The use of DNA barcoding to monitor the marine mammal biodiversity along the French Atlantic coast

In the last ten years, 14 species of cetaceans and five species of pinnipeds stranded along the Atlantic coast of Brittany in the North West of France. All species included, an average of 150 animals strand each year in this area. Based on reports from the stranding network operating along this coast, the most common stranding events comprise six cetacean species (Delphinus delphis, Tursiops truncatus, Stenella coeruleoalba, Globicephala melas, Grampus griseus, Phocoena phocoena)and one pinniped species (Halichoerus grypus). Rare stranding events include deep-diving or exotic species, such as arctic seals. In this study, our aim was to determine the potential contribution of DNA barcoding to the monitoring of marine mammal biodiversity as performed by the stranding network.We sequenced more than 500 bp of the 5’ end of the mitochondrial COI gene of 89 animals of 15 different species (12 cetaceans, and three pinnipeds). Except for members of the Delphininae, all species were unambiguously discriminated on the basis of their COI sequences. We then applied DNA barcoding to identify some “undetermined” samples. With again the exception of the Delphininae, this was successful using the BOLD identification engine. For samples of the Delphininae, we sequenced a portion of the mitochondrial control region (MCR), and using a non-metric multidimentional scaling plot and posterior probability calculations we were able to determine putatively each species. We then showed, in the case of the harbour porpoise, that COI polymorphisms, although being lower than MCR ones, could also be used to assess intraspecific variability. All these results show that the use of DNA barcoding in conjunction with a stranding network could clearly increase the accuracy of the monitoring of marine mammal biodiversity.     

Employing DNA barcoding as taxonomy and conservation tools for fish species censuses at the southeastern Mediterranean,a hot-spot area for biological invasion

This study evaluates the utility of DNA barcoding (mitochondrial cytochrome oxidase subunit I; COI) as a biodiversity and conservation applied tool for identifying fish fauna from the southeastern Mediterranean (the continental coast of Israel), a hot-spot area for biological invasion, also with an eye to establish the foundation for follow-up studies that will use environmental DNA (eDNA) tracks of native and invasive species, and for the application of eDNA concepts and methodologies in nature conservation. We established a dataset of 280 DNA barcodes, representing 110 marine fish species (all identified by a taxonomist), belonging to 75 native and 35 Lessepsian migratory species that were tested within and against the BOLD system database. Most of the query sequences showed 98% matches with reference DNA barcodes in the BOLD system excluding two Trachurus species, three Dasyatis pastinaca and two Rhinobatos rhinobatos individuals. Relatively high intraspecific genetic distances were observed in two Elasmobranchii species (8.83%–18%), suggesting possible cryptic species. In contrast, relatively low interspecific genetic distances were found between two Actinopterygii species (1.54%). Gobiidae family members were observed as being the most scattered on the Bayesian tree. Out of the 110 fish species, 48 were taxonomically discordant with the BOLD BINs (4 at the family level, 15 at the genus level and 29 at the species level), 53 were concordant and 9 were singletons. Discordancy was noted for some Diplodus and Fistularia species during the BOLD BIN analysis. Apogon queketti, Champsodon nudivittis and Cheilodipterus novemstriatus were uploaded to the BOLD system for the first time. We elucidated 177 haplotypes, 122 for the native fish species and 55 for the Lessepsian species. The results will allow the evaluation of invasive species success and will help developing improved policies for the conservation of Mediterranean biodiversity.     

Soil fungal diversity in three nature reserves of Jiuzhaigou County,Sichuan Province,China

Jiuzhaigou County is located at the southern transition zone of Sichuan Basin and the Qinghai–Tibet Plateau and is the site of three famous nature reserves, namely, the Jiuzhaigou Nature Reserve (JZNR), Baihe Nature Reserve (BHNR) and Wujiao Nature Reserve (WJNR). The soil fungal diversity in this region has not yet been investigated. In this study, we collected 25 soil samples from these three nature reserves. Soil fungi were isolated using the soil dilution plate technique and Rose Bengal agar medium. The culturable soil fungal density based on analysis of the 25 samples ranged from 2.18 log to 4.38 log CFU g^?1 dry weight soil, with the fungal density being highest in samples from JZNR and lowest in those from BHNR. Based on morphological characters and the results of phylogenetic analysis of the internal transcribed spacer (ITS) of the rDNA operon, we identified 38 genera (two genera could not be identified) belonging to Ascomycota, Zygomycota and Basidiomycota. The dominant genera were Penicillium, Humicola, Aspergillus and Trichoderma. The species richness index S, biodiversity index H′ and evenness index E of the 25 sampling sites were in the range 10–29, 1.96–3.05 and 0.74–0.95, respectively. The highest mean values of the S, H′ and E indices were in soil samples from BHNR, where the values of these indices were 20.00, 2.66 and 0.90, respectively. These results indicate that the diversity of culturable fungi in these three nature reserves was high. Furthermore, a total 14 Trichoderma isolates were tested for their antagonism activity against mycelium growth of three pathogens: Bipolaris maydis, Curvularia lunata, Rhizoctonia solani. The results showed that six Trichoderma isolates had good antagonistic effects on the three pathogenic fungi.     

Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ～ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.     

Natural hybridization and introgression among sympatrically distributed Rhododendron species in Guizhou,China

Natural hybridizations occur among Rhododendron delavayi, R. decorum and R. irroratum, however, there was little study that had addressed the interbreeding behaviors when the three species grew in the same geographic areas. In this study, 37 accessions, containing the three species and the putative hybrids, were obtained from Baili Rhododendron Nature Reserve, Guizhou, China. Examinations of hybridization patterns with AFLP markers have led to a total of 107 diagnostic DNA fragments, which were analyzed with Principal Co-ordinate, Structure and NewHybirds analyses. The data confirmed that the existence of hybrids originated from the interbreeding between R. delavayi and R. irroratum in Baili Rhododendron Nature Reserve. R. decorum did not appear to be involved in the hybridization. Furthermore, most hybrids detected were a result of backcrossing, indicating that this hybrid differed from F1 dominant hybrids between R. delavayi and R. irroratum found in previous studies.     

A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva,Gingivitis or Mild Periodontitis

Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.     

The contribution of standing waters to aquatic biodiversity: the case of water beetles in southeastern Iberia

The southeast of the Iberian Peninsula is a recognized area of high aquatic biodiversity, water beetles being one of the most well-known groups contributing to such biodiversity. The standing waters of this area show a high habitat diversity, occurring fresh, saline, temporary, permanent, karstic, endorheic, and artificial small water bodies. Despite this, there has been no attempt to analyze their contribution to local biodiversity. In this work, we identify the species inhabiting standing waters, analyze their contribution to the checklist from the study area, and recognize the species exclusive to this kind of habitat. We also highlight the most threatened species, identify the habitats which constitute biodiversity hotspots for this group, explore patterns of water beetle assemblage composition, and identify indicator species associated with each habitat type. We collected 125 species, 55 of them being typical to lentic habitats, in the set of 26 sampled standing water bodies, which means the 57% of the 218 species recorded in the most recent checklist for the study area. A total of 10 species are Iberian endemics and four can be considered threatened, Ochthebius irenae falling in both categories. Natural ponds showed the highest species richness (91 species), while saline water bodies (endorheic lagoons and continental salt-pans) contributed the most threatened species: Nebrioporus baeticus, Ochthebius delgadoi, Ochthebius tudmirensis and Ochthebius irenae. The most representative species for continental salt-pans was Ochthebius notabilis, for endorheic lagoons Ochthebius marinus and Hygrotus pallidulus, while Hydroglyphus geminus played this part in rice fields. Our results suggest that rice fields, endorheic lagoons, and continental salt-pans have specific water beetles assemblages, which could be used in bioassessment and conservation studies. In general, standing waters are seriously threatened in this area, particularly as a result of intensive agricultural activities. Thus, taking into account these ecosystems hold a high number of species, their continued degradation is likely to result in a significant loss of biodiversity, including key populations of a number of threatened and endemic species.     

Gutsy genetics: identification of digested piscine prey items in the stomach contents of sympatric native and introduced warmwater catfishes via DNA barcoding

A major focus of ecology is understanding trophic relationships and energy flows in natural systems, associated food web dynamics and changes in food webs due to introduced species. Predator-prey interactions are often assessed by examining stomach contents. However, partially digested remains may be difficult to accurately identify by traditional visual analysis. Here we evaluate the effectiveness of DNA barcoding to identify digested piscine prey remains in invasive Blue Catfish Ictalurus furcatus, non-native, but established Channel Catfish Ictalurus punctatus and native White Catfish Ameiurus catus from Chesapeake Bay, USA. Stomach contents were examined and piscine prey items were scored as lightly digested, moderately digested or severely digested. A 652 base pair region of the cytochrome c oxidase subunit I (COI-5P) mitochondrial DNA gene was sequenced for each prey item. Edited barcode sequences were compared to locally-caught and validated reference sequences in BOLD (Barcode of Life Database). A large majority of prey items were sufficiently digested to limit morphological identification (9.4 % to species and an additional 12.1 % to family). However, overall barcoding success was high (90.3 %) with little difference among the digestion classifications. Combining morphological and genetic identifications, we classified 91.6 % of fish prey items to species. Twenty-three fish species were identified, including species undergoing active restoration efforts (e.g., Alosa spp.) and commercially important species, e.g., Striped Bass Morone saxatilis, White Perch Morone americana, American Eel Anguilla rostrata and Menhaden Brevoortia tyrannus. We found DNA barcoding highly successful at identifying all but the most heavily degraded prey items and to be an efficient and effective method for obtaining diet information to strengthen the resolution of trophic analyses including diet comparisons among sympatric native and non-native predators.     

Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.     

DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers

Excessive ingestion of mercury—a health hazard associated with consuming predatory fishes—damages neurological, sensory-motor and cardiovascular functioning. The mercury levels found in Bigeye Tuna (Thunnus obesus) and bluefin tuna species (Thunnus maccoyii, Thunnus orientalis, and Thunnus thynnus), exceed or approach levels permissible by Canada, the European Union, Japan, the US, and the World Health Organization. We used DNA barcodes to identify tuna sushi samples analysed for mercury and demonstrate that the ability to identify cryptic samples in the market place allows regulatory agencies to more accurately measure the risk faced by fish consumers and enact policies that better safeguard their health.     

Molecular identification and first documentation of seven species of Carpophilus Stephens (Nitidulidae: Carpophilinae) in oil palm ecosystem,Peninsular Malaysia

Sap beetles are commonly found in many agricultural systems worldwide. In Malaysia, sap beetles are less documented despite their abundance in oil palm plantations. The similarities in the morphology of the sap beetle makes the identification process difficult from the same genus. This study aims to determine of composition of the sap beetle species from the genus Carpophilus, to identify the species of sap beetles and to document the DNA barcode for those species collected from the oil palm plantation, which has never been done before. DNA of seven species of sap beetles within the subfamily Carpophilinae have been successfully amplified using cytochrome oxidase subunit I marker (COI) namely C. hemipterus (Linnaeus), C. maculatus Murray, C. mutilatus Erichson, C. marginellus Motschulsky, C. sp. 1, C. nepos Murray and C. obsoletus Erichson represented 21, 12, 33, 8, 9, 6 and 11%, respectively from the total number collected. The sequences were analysed and a tree was constructed based on Neighbor-Joining (NJ) analysis with Meligethes thalassophilus chosen as an outgroup. All of the samples showed 90–100% similarities in BOLD and BLAST analyses. NJ trees constructed show that each sap beetle species collected from the study site located at their specific lineages on the tree. Interestingly, the DNA barcoding described in this study is the first to document barcoding data of sap beetles genus Carpophilus in Malaysia and this can be a helpful tool in updating the taxonomic and molecular status of the sap beetles in Malaysia.     

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.     

Functional group classification and target species selection for Yancheng Nature Reserve, China

Yancheng Nature Reserve in east China is of great importance to the protection of biodiversity. It is a principal refuge for Red-crowned Cranes (Grus japonensis) and many other rare species, an important station for migrant waterfowls in the Asian Pacific, and a captive breeding base for Milu (Elaphurus davidianus). However, since the late 1980s, serious losses of biodiversity have been emerging in the protected area. In this study, to value objectively and improve management of the Yancheng Nature Reserve, a new approach for conservation management is proposed by virtue of the theories of self-organizing systems, functional group classification and target species selection. According to this approach, biological components in Yancheng Nature Reserve are classified into 10 supporting functional groups, eight target functional groups and 11 subordinate functional groups. Eight target species were selected from those target functional groups, i.e., G. japonensis, E. davidianus, Hydropotes inermis, Larus saundersi, Centropus toulou, Felis bengalensis, Paradoxornis heudei, and Platalea minor. Past observations in Yancheng Nature Reserve illustrate that the new approach is feasible, and this new approach will also benefit the management of the nature reserve.     

Agricultural encroachment and lack of enforcement threaten connectivity of range-wide jaguar (Panthera onca) corridor

The purpose of protected areas is to safeguard natural habitats and their biodiversity. However, few protected areas are large enough for the long-term conservation of resident species, making the landscapes surrounding protected areas equally important for species conservation. This is especially true for wide-ranging carnivores such as the jaguar (Panthera onca), for which conservation strategies stress the maintenance of range-wide habitat connectivity via protected areas and dispersal corridors. We used 587 interviews with local people in 441 36-km² sampling units to assess the presence of jaguar and six prey species in a critical corridor in Central America, linking Bosawas Biosphere Reserve and Wawashan Nature Reserve in Nicaragua. We analyzed these data using single-season false-positive detection models in a site occupancy framework. We found that agricultural encroachment, particularly pervasive within a protected area, was the main factor limiting the presence of jaguar and three large-bodied prey species: white-lipped peccary (Tayassu pecari), collared peccary (Pecari tajacu), and red brocket deer (Mazama americana). We suggest that improperly-managed protected areas can suffer the loss of apex predators and their prey, thereby jeopardizing range-wide conservation strategies for threatened species.     

New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species,Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.     

DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.     

Plant DNA barcodes can accurately estimate species richness in poorly known floras

Background

Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (∼70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology.

Methodology/Principal Findings

Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species.

Conclusions/Significance

We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.