David Porco

DNA Barcoding Reveals Cryptic Diversity in Lumbricus terrestris L., 1758 (Clitellata): Resurrection of L. herculeus (Savigny, 1826)

The widely studied and invasive earthworm, Lumbricus terrestris L., 1758 has been the subject of nomenclatural debate for many years. However these disputes were not based on suspicions of heterogeneity, but rather on the descriptions and nomenclatural acts associated with the species name. Large numbers of DNA barcode sequences of the cytochrome oxidase I obtained for nominal L. terrestris and six congeneric species reveal that there are two distinct lineages within nominal L. terrestris. One of those lineages contains the Swedish population from which the name-bearing specimen of L. terrestris was obtained. The other contains the population from which the syntype series of Enterion herculeum Savigny, 1826 was collected. In both cases modern and old representatives yielded barcode sequences allowing us to clearly establish that these are two distinct species, as different from one another as any other pair of congeners in our data set. The two are morphologically indistinguishable, except by overlapping size-related characters. We have designated a new neotype for L. terrestris. The newly designated neotype and a syntype of L. herculeus yielded DNA adequate for sequencing part of the cytochrome oxidase I gene (COI). The sequence data make possible the objective determination of the identities of earthworms morphologically identical to L. terrestris and L. herculeus, regardless of body size and segment number. Past work on nominal L. terrestris could have been on either or both species, although L. herculeus has yet to be found outside of Europe.

Biological invasions in soil: DNA barcoding as a monitoring tool in a multiple taxa survey targeting European earthworms and springtails in North America

Biological invasions are increasingly recognized as a potent force altering native ecosystems worldwide. Many of the best documented cases involve the massive invasions of North America by plant and animal taxa native to Europe. In this study, we use DNA barcoding to survey the occurrence and genetic structure of two major groups of soil invertebrates in both their native and introduced ranges: Collembola and earthworms. Populations of ten species of earthworms and five species of Collembola were barcoded from both continents. Most of these species exhibited a similar genetic structure of large and stable populations in North America and Europe, a result supporting a scenario of multiple invasions. This was expected for earthworm species involved in human economic activities, but not foreseen for Collembola species de facto unintentionally introduced. This study also establishes that invasive species surveys employing DNA barcoding gain additional resolution over those based on morphology as they allow evaluation of cryptic lineages exhibiting different invasion histories.

DNA barcodes for soil animal taxonomy

A biodiversidade das comunidades do solo continua muito pouco conhecida e entendida. Axa0biologia do solo e fortemente afetada pela crise taxonomica, e a maior parte dos grupos de animais dessa biota sofre forte impedimento taxonomico. Oxa0objetivo deste trabalho foi determinar como o codigo de barras de DNA - um metodo novo que usa uma etiqueta microgenomica para identificacao e discriminacao de especies - permite uma melhor avaliacao da taxonomia da biota edafica. Foram analisadas 1.152xa0sequencias de codigos de barras de dois grupos principais de animais, colembolos e minhocas, que apresentaram ampla amostragem taxonomica e geografica. Alem de refletir fortemente o impedimento taxonomico de ambos os grupos, com um grande numero de linhagens divergentes no nivel da especie que ainda nao esta descrita, os resultados tambem destacam um alto (15%) nivel de diversidade criptica dentro de especies conhecidas de minhocas e colembolos. Esses resultados apoiam estudos locais recentes que usaram metodos similares. Considerando as dificuldades taxonomicas enfrentadas para identificar os animais de solo, ferramentas de identificacao usando DNA podem facilitar e melhorar a exploracao da biodiversidade e a sua descricao. Asxa0campanhas de codigo de barras de DNA estao se desenvolvendo rapidamente com animais do solo, e a comunidade de biologos e incitada a adotar esses metodos.

Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola.

Here, we describe a simple method adapted for high‐throughput protocols allowing voucher specimen recovery for Collembola and by extension for other soft‐bodied small arthropods. A standard extraction protocol was tested to examine the effects of lysis duration (1, 2, 4, 12u2003h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1u2003h of lysis, while voucher condition was fine for up to 12u2003h. The lysis step substantially shortens the clearing process necessary for morphological examination.

Re‐integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA‐based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.

Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae): the first eukaryotic species description combining transcriptomic, DNA barcoding and micro-CT imaging data

Abstract We demonstrate how a classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. A cave-dwelling centipede, Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae), found in a remote karst region in Knin, Croatia, is the first eukaryotic species for which, in addition to the traditional morphological description, we provide a fully sequenced transcriptome, a DNA barcode, detailed anatomical X-ray microtomography (micro-CT) scans, and a movie of the living specimen to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a new species for the first time, we create a high-resolution morphological and anatomical dataset that allows virtual reconstructions of the specimen and subsequent interactive manipulation to test the recently introduced ‘cybertype’ notion. In addition, the transcriptome was recorded with a total of 67,785 scaffolds, having an average length of 812 bp and N50 of 1,448 bp (see GigaDB). Subsequent annotation of 22,866 scaffolds was conducted by tracing homologs against current available databases, including Nr, SwissProt and COG. This pilot project illustrates a workflow of producing, storing, publishing and disseminating large data sets associated with a description of a new taxon. All data have been deposited in publicly accessible repositories, such as GigaScience GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability.

The centipede genus Eupolybothrus Verhoeff, 1907 (Chilopoda: Lithobiomorpha: Lithobiidae) in North Africa, a cybertaxonomic revision, with a key to all species in the genus and the first use of DNA barcoding for the group.

Abstract The centipede genus Eupolybothrus Verhoeff, 1907 in North Africa is revised. A new cavernicolous species, Eupolybothrus kahfi Stoev & Akkari, sp. n., is described from a cave in Jebel Zaghouan, northeast Tunisia. Morphologically, it is most closely related to Eupolybothrus nudicornis (Gervais, 1837) from North Africa and Southwest Europe but can be readily distinguished by the long antennae and leg-pair 15, a conical dorso-median protuberance emerging from the posterior part of prefemur 15, and the shape of the male first genital sternite. Molecular sequence data from the cytochrome c oxidase I gene (mtDNA–5’ COI-barcoding fragment) exhibit 19.19% divergence between Eupolybothrus kahfi and Eupolybothrus nudicornis, an interspecific value comparable to those observed among four other species of Eupolybothrus which, combined with a low intraspecific divergence (0.3–1.14%), supports the morphological diagnosis of Eupolybothrus kahfi as a separate species. This is the first troglomorphic myriapod to be found in Tunisia, and the second troglomorph lithobiomorph centipede known from North Africa. Eupolybothrus nudicornis is redescribed based on abundant material from Tunisia and its post-embryonic development, distribution and habitat preferences recorded. Eupolybothrus cloudsley-thompsoni Turk, 1955, a nominal species based on Tunisian type material, is placed in synonymy with Eupolybothrus nudicornis. To comply with the latest technological developments in publishing of biological information, the paper implements new approaches in cybertaxonomy, such as fine granularity XML tagging validated against the NLM DTD TaxPub for PubMedCentral and dissemination in XML to various aggregators (GBIF, EOL, Wikipedia), vizualisation of all taxa mentioned in the text via the dynamically created Pensoft Taxon Profile (PTP) page, data publishing, georeferencing of all localities via Google Earth, and ZooBank, GenBank and MorphBank registration of datasets. An interactive key to all valid species of Eupolybothrus is made with DELTA software.

Phylogeny of Collembola based on cuticular compounds:inherent usefulness and limitation of a character type

The phylogeny of Collembola, originally discussed from a morphological point of view, has more recently benefited from novel insights brought by molecular analyses. Both morphological and molecular characters produced a well-resolved phylogenetic hypothesis including all orders, most families, and a large number of genera. However, several conflicting points exist between molecular and morphological data, and new characters are clearly needed to resolve these inconsistencies. In this study the usefulness of a new character type not previously used in the phylogenetic study of Collembola was tested: the epicuticular chemical compounds. Our phylogenetic analysis was based on 380 compounds from 26 Collembola species. The results show good resolution for terminal branches but not for internal nodes. This is probably due to the partial involvement of epicuticular lipids in ecological functions such as water conservation and sexual attraction. Thus, this character type is appropriate for reconstructing phylogenetic relationships among recently diversified groups.

Cuticular Compounds Bring New Insight in the Post-Glacial Recolonization of a Pyrenean Area: Deutonura deficiens Deharveng, 1979 Complex, a Case Study

Background In most Arthropod groups, the study of systematics and evolution rely mostly on neutral characters, in this context cuticular compounds, as non-neutral characters, represent an underexplored but potentially informative type of characters at the infraspecific level as they have been routinely proven to be involved in sexual attraction. Methods and Findings The collembolan species complex Deutonura deficiens was chosen as a model in order to test the utility of these characters for delineating four infraspecific entities of this group. Specimens were collected for three subspecies (D. d. deficiens, D. d. meridionalis, D. d. sylvatica) and two morphotypes (D. d. sylvatica morphoype A and B) of the complex; an additional species D. monticola was added. Cuticular compounds were extracted and separated by gas chromatography for each individual. Our results demonstrate that cuticular compounds succeeded in separating the different elements of this complex. Those data allowed also the reconstruction of the phylogenetic relationships among them. Conclusions The discriminating power of cuticular compounds is directly related to their involvement in sexual attraction and mate recognition. These findings allowed a discussion on the potential involvement of intrinsic and paleoclimatic factors in the origin and the diversification of this complex in the Pyrenean zone. This character type brings the first advance from pattern to process concerning the origin of this species complex.

Two new Megalothorax species of the minimus group (Collembola, Neelidae).

Abstract Two new Megalothorax species, Megalothorax potapovi sp. n. from the Russian Far East and Megalothorax sanguineus sp. n. from the French Pyrénées are described. The two new species have a set of morphological characters (including a smooth mucro) that places them among the minimus group sensu Schneider and D’Haese (2013). Megalothorax potapovi characteristics include dorsal protuberance on forehead, peculiar chaetotaxy of antenna III and strong lanceolate chaetae on body. Megalothorax sanguineus characteristics include strong red pigmentation, large network of integumentary channels on head and elongated apex of the two postero-distal spines of dens. The DNA barcodes ( cytochrome oxidase subunit I–COI) of the two species are also provided and analyzed among a broader sampling of the genus in order to support further their specific status. A special focus is given to the labral morphological characteristics. Pseudopores-like elements are reported for the first time in the genus. Positions of the τ-chaetae near the dorsal sensory field of thorax II are compared for several species of the genus.