Alexander M. Weigand

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.

Drastic underestimation of amphipod biodiversity in the endangered Irano-Anatolian and Caucasus biodiversity hotspots

Biodiversity hotspots are centers of biological diversity and particularly threatened by anthropogenic activities. Their true magnitude of species diversity and endemism, however, is still largely unknown as species diversity is traditionally assessed using morphological descriptions only, thereby ignoring cryptic species. This directly limits evidence-based monitoring and management strategies. Here we used molecular species delimitation methods to quantify cryptic diversity of the montane amphipods in the Irano-Anatolian and Caucasus biodiversity hotspots. Amphipods are ecosystem engineers in rivers and lakes. Species diversity was assessed by analysing two genetic markers (mitochondrial COI and nuclear 28S rDNA), compared with morphological assignments. Our results unambiguously demonstrate that species diversity and endemism is dramatically underestimated, with 42 genetically identified freshwater species in only five reported morphospecies. Over 90% of the newly recovered species cluster inside Gammarus komareki and G. lacustris; 69% of the recovered species comprise narrow range endemics. Amphipod biodiversity is drastically underestimated for the studied regions. Thus, the risk of biodiversity loss is significantly greater than currently inferred as most endangered species remain unrecognized and/or are only found locally. Integrative application of genetic assessments in monitoring programs will help to understand the true magnitude of biodiversity and accurately evaluate its threat status.

Positive selection on panpulmonate mitogenomes provide new clues on adaptations to terrestrial life

BackgroundTransitions from marine to intertidal and terrestrial habitats resulted in a significant adaptive radiation within the Panpulmonata (Gastropoda: Heterobranchia). This clade comprises several groups that invaded the land realm independently and in different time periods, e.g., Ellobioidea, Systellomatophora, and Stylommatophora. Thus, mitochondrial genomes of panpulmonate gastropods are promising to screen for adaptive molecular signatures related to land invasions.ResultsWe obtained three complete mitochondrial genomes of terrestrial panpulmonates, i.e., the ellobiid Carychium tridentatum, and the stylommatophorans Arion rufus and Helicella itala. Our dataset consisted of 50 mitogenomes comprising almost all major panpulmonate lineages. The phylogenetic tree based on mitochondrial genes supports the monophyly of the clade Panpulmonata. Terrestrial lineages were sampled from Ellobioidea (1 sp.) and Stylommatophora (9 spp.). The branch-site test of positive selection detected significant non-synonymous changes in the terrestrial branches leading to Carychium (Ellobiodea) and Stylommatophora. These convergent changes occurred in the cob and nad5 genes (OXPHOS complex III and I, respectively).ConclusionsThe convergence of the non-synonymous changes in cob and nad5 suggest possible ancient episodes of positive selection related to adaptations to non-marine habitats. The positively selected sites in our data are in agreement with previous results in vertebrates suggesting a general pattern of adaptation to the new metabolic requirements. The demand for energy due to the colonization of land (for example, to move and sustain the body mass in the new habitat) and the necessity to tolerate new conditions of abiotic stress may have changed the physiological constraints in the early terrestrial panpulmonates and triggered adaptations at the mitochondrial level.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

A simple centrifugation protocol leads to a 55-fold mitochondrial DNA enrichment and paves the way for future mitogenomic research

1. DNA (meta)barcoding is increasingly used to study and monitor biodiversity and available for standardised assessments. However, it suffers from PCR bias which can lead to the loss of taxonomic groups. PCR-free techniques such as metagenomics are therefore thought to be more suited for biodiversity assessments but are currently limited by incomplete reference libraries. 2. The technique of ‘mitogenome-skimming’ or ‘mitogenomics’, where complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria prior to DNA extraction and sequencing, roughly 99 % of reads are of non-mitochondrial origin and therefore mostly useless for species identification. 3. Here, we present a simple centrifugation protocol that leads to a 55-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’-communities – comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each – we recovered whole mitochondrial genomes with a minimum coverage of 2417-fold. In addition, we ‘skimmed’ the whole mitochondrial genome of the acanthocephalan endoparasite Pomphorhynchus laevis (444-fold coverage) obviously being present in G. roeselii. 4. Correlation between body mass of specimens in each community and generated sequence number was moderate in samples enriched for mitochondria. Potential problems arise with species demonstrating a high weight, but produce less than expected mitochondrial reads (e.g. shelled organisms). The developed protocol will greatly speed up building reference libraries for whole mitochondrial genomes, as dozens of species can be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without enrichment for mitochondria.

A simple centrifugation protocol for metagenomic studies increases mitochondrial DNA yield by two orders of magnitude

DNA (meta)barcoding is applied to study biodiversity and is available for standardized biodiversity assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries. The technique of “mitogenome-skimming” or “mitogenomics,” in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99% of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification. Here, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’-communities—comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each—we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis. The enrichment protocol will speed up building reference libraries for whole mitochondrial genomes, as dozens of species can be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments, using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

Genome-wide single-nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species-The case of the Ancylus fluviatilis species complex

Abstract DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high‐resolution genome‐wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome‐wide data for species delimitation in such groups.

A new cave centipede from Croatia, Eupolybothrus liburnicus sp. n., with notes on the subgenus Schizopolybothrus Verhoeff, 1934 (Chilopoda, Lithobiomorpha, Lithobiidae)

Abstract A new species of Eupolybothrus Verhoeff, 1907 discovered in caves of Velebit Mountain in Croatia is described. E. liburnicus sp. n. exhibits a few morphological differences from its most similar congeners, all of which are attributed to the subgenus Schizopolybothrus Verhoeff, 1934, and two approaches to species delimitation using the COI barcode region identify it as distinct from the closely allied E. cavernicolus Stoev & Komerički, 2013. E. spiniger (Latzel, 1888) is redescribed and a lectotype is designated for it as well as E. caesar (Verhoeff, 1899) to stabilize their respective taxonomic status. The subspecies E. acherontis wardaranus Verhoeff, 1937, previously suspected to be a synonym of E. caesar (Verhoeff, 1899), is redescribed and its taxonomy revised after the study of type material whereas the identity of E. acherontis (Verhoeff, 1900) described from a female from southwest Trebinje (Bosnia and Herzegovina) remains unknown. Type material of E. stygis (Folkmanova, 1940) is confirmed to be lost and future designation of neotypes from topotypic specimens is necessary to stabilize its taxonomy. The importance of setal arrangement on the intermediate and 14th tergites and the sexual modifications on the male 15th prefemur for species identification is discussed in the light of present findings, and a review of the species of E. (Schizopolybothrus) that display these traits is also provided.

The complete mitochondrial genome of a cryptic amphipod species from the Gammarus fossarum complex

Abstract The freshwater amphipod Gammarus fossarum is widely distributed throughout Europe and an important species for stream biomonitoring. It is known to consist of several cryptic species. We here report the complete mitochondrial genome of G. fossarum clade 11/type B with a length of 15,989 bp, encoding for 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. Protein-coding and ribosomal genes have a similar arrangement as in other gammarid amphipods. A phylogenetic analysis clarifies the placement of G. fossarum within the Gammaridae.