Unexpectedly high levels of cryptic diversity uncovered by a complete DNA barcoding of reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the signifi- cance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra ' s most impor- tant vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-bar-coded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps.The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8-54.4%. This has implications in the species ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework

DNA Barcoding of the reptiles of the Socotra Archipelago

Trabajo presentado en las XV Jornades Herpetològiques Catalanes, celebradas en Sant Feliu de Codines del 10 al 12 de octubre de 2012.A decade ago, DNA barcoding was proposed as a fast, cost-efficient and simple taxonomic method based on the use of a unique, short and standardized gene region for identifying existing species and speeding the discovery of new ones. However, due to technical problems, few DNA barcoding studies in squamate reptiles have been conducted so far. In this work, using three sets of primers, we successfully DNA barcoded 380 individuals of all presently recognized species of reptiles in the Socotra Archipelago (31 species, 12 genera), classified as a UNESCO World Heritage Site. Although no barcoding gap exists in the whole dataset, specimen identification success rate of DNA barcoding is moderate to high (68-94%) based on distance-based and tree-based techniques. The low levels of inter-specific genetic divergence observed between some species are consistent with paraphyly of Pristurus sokotranus. On the other hand, the high levels of intra-specific variability found within some species suggest cryptic diversity. Depending on the species delimitation approaches, between 4 and 37 more species than those presently recognized should be taken into account for future conservation efforts. Through this study we conclude that DNA barcoding is very useful for specimen identification, but for species discovery and description it will only be effective if incorporated into a more integrative taxonomic framework.N

Desvendando uma diversidade extraordinária – ADN Barcoding dos répteis de Socotra

Trabajo presentado en el XIII Iberian Congress of Herpetology (XIII Congresso Luso-Espanhol de Herpetologia, XIII Congreso Luso-Español de Herpetología), celebrado en Aveiro (Portugal) del 30 de septiembre al 4 de octubre de 2014.[EN] DNA barcoding is based on the idea of using a short genetic sequence from a standard marker (cytochrome c oxidase 1 gene, COI) for species identification. It has the advantage of facilitating species identification and accelerating discovery of candidate/cryptic species in a largely reliably and cost‐efficient way, with many implications in conservation and management. However, it presents some technical problems in amplification, especially with reptiles; and methodological problems, such as difficulty in threshold delimitation for barcode gaps and minimum number of sequences per species, scale‐dependency, and limitations in phylogenetic inference. Recent barcoding campaigns of reptiles have been successful and so we urged to apply it to Socotra due to the significance of this region, classified as Natural World Heritage, and the conservation interest of their unique reptile fauna, presently with 29 endemic species. After several campaigns of extensive sampling in the archipelago with stations each 10 x 10 km, more than 1300 tissues were collected. We have successfully sequenced, using three sets of primers, 380 individuals of all presently recognised species with a best match success of 99%, and with all species barcode success of about 72% of specimen identification. We detected one case of paraphyly and also high levels of instraspecific genetic variability, sometimes higher than values of interspecific genetic variability. The result of GMYC species delimitation analysis at different levels, as well as a barcode gap analysis using different thresholds, unveiled relatively high levels of cryptic diversity, suggesting that between 4 and 37 more entities than those presently recognised should be taken into account for conservation planning. Published and preliminary results using both mitochondrial and nuclear markers and the high levels of genetic variability in COI detected in this study, especially within Hemidactylus and Pristurus, including one case of paraphyly, suggest that the taxonomy of some Socotra reptiles should be revised using an integrative approach combining molecular and morphological data.[PT] O barcoding de ADN é baseado na ideia de usar uma sequência genética curta de um marcador padrão (gene citocromo c oxidase 1, COI) para a identificação de espécies. Tem a vantagem de facilitar a identificação das espécies e acelerar a descoberta de espécies candidatas/ crípticas de uma forma bastante fiável e economicamente eficiente, com várias implicações na conservação e gestão. No entanto, apresenta alguns problemas técnicos na amplificação, especialmente de répteis; e problemas metodológicos, como a dificuldade na delimitação dos limiares do intervalo do barcode e do número mínimo de sequências de cada espécie, a dependência da escala e limitações nas inferências filogenéticas. Campanhas recentes de barcoding de répteis foram bem‐sucedidas e por isso apressámo‐nos para aplicá‐lo em Socotra, devido à importância desta região, classificada como Património Mundial Natural, e a importância da conservação da sua herpetofauna única, actualmente com 29 espécies endémicas. Depois de várias campanhas de amostragem intensas no arquipélago, com estações a cada 10 x 10 km, mais de 1300 tecidos foram recolhidos. Sequenciámos com sucesso, utilizandose três conjuntos de primers, 380 indivíduos de todas as espécies actualmente reconhecidas, com um sucesso óptimo de reconhecimento de 99%, e com um sucesso de barcoding de todas as espécies de cerca de 72% na identificação dos espécimes. Detectámos um caso de parafilia e também elevados níveis de variabilidade genética intra‐específica, por vezes maiores que os valores de variabilidade genética interespecífica. O resultado da análise de delimitação de espécies GMYC a diferentes níveis, bem como a análise de intervalos de barcoding usando diferentes limiares, revelou níveis relativamente altos de diversidade críptica, sugerindo que devem ser tidos em conta para a gestão da conservação entre mais 4 a 37 entidades do que aquelas que são actualmente reconhecidas. Os resultados previamente publicados e preliminares, utilizando dois marcadores mitocondriais e nucleares, e os altos níveis de variabilidade genética em COI detectados neste estudo, especialmente dentro de Hemidactylus e Pristurus, incluindo um caso de parafilia, sugerem que a taxonomia de alguns répteis de Socotra deveria ser revista através de uma aproximação integrativa combinando dados moleculares e morfológicos.N

Unexpectedly high levels of cryptic diversity uncovered by a complete DNA barcoding of reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the signifi- cance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra ' s most impor- tant vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-bar-coded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps.The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8-54.4%. This has implications in the species ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework

Bayesian COI tree for all the reptiles of the Socotra Archipelago.

<p>Species delimitations using three distance thresholds with higher taxonomic accuracy (3%, 6% and 9%) and GMYC using three different datasets: A—including all 380 barcoded sequences in the same analysis; B—including independent analyses for Serpentes, Scincoidea, Lacertoidea and Gekkota; C—including independent analyses for the following families: Leptotyphlopidae, Scincidae, Lacertidae, Sphaerodactylidae, Phyllodactylidae, Gekkonidae; *indicates clusters that were depicted as in A because taxa are monoespecific or monogeneric. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149985#sec002" target="_blank">Material and Methods</a> for further details. Black dots indicate posterior probability values ≥0.95. Bootstrap values ≥70% of the ML analysis are shown next to the nodes.</p

Taxonomic accuracy.

<p>Taxonomic accuracy.</p

Sampling site and localities.

<p>Map showing the geographic situation of the Socotra Archipelago and the sampling localities for all the 380 barcoded individuals included in this study. Maps were drawn using DIVA-GIS v.7.5 (available at <a href="http://www.diva-gis.org" target="_blank">http://www.diva-gis.org</a>; digital elevation model freely available at <a href="http://earthobservatory.nasa.gov/" target="_blank">http://earthobservatory.nasa.gov/</a>).</p

Specimen identification success.

<p>Values for both distance-based (‘best match’; BM, ‘best close match’; BCM, and ‘all species barcodes’; ASB—using different distance thresholds) and tree-based (Hebert et al. 2003a; Meier et al. 2006) approaches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149985#pone.0149985.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149985#pone.0149985.ref034" target="_blank">34</a>].</p

Genetic divergences.

<p>Genetic divergences.</p