached more equilibrated densities during the latter. The major, indirect implications of these functional changes are discussed.     

Isolation and characterization of polymorphic microsatellite markers for the endangered Korean freshwater fish Hemibarbus mylodon

Korean barbel, Hemibarbus mylodon, is an endangered freshwater species endemic to Korea. In order to undertake a conservation program for this species in Korea, it is essential to evaluate its genetic diversity and population structure. For this purpose, we developed 10 polymorphic microsatellite markers and examined their allelic variation using a total of 60 wild individuals collected from three different localities. The number of alleles per locus ranged from three to 26. The expected and observed heterozygosities per locus ranged from 0.19 to 0.94 and from 0.20 to 0.98, respectively. Significant departures from Hardy–Weinberg equilibrium expectations occurred in four loci. These markers will be useful for the assessment of genetic diversity for this species.     

Conservation of rare and endangered plants using in vitro methods

Summary Many botanic gardens now have tissue culture laboratories for the micropropagation of plants that are difficult to propagate by conventional horticultural techniques. In many cases the work centers on rare and endangered species. Examples of the use of different techniques including micropropagation, in vitro seed germination, dual culture with symbiotic fungi, and regeneration from callus are discussed with reference to their application to plant germplasm conservation. Presented in the Session-in-Depth Cell Culture of Endangered Species at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Changes in fish assemblages in catchments in north‐eastern Spain: biodiversity,conservation status and introduced species

1. North‐eastern Spain is a hot spot for the introduction of alien fish species, and its native fish fauna is one of the most endangered worldwide. We used an extensive data set from 2002 to 2003 and historical information from the area to characterize fish diversity and establish conservation priorities in river catchments. 2. Diversity indices were used to characterize fish diversity at the basin scale. An index of conservation status was applied for each species, which considers the occurrence, abundance and endemicity of each taxon. We used indirect ordination methods to test the relationship among basin features and to identify those variables most correlated with each other. To identify physical, biotic and environmental characteristics that seem to make a basin particularly susceptible to invasion, we performed a step‐wise multiple regression to examine the relationship between the number of native, translocated and introduced fish species (including the original native species richness of each basin), and landscape variables. 3. Over a period of approximately 50 years, the mean range size of native fish species has decreased by 60%. The greatest decline occurred in Gasterosteus gymnurus, Anguilla anguilla and Salaria fluviatilis, for which species over 75% of the original distribution area has been lost. The species with the highest conservation index were Gasterosteus gymnurus and Salaria fluviatilis. 4. Basin area and the catchment type explained 70% of variation in native species richness, whereas the number of dams and basin area accounted for more than 80% of variation in the number of introduced species. 5. The original native species richness and the number of introduced species at basin scale were not related, and thus there was no evidence of “biotic resistance” to invasion. The restoration of natural hydrologic processes and the development of specific management tools to protect native species, such as the prioritization of areas for fish conservation and the eradication of local populations of exotic species, are required to restore native fish fauna in these catchments.     

Browsed twig environmental DNA: diagnostic PCR to identify ungulate species

Ungulate browsing can have a strong effect on ecological processes by affecting plant community structure and composition, with cascading effects on nutrient cycling and animal communities. However, in the absence of direct observations of foraging, species‐specific foraging behaviours are difficult to quantify. We therefore know relatively little about foraging competition and species‐specific browsing patterns in systems with several browsers. However, during browsing, a small amount of saliva containing buccal cells is deposited at the bite site, providing a source of environmental DNA (eDNA) that can be used for species identification. Here, we describe extraction and PCR protocols for a browser species diagnostic kit. Species‐specific primers for mitochondrial DNA were optimized and validated using twigs browsed by captive animals. A time series showed that about 50% of the samples will amplify up to 12 weeks after the browsing event and that some samples amplify up to 24 weeks after browsing (12.5%). Applied to samples of natural browsing from an area where moose (Alces alces), roe deer (Capreolus capreolus), fallow deer (Cervus dama) and red deer (Cervus elaphus) are sympatric, amplification success reached 75%. This method promises to greatly improve our understanding of multispecies browsing systems without the need for direct observations.     

Data on molecular taxonomy and genetic diversification of the European Hermit beetles, a species complex of endangered insects (Coleoptera: Scarabaeidae, Cetoniinae, Osmoderma)

A molecular analysis was carried out on the European hermit beetles (the Osmoderma eremita species complex) to explore their genetic diversification and the robustness of previous morphologically based taxonomic arrangements. Complete sequences of mtDNA cytochrome C oxidase I gene were obtained from 26 individuals. Mean levels of interspecific sequence divergence ranged from 0.044 to 0.186. The results indicate a clear-cut distinction between two clades. The first one includes the W-European O. eremita Scopoli 1763 ; and the two Italian endemic taxa Osmoderma italicum Sparacio, 2000 and Osmoderma cristinae Sparacio, 1994 ; from southern peninsular Italy and Sicily, respectively. The second one includes the widespread E-European Osmoderma barnabita ; and the southern Balcanic Osmoderma lassallei Baraud and Tauzin, 1991 from Greece and European Turkey. Within the two clades, molecular and morphological data well support a specific rank for O. lassallei and O. barnabita on one side, and for O. eremita and O. cristinae on the other side, while the taxonomic position of O. italicum , more closely related to O. eremita , is still uncertain. Current geographical distribution, interspecific genetic diversification, and very low levels of intraspecific genetic divergence in western European populations of O. eremita sensu stricto are hypothesized to be the result of multiple speciation events (mainly occurred in refugial forest areas of the Italian and Balkan peninsulas and Sicily before and during the Pleistocene glacial peaks), followed by fast post-glacial northward and westward expansion of some species.     

Exploring biodiversity in a metropolitan area in the Mediterranean region: The urban and suburban flora of Rome (Italy)

This work provides an overview of plant diversity in the municipality of Rome (Italy) through an assessment of the flora in urban and suburban sectors of the city. It is aimed at providing the knowledge required to support proactive action for plant conservation. On the basis of a literature-derived catalogue and of an extensive survey campaign, the flora was investigated in terms of conservation interest; habitat types and locations that require protection measures were identified according to the occurrence of valuable native plants. Valuable species exclusive of the urban sector are threatened by the compaction trend affecting the urban fabric. Such species occur mainly in ruderal environments and fallows of archaeological sites and urban parks, near river courses and in remnants of natural forests. Valuable species exclusive of the suburban sector are threatened by urban sprawl. They occur prevalently in wet environments of the subcoastal strip and in coastal sands, Mediterranean maquis, tuffaceous gorges, sulphur springs and archaeological sites. The results highlight the need to preserve the complexity of the land mosaic, especially within the urban matrix, and to strengthen the existing environmental protection tools in the suburban area against foreseeable land cover changes.