Jonas J. Astrin

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes

BackgroundThe identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous.ResultsWe tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae.ConclusionOur results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

Molecular taxonomy in pholcid spiders (Pholcidae, Araneae): evaluation of species identification methods using CO1 and 16S rRNA

The identification of species using molecular characters is a promising approach in alpha taxonomy and in any discipline depending on reliable assignment of specimens. Previous studies have shown the feasibility of the method, but considerable controversy persists. In this study, we use pholcid spiders in an effort to address two main issues. First, we evaluate and calibrate molecular species (re‐)identification within a closely related group of organisms by using specimens that are morphologically unambiguously either conspecific or not. Species limits hypothesized a priori based on morphology were almost universally reconstructed by both mitochondrial markers used. Second, we focus on species identification methodology in a morphology‐calibrated scenario, i.e. on how to assess the quality of a dataset and of the method used to obtain distance estimates (e.g. choice of markers, alignment strategy, type of distance data). We develop a number of statistical estimators permitting the measurement and communication of the clarity of species boundaries in a dataset and discuss their benefits and drawbacks. We propose that box plots rather than histograms are the superior tool for graphically illustrating taxonomic signal and that the median is a more appropriate measure of central tendency than the mean. Applying the suggested tools to our data, we propose that in molecular species identification, indel‐related alignment uncertainties may often be even advantageous (by accentuating taxonomy‐relevant information) and we conclude that — at least for our dataset — 16S is better suited to taxonomy than CO1.

Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera : Curculionidae)

A phylogeny is presented for the western Palaearctic representatives of the weevil subfamily Cryptorhynchinae using a combination of phenotypic and genotypic characters. This phylogeny is the first for the extremely species-rich Cryptorhynchinae to use molecular data (mitochondrial CO1 and 16S as well as nuclear ribosomal 28S). The results of this study show the need for molecular tools within this morphologically cryptic group of weevils and provide a scaffold based on which genus assignment can be tested. The present study mostly corroborates the current subdivision into genera (but many of the subgeneric groups are questioned). Three new genera are described: Montanacalles gen. nov. (type species: Kyklioacalles nevadaensis Stuben, 2001), Coloracalles gen. nov. (type species: Acalles humerosus Fairmaire, 1862) and Elliptacalles gen. nov. (type species: Acalles longus Desbrochers, 1892). Relevant external characters and the male genitalia of all discussed taxa are illustrated. Three species are transferred to different genera: Kyklioacalles aubei (Boheman, 1837) (formerly: Acalles), Ruteria major (Solari A. & F., 1907) and Ruteria minosi (Bahr & Bayer, 2005) (both formerly Echinodera).

The importance of biobanking in molecular taxonomy, with proposed definitions for vouchers in a molecular context

DNA barcoding and molecular or integrative taxonomy projects are among the most valuable sources for biobank specimens of wild organisms, thanks to – among other aspects – the high level of specimen diversity and thanks to a thorough taxonomic coverage. Specimens used to build barcoding reference libraries tend to be accompanied by deeper and higher-quality data than samples from many other sources, as they are often contributed by taxonomists, and identifications are cross-checked through barcode analysis. Vouchering of morphological specimens in natural history collections is a prerequisite for proper barcoding, which is advantageous for biobanking as well, as biobank samples should always be linked to specimen vouchers. As a further added value, barcoding provides an inherent, molecular species ID tag to the processed biobank sample. n nBanked barcoding samples can greatly catalyze taxonomy, as well as many other fields of application, such as the emerging large genome sequencing projects that are constantly increasing the demand for well-preserved samples from a multitude of different species (see Wong et al. 2012). n nConsidered from the opposite perspective of the synergy, barcoding can benefit greatly from biobanking as well. Biobanking enables the expansion of barcoding datasets with biobanked samples from other projects. It also offers the possibility to add new barcoding markers any time in the future, e.g. scaling up to ‘next-generation barcoding’ (e.g. Taylor and Harris 2012) if feasible (manageability of data, NGS and data handling cost, performance in mixed samples, etc.), without the necessity of repeating the time-consuming and expensive steps of sample collection, data collection and identification, and vouchering. n nFinally and most importantly, biobanks offer barcoding projects the possibility to adequately voucher their molecular samples and to warrant reproducibility of results. n nResearchers involved in barcoding projects should make sure their samples are properly vouchered – morphologically AND molecularly. They can do this by depositing their samples at a dedicated natural history collection. Increasingly, these repositories are establishing biobanks / DNA banks / tissue banks for curated long-term, ultra cold conservation of molecular samples, are adopting standard operating procedures and making their samples available online e.g. through biobank networks like the DNA Bank Network (http://www.dnabank-network.org/) or soon also the Global Genome Biodiversity Network (http://ggbn.org/). Those museums and natural history collections that implement these features and commit themselves to provide the community with proper biobanks (although maybe called differently) offer a very efficient and elegant way to both draw on and to deposit morphological-molecular ‘tandem’ samples. Often underappreciated by public and policy-makers (Suarez and Tsutsui 2004), natural history collections holding and curating specimen vouchers and/or cross-referenced molecular vouchers and their data play a “major role in organizing systematic knowledge in the molecular age” (Whitfield and Cameron 1994). n nAlthough it has been pointed out before (e.g. Hafner 1994), the importance of vouchering molecular samples is not yet fully apprehended in the scientific community (perhaps because of the way taxonomy has been traditionally carried out). n nWe would like to encourage authors, editors and reviewers of scientific papers to give also molecular vouchers the attention they deserve. n nVouchers – morphological and molecular alike – not only form the connection between study data and taxonomic identification. They are much more: vouchers link the data collected in individual studies with the immense wealth of data that can still be (or already have been) collected through the vouchers: repetitively or in an additive manner. Put short, vouchers link individual studies with other studies and inferences, past or future. n nIt becomes obvious that it is only through adequate vouchering that we can make organismic biology meaningful, warranting reproducibility and embedding our research into existing and emerging knowledge. n nIn a laudable approach to increasing semantic accuracy regarding the voucher concept, Pleijel et al. (2008) suggest a terminology for those specimen vouchers used to produce molecular (sub-)samples. These are coined ‘genophores’ (although of course molecular samples lend themselves to more than genetic analysis), and for mnemonic ease follow the taxonomic nomenclatorial codes in style: n na hologenophore is the specimen voucher from which the molecular sample is directly derived, an isogenophore is a different specimen with a clonal relationship to the study organism, while a progenophore represents a voucher that is linked to the specimen sampled for molecular analysis by a parent-descendant or sibling relationship. A paragenophore is a putatively conspecific specimen voucher collected together with the ‘molecular’ specimen. The same applies to the syngenophore, except that it is collected at another place or time. n nThese genealogy-based distinctions made by Pleijel et al. (2008) are helpful for categorizing a specimen voucher in its relation to a molecular voucher and we endorse their use in this context. The function/purpose or the nature of vouchers was deliberately not addressed by Pleijel and colleagues. However, especially in the context of molecular samples, we perceive the necessity to do so, as varying uses of terms can be observed (e.g. “DNA voucher” used synonymously for the DNA source or for the isolated DNA). Different use of terms makes it difficult to extract data from biological collection databases or from the literature in a semantically meaningful way. Therefore, in the following we propose some voucher, sample and repository definitions, with special focus on a molecular context. n n nspecimen voucher: a specimen serving as the basis for taxonomic identification and possibly also for other queries. A specimen voucher is often, but not necessarily a whole organism, or part of it (it can be a trace or ichnofossil, scats, eggs, images, etc.). n nNarrower terms: morphological voucher, acoustic voucher, e-voucher, etc. n n nmorphological voucher: a specimen that allows the inspection of morphological characters. n n ne-voucher: digital objects that serve as vouchers (morphological, acoustic, etc.), e.g. sound recordings, audiovisual material, images, etc. n n n n nmolecular voucher: a sample that is deliberately preserved and curated in a way that will conserve its molecular properties for analysis. A molecular voucher should always be linked to a specimen voucher (which sometimes can be the same object if sufficient characters remain, see tissue voucher). n nNarrower terms: biobank voucher, DNA voucher, tissue voucher, RNA voucher, protein voucher, genomic sample, etc. n n ntissue voucher: tissue subsampled from a specimen – or the entire specimen –, preserved (usu. frozen) to keep its molecular properties (either fixed tissue or viable cells) for future analysis n n nDNA voucher: the isolated and preserved, frozen or dried (usu. genomic) DNA. As a derived sample, a DNA voucher should not – if anyhow possible – function as specimen voucher. n n nbiobank voucher: any molecular voucher curated in a biobank. A biobank voucher is a biobank sample that links to other physical objects or data (other than their metadata), i.e. most biobank samples are (biobank) vouchers, as they usually link to a separate specimen voucher n n ngenomic sample: preserved sample containing (isolated or as a constituent) a high percentage of an organism’s genome in widely unfragmented form n n n n nbiobank: a curated collection/repository of biological materials that warrants long-term integrity at molecular level, authenticity, availability and rights management of its samples by adhering to standard operating procedures (SOPs). n nNarrower terms: DNA bank, tissue bank, biodiversity biobank, etc. n n nbiodiversity biobank: term currently used to refer to a biobank holding non-human samples n n ngenomic collection: a molecular collection holding genomic samples

Exploring diversity in cryptorhynchine weevils (Coleoptera) using distance-, character- and tree-based species delineation

Species boundaries are studied in a group of beetles, the western Palaearctic Cryptorhynchinae. We test for congruence of traditionally identified morphospecies with species inferred through parsimony networks, distance-based clustering and the ultrametric tree-based generalized mixed yule-coalescent (GMYC) approach. For that purpose, we sequenced two variable fragments of mitochondrial DNA (CO1 and 16S) for a total of 791 specimens in 217 species of Cryptorhynchinae. Parsimony networks, morphology-calibrated distance clusters and the different tree-based species inferences all achieved low congruence with morphospecies, at best 60%. Although the degree of match with morphospecies was often similar for the different approaches, the composition of clusters partially varied. A barcoding gap was absent in morphospecies-oriented distances as well as for GMYC species clusters. This demonstrates that not only erroneous taxonomic assignments, incomplete lineage sorting, hybridization, or insufficient sampling can compromise distance-based identification, but also differences in speciation rates and uneven tree structure. The initially low match between morphospecies and the different molecular species delineation methods in this case study shows the necessity of combining the output of various methods in an integrative approach. Thereby we obtain an idea about the reliability of the different results and signals, which enables us to fine-tune sampling, delineation technique and data collection, and to identify species that require taxonomic revision.

Pholcid spider molecular systematics revisited, with new insights into the biogeography and the evolution of the group

We analysed seven genetic markers sampled from 165 pholcids and 34 outgroups in order to test and improve the recently revised classification of the family. Our results are based on the largest and most comprehensive set of molecular data so far to study pholcid relationships. The data were analysed using parsimony, maximum‐likelihood and Bayesian methods for phylogenetic reconstruction. We show that in several previously problematic cases molecular and morphological data are converging towards a single hypothesis. This is also the first study that explicitly addresses the age of pholcid diversification and intends to shed light on the factors that have shaped species diversity and distributions. Results from relaxed uncorrelated lognormal clock analyses suggest that the family is much older than revealed by the fossil record alone. The first pholcids appeared and diversified in the early Mesozoic about 207u2003Ma ago (185–228u2003Ma) before the breakup of the supercontinent Pangea. Vicariance events coupled with niche conservatism seem to have played an important role in setting distributional patterns of pholcids. Finally, our data provide further support for multiple convergent shifts in microhabitat preferences in several pholcid lineages. Our findings suggest that both adaptive and non‐adaptive speciation may have played an important role in the diversification of pholcid lineages.

Increased sampling blurs morphological and molecular species limits: revision of the Hispaniolan endemic spider genus Tainonia (Araneae : Pholcidae)

The genus Tainonia comprises unusually large pholcids endemic to Hispaniola. Previously, only the type species had been formally described, represented in collections by no more than 12 adult specimens. However, the existence of more species has been hypothesised based on a few further individuals. The present paper is based on a sample of 205 mostly newly collected adult specimens from 18 localities in the Dominican Republic and four localities in Haiti. The increased sampling reveals a wide range of variation, including intermediate levels of divergence that often blur rather than clarify species limits. Therefore, although not all taxonomic questions can be settled here, morphological (including morphometric) and molecular (mitochondrial 16S, CO1) data strongly support two new species: one in La Visite National Park, Haiti (T. visite, sp. nov.) and another on Samana Peninsula and parts of the eastern Dominican Republic (T. samana, sp. nov.). Species limits among the other populations are more difficult to support or reject. Specimens from Bayahibe (eastern Dominican Republic) and from La Cienaga (Cordillera Central) are each assigned species status on the basis of consistent morphological differences (T. bayahibe, sp. nov., T. cienaga, sp. nov.), but no molecular data are available due to lack of specimens. All other specimens are provisionally assigned to a possibly paraphyletic T. serripes (Simon). There is considerable morphological variation within this widely distributed group of populations but this variation is rather continuous and molecular distances fill most of the range between morphologically unambiguous conspecifics and unambiguous heterospecifics.

How to tackle the molecular species inventory for an industrialized nation-lessons from the first phase of the German Barcode of Life initiative GBOL (2012-2015).

Biodiversity loss is mainly driven by human activity. While concern grows over the fate of hot spots of biodiversity, contemporary species losses still prevail in industrialized nations. Therefore, strategies were formulated to halt or reverse the loss, driven by evidence for its value for ecosystem services. Maintenance of the latter through conservation depends on correctly identified species. To this aim, the German Federal Ministry of Education and Research is funding the GBOL project, a consortium of natural history collections, botanic gardens, and universities working on a barcode reference database for the countrys fauna and flora. Several noticeable findings could be useful for future campaigns: (i) validating taxon lists to serve as a taxonomic backbone is time-consuming, but without alternative; (ii) offering financial incentives to taxonomic experts, often citizen scientists, is indispensable; (iii) completion of the libraries for widespread species enables analyses of environmental samples, but the process may not hold pace with technological advancements; (iv) discoveries of new species are among the best stories for the media; (v) a commitment to common data standards and repositories is needed, as well as transboundary cooperation between nations; (vi) after validation, all data should be published online via the BOLD to make them searchable for external users and to allow cross-checking with data from other countries.

Molecular phylogeny of Echinodera and Ruteria (Coleoptera : Curculionidae : Cryptorhynchinae) and the parallel speciation of Canary Island weevils along replicate environmental gradients

A molecular phylogeny for the western Palaearctic weevil genus Echinodera Wollaston, 1863 and the former genus Ruteria Roudier, 1954 is presented, combining two mitochondrial genes (CO1 and 16S) in a Bayesian analysis. Special consideration is given to the species of Echinodera from the Canary Islands. Between islands, these are represented by multiple vicariant species that have undergone parallel speciation along replicate environmental gradients on the respective islands. Based on the phylogenetic tree and further data, a number of taxonomic changes is presented: two new species are described, Echinodera montana, sp. nov. from the Canaries (Fuerteventura) and Echinodera bargouensis, sp. nov. from Tunisia. Five species are declared to be synonyms: Echinodera gomerensis Stuben, 2000, syn. nov.u2009=u2009Echinodera praedicta Germann & Stuben, 2006, syn. nov.u2009=u2009Echinodera pseudohystrix Stuben, 2000; Ruteria bellieri epirica Wolf, 2001, syn. nov.u2009=u2009Echinodera tyrrhenica Caldara, 1978, syn. nov.u2009=u2009Acalles bellieri Reiche, 1860; Echindera troodosi Wolf, 2010, syn. nov.u2009=u2009Echinodera cyprica Stuben, 2010. The subgenus Echinodera (Dieckmannia) Stuben, 1998 is a synonym of Echinodera s. str. The genus Ruteria is again declared a subgenus of Echinodera: Echinodera (Ruteria) Roudier, 1954 stat. rev. Two species are transferred to a different subgenus: Echinodera (Ruteria) incognita (Hoffmann, 1956) and Echinodera (Ruteria) cognita Stuben, 2006 (both formerly Echinodera s. str.).

Towards a DNA Barcode Reference Database for Spiders and Harvestmen of Germany

As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individuals/species). This covers about 60% of the spider fauna and more than 70% of the harvestmen fauna recorded for Germany. The overwhelming majority of species could be readily identified through DNA barcoding: median distances between closest species lay around 9% in spiders and 13% in harvestmen, while in 95% of the cases, intraspecific distances were below 2.5% and 8% respectively, with intraspecific medians at 0.3% and 0.2%. However, almost 20 spider species, most notably in the family Lycosidae, could not be separated through DNA barcoding (although many of them present discrete morphological differences). Conspicuously high interspecific distances were found in even more cases, hinting at cryptic species in some instances. A new program is presented: DiStats calculates the statistics needed to meet DNA barcode release criteria. Furthermore, new generic COI primers useful for a wide range of taxa (also other than arachnids) are introduced.