eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. RESULTS: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. CONCLUSIONS: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg.

Getting rid of 'rain' and 'stars': Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets ('stars'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets ('stars') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).

Mitonuclear discordance and patterns of reproductive isolation in a complex of simultaneously hermaphroditic species, the Allolobophora chlorotica case study.

Historical events of population fragmentation, expansion and admixture over geological time may result in complex patterns of reproductive isolation and may explain why, for some taxa, the study of mitochondrial (mt) and nuclear (nu) genetic data results in discordant evolutionary patterns. Complex patterns of taxonomic diversity were recently revealed in earthworms for which distribution is largely the result of paleogeographical events. Here, we investigated reproductive isolation patterns in a complex of cryptic species of earthworms in which discordant patterns between mt and nu genetic lineages were previously revealed, the Allolobophora chlorotica aggregate. Using four nu microsatellite markers and a fragment of the cytochrome c oxidase subunit I mt gene, we carried out a parentage analysis to investigate the mating patterns (i) between individuals belonging to two divergent mt lineages that cannot be distinguished with nu markers and (ii) between individuals belonging to lineages that are differentiated both at the mt and nu levels. Amongst the 157 field-collected individuals, 66 adults were used in cross-breeding experiments to form 22 trios based on their assignment to a mt lineage, and 453 obtained juveniles were genotyped. We showed that adults that mated with both their potential mates in the trio produced significantly more juveniles. In crosses between lineages that diverged exclusively at the mt level, a sex-specific pattern of reproduction characteristic to each lineage was observed, suggesting a possible conflict of interest concerning the use of male/female function between mating partners. In crosses between lineages that diverged both at the mt and nu levels, a high production of cocoons was counterbalanced by a low hatching rate, suggesting a post-zygotic reproductive isolation. Different degrees of reproductive isolation, from differential sex allocation to post-zygotic isolation, were thus revealed. Lineages appear to be at different stages in the speciation process, which likely explain the observed opposite patterns of mitonuclear congruence.

eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity.

LAMP assays are becoming increasingly popular in the field of invasive species detection but are still underused in eDNA-based monitoring. Here, we propose a LAMP assay designed to detect the North American crayfish species Pacifastacus leniusculus in water samples from streams. The presence of P. leniusculus was detected through this new LAMP assay in all but one of the nine sites sampled. No correlation was found between ddPCR absolute concentration measurements and the number of LAMP-positive technical replicates. However, we showed that using dependent technical replicates could significantly enhance the detection sensitivity of the LAMP assay. Applied to other assays, it could improve sensitivity and thus allow for a more efficient use of eDNA-based LAMP assays for invasive species detection in aquatic ecosystems.

A new European species of Ceratophysella (Collembola, Hypogastruridae) revealed by morphological data and DNA barcodes.

A new species, Ceratophysella stachi, from Denmark, Germany, Luxembourg, Norway, Poland, and Ukraine is described based on morphological data and DNA barcodes. It belongs to a small European group of species with type B chaetotaxy and strong tegumentary granulation with distinct fields of coarse granules: C. granulata Stach, 1949, C. lawrencei (Gisin, 1963), C. neomeridionalis (Nosek & Cervek, 1970), C. scotica (Carpenter & Evans, 1899), and C. silvatica Rusek, 1964. It differs from all of them in the chaetotaxy of lateral parts of thoracic terga II-III (setae m6 present and one additional seta outside lateral sensillum m7 present or absent) that is exceptional within the whole C. armata-group. Notes on closely related species C. granulata are also given.

A new member of the genus Isotomurus from the Kuril Islands (Collembola: Isotomidae): returning to the problem of "colour pattern species".

Colour pattern is the most common character to identify species in several large genera of Collembola. Its use often raises problems due to various and poorly investigated extent of chromatic variability among species. Isotomurus festus sp. nov. is here described from Kunashir Isl. (the Kuriles, the Far East of Russia). The species, a member of the 'antennalis' group, is characterized by the lack of trichobothria and slender claws, but is greatly variable in coloration. DNA barcoding (COI) results supports that all the colour forms encountered belong to the same species. While colour pattern has been shown to be the most reliable character for species identification in several Entomobryidae genera, it might not be the case in Isotomurus Börner, 1903, the sole large Isotomidae genus where colour pattern is routinely used for taxonomy.

Correction: Barcoding Eophila crodabepis sp. nov. (Annelida, Oligochaeta, Lumbricidae), a Large Stripy Earthworm from Alpine Foothills of Northeastern Italy Similar to Eophila tellinii (Rosa, 1888).

[This corrects the article DOI: 10.1371/journal.pone.0151799.].

Barcoding Eophila crodabepis sp. nov. (Annelida, Oligochaeta, Lumbricidae), a Large Stripy Earthworm from Alpine Foothills of Northeastern Italy Similar to Eophila tellinii (Rosa, 1888).

A new Italian earthworm morphologically close to the similarly large and anecic Eophila tellinii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750 km2 from East to West on the Asiago Plateau and Vittorio Veneto Hills, from North to South on mounts Belluno Prealps (Praderadego and Cesen), Asiago, Grappa and onto the Montello foothills. This range abuts that of Eophila tellinii in northern Friuli Venezia Giulia region. Known localities of both E. tellinii and E. crodabepis sp. nov. are mapped. mtDNA barcoding definitively separates the new western species from classical Eophila tellinii (Rosa, 1888).

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

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.

The puzzling distribution of Heteromurus (Verhoeffiella) absoloni Kseneman, 1938 (Collembola: Entomobryidae: Heteromurinae) resolved: detailed redescription of the nominal species and description of a new species from Catalonia (Spain).

The species Heteromurus (Verhoeffiella) absoloni Kseneman, 1938 is redescribed in detail and characterized by its barcode, based on specimens from its type locality in Montenegro. A neotype is designated. Dorsal S-chaetotaxy is given for the first time in the subgenus Verhoeffiella. Chaeta morphology and distribution are thoroughly analyzed, in particular on antennae where 12 chaetal types are recognized. Several morphological features are newly described for the genus and for Heteromurinae. The widely disjunct distribution of the species is approached through morphological and molecular comparison of specimens from the type locality in Montenegro and from the Catalan population. We established that this last record is a new species described here as  Heteromurus (Verhoeffiella) gamae sp. nov. New combination is proposed Heteromurus (Verhoeffiella) constantinellus (Curcic & Lucic in Lucic, Curcic & Mitic 2007) comb. nov. A table of all species of the subgenus is provided. The taxonomic status of Verhoeffiella and the problems of species discriminations in the subgenus are discussed.

A new species of Deutonura (Collembola: Neanuridae: Neanurinae) from north-eastern Algeria, and characterisation of two intraspecific lineages by their barcodes.

A new species of Deutonura, D. zana sp. nov., is described from north-eastern Algeria. It is morphologically similar in most characters to D. deficiens meridionalis and to D. luberonensis, both members of the D. phlegraea group, differing from the former by the absence of chaeta O on head, and from the later by the separation of tubercles Di and De on Th. I. The muscular insertion pattern of the new species is figured, and suggested as a potential new character for the taxonomy of Neanurinae. Deutonura zana sp. nov. is well characterised by its barcode sequence. Within the new species as morphologically defined, two groups of COI haplotypes, in individuals indistinguishable morphologically, are reported from two distinct mountain ranges. The need of a morphological assessment of demes diverging at significant infra-specific level in their barcodes is stressed.

Barcoding the Collembola of Churchill: a molecular taxonomic reassessment of species diversity in a sub-Arctic area.

Although their functional importance in ecosystems is increasingly recognized, soil-dwelling micro-arthropods are usually poorly known in comparison with their above-ground counterparts. Collembola constitute a significant and species-rich component of the soil biodiversity, but it remains a woefully understudied group because of the taxonomic impediment. The ever-increasing use of molecular taxonomic tools, such as DNA barcoding, provides a possible solution. Here, we test the use of this approach through a diversity survey of Collembola from the vicinity of Churchill, Manitoba, Canada, and compare the results with previous surveys in the same area and in other sub-Arctic regions. The systematic barcoding campaign at Churchill revealed a diverse collembolan fauna consisting of 97 species-level MOTUs in six types of habitats. If all these MOTUs are confirmed as species, this richness would be far higher than prior records for Arctic Canada and could lead to reconsider the actual diversity of the group in Arctic environments.

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

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 Komericki & 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.

Cryptic diversity in the ubiquist species Parisotoma notabilis (Collembola, Isotomidae): a long-used chimeric species?

Parisotoma notabilis is the most common species of Collembola in Europe and is currently designated as ubiquist. This species has been extensively used in numerous studies and is considered as well characterized on a morphological ground. Despite the homogeneity of its morphology, the sequencing of the barcoding fragment (5' end of COI) for several populations throughout Europe and North America revealed four distinct genetic lineages. The divergence found between these lineages was similar to the genetic distance among other species of the genus Parisotoma included in the analysis. All four lineages have been confirmed by the nuclear gene 28S. This congruence between mitochondrial and nuclear signals, as well as the geographical distribution pattern of lineages observed in Europe, supports the potential specific status of these lineages. Based on specimens from the type locality (Hamburg), the species name was successfully assigned to one of these lineages. This finding raises several problems as Parisotoma notabilis has been widely used in many ecological studies. Accumulation of new data for the different lineages detected, especially ecological information and life history traits, is needed to help resolve this situation.

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, 12 h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1 h of lysis, while voucher condition was fine for up to 12 h. The lysis step substantially shortens the clearing process necessary for morphological examination.

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.

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.

The centipede genus Eupolybothrus Verhoeff, 1907 in North Africa is revised. A new cavernicolous species, Eupolybothruskahfi Stoev & Akkari, sp. n., is described from a cave in Jebel Zaghouan, northeast Tunisia. Morphologically, it is most closely related to Eupolybothrusnudicornis (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 Eupolybothruskahfi and Eupolybothrusnudicornis, 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 Eupolybothruskahfi 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. Eupolybothrusnudicornis is redescribed based on abundant material from Tunisia and its post-embryonic development, distribution and habitat preferences recorded. Eupolybothruscloudsley-thompsoni Turk, 1955, a nominal species based on Tunisian type material, is placed in synonymy with Eupolybothrusnudicornis. 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.

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.

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.