BackgroundRates of molecular evolution are known to vary across taxa and among genes, and this requires rate calibration for each specific dataset based on external information. Calibration is sensitive to evolutionary model parameters, partitioning schemes and clock model. However, the way in which these and other analytical aspects affect both the rates and the resulting clade ages from calibrated phylogenies are not yet well understood. To investigate these aspects we have conducted calibration analyses for the genus Carabus (Coleoptera, Carabidae) on five mitochondrial and four nuclear DNA fragments with 7888 nt total length, testing different clock models and partitioning schemes to select the most suitable using Bayes Factors comparisons.ResultsWe used these data to investigate the effect of ambiguous character and outgroup inclusion on both the rates of molecular evolution and the TMRCA of Carabus. We found considerable variation in rates of molecular evolution depending on the fragment studied (ranging from 5.02% in cob to 0.26% divergence/My in LSU-A), but also on analytical conditions. Alternative choices of clock model, partitioning scheme, treatment of ambiguous characters, and outgroup inclusion resulted in rate increments ranging from 28% (HUWE1) to 1000% (LSU-B and ITS2) and increments in the TMRCA of Carabus ranging from 8.4% (cox1-A) to 540% (ITS2). Results support an origin of the genus Carabus during the Oligocene in the Eurasian continent followed by a Miocene differentiation that originated all main extant lineages.ConclusionsThe combination of several genes is proposed as the best strategy to minimise both the idiosyncratic behaviors of individual markers and the effect of analytical aspects in rate and age estimations. Our results highlight the importance of estimating rates of molecular evolution for each specific dataset, selecting for optimal clock and partitioning models as well as other methodological issues potentially affecting rate estimation.

Metabarcoding of complex metazoan communities is increasingly being used to measure biodiversity in terrestrial, freshwater and marine ecosystems, revolutionizing our ability to observe patterns and infer processes regarding the origin and conservation of biodiversity. A fundamentally important question is which genetic marker to amplify, and although the mitochondrial cytochrome oxidase subunit I (COI) gene is one of the more widely used markers in metabarcoding for the Metazoa, doubts have recently been raised about its suitability. We argue that (a) the extensive coverage of reference sequence databases for COI; (b) the variation it presents; (c) the comparative advantages for denoising protein-coding genes; and (d) recent advances in DNA sequencing protocols argue in favour of standardizing for the use of COI for metazoan community samples. We also highlight where research efforts should focus to maximize the utility of metabarcoding.

Short running title:High-throughput sequencing to unveil soil mesofauna Summary 1. Biological communities inhabiting the soil are among the most diversified, complex and yet most poorly studied of terrestrial ecosystems. The greatest knowledge gaps apply to the arthropod mesofauna (0.1-2 mm body size) because conventional morphological and molecular approaches are in many cases insufficient for the characterisation of these complex communities. The development of high-throughput sequencing (HTS) methodologies is required to solve current impediments and to further advance our understanding of belowground biodiversity. We propose a Flotation-Berlese-Flotation (FBF) protocol for sampling and specimenprocessing to obtain 'clean' DNA extractions of arthropod mesofauna from the soil. In addition, we developed and tested HTS protocols for the characterization of arthropod communities from these bulk DNA extractions using cox1 metabarcoding and shotgun metagenomic sequencing on the MiSeq Illumina platform. 3.The FBF protocol provided DNA of soil arthropods from sufficiently large volumes of soil and free from contaminating bacteria and inhibitors. Metabarcoding and metagenomic sequencing on two deep soil samples from Iberian grasslands revealed >100 species of Acari and Collembola from 28 families. Genome assembly straight from shotgun sequencing of bulk specimens produced partial and full mitogenomes for 54 species with average length of >6000 bp. Metabarcoding and metagenomic sequencing resulted in closely congruent OTUs but species numbers were highest with metabarcoding, while ~73% of species were confirmed by matching shotgun sequence reads and ~48% by contig assembly from those shotgun reads. Accepted ArticleThis article is protected by copyright. All rights reserved. 4.In combination, the FBF protocol together with the PCR-based and shotgun sequencing pipelines addressed most of the challenges of studying soil arthropod mesofauna on the MiSeq Illumina platform. They are powerful, cost-efficient tools for characterising soil diversity in a phylogenetic and community ecology context. These methodological developments of HTS approaches for the study of mesofauna will accelerate ecological and evolutionary studies, biomonitoring of soil arthropods, and progress in both theoretical and applied soil science.

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Aim  In aquatic ecosystems, standing (lentic) and running (lotic) waters differ fundamentally in their stability and persistence, shaping the comparative population genetic structure, geographical range size and speciation rates of lentic versus lotic lineages. While the drivers of this pattern remain incompletely understood, the suite of traits making up the ability of a species to establish new populations is instrumental in determining such differences. Here we explore the degree to which the association between habitat type and geographical range size results from differences in dispersal ability or fundamental niche breadth in the members of the Enochrus bicolor complex, an aquatic beetle clade with species across the lentic–lotic divide.Location  Western Mediterranean, with a special focus on North Africa, the Iberian Peninsula and Sicily.Methods  DNA sequences for four loci were obtained from species of the E. bicolor complex and analysed using phylogenetic inference. Dispersal and establishment abilities were assessed in lentic–lotic species pairs of the complex, using flight wing morphometrics and thermal tolerance ranges as surrogates, respectively.Results  There were clear differences in range size between the lotic and lentic taxa of the complex, which appears to have had a lotic origin with two transitions to standing waters. Only small differences were observed in temperature tolerance and acclimation ability between the two lotic–lentic sister species studied. By contrast, wing morphometrics revealed clear, consistent differences between lotic and lentic Enochrus species pairs, the latter having a higher dispersal capacity.Main conclusions  We hypothesize that there have been two habitat shifts from lotic to lentic waters, which have allowed marked expansions in geographical range size in western Mediterranean species of the E. bicolor complex. Differences in dispersal rather than in establishment ability appear to underlie differences in geographical range extent, as transitions to lentic waters were associated with changes in wing morphology, but not in thermal tolerance range. In this lineage of water beetles, selection for dispersal in geologically short‐lived lentic systems has driven the evolution of larger range sizes in lentic taxa compared with those of their lotic relatives.

Geographic isolation substantially contributes to species endemism on oceanic islands when speciation involves the colonisation of a new island. However, less is understood about the drivers of speciation within islands. What is lacking is a general understanding of the geographic scale of gene flow limitation within islands, and thus the spatial scale and drivers of geographical speciation within insular contexts. Using a community of beetle species, we show that when dispersal ability and climate tolerance are restricted, microclimatic variation over distances of only a few kilometres can maintain strong geographic isolation extending back several millions of years. Further to this, we demonstrate congruent diversification with gene flow across species, mediated by Quaternary climate oscillations that have facilitated a dynamic of isolation and secondary contact. The unprecedented scale of parallel species responses to a common environmental driver for evolutionary change has profound consequences for understanding past and future species responses to climate variation.

High-throughput DNA methods hold great promise for the study of taxonomically intractable mesofauna of the soil. Here, we assess species diversity and community structure in a phylogenetic framework, by sequencing total DNA from bulk specimen samples and assembly of mitochondrial genomes. The combination of mitochondrial metagenomics and DNA barcode sequencing of 1494 specimens in 69 soil samples from three geographic regions in southern Iberia revealed >300 species of soil Coleoptera (beetles) from a broad spectrum of phylogenetic lineages. A set of 214 mitochondrial sequences longer than 3000 bp was generated and used to estimate a well-supported phylogenetic tree of the order Coleoptera. Shorter sequences, including cox1 barcodes, were placed on this mitogenomic tree. Raw Illumina reads were mapped against all available sequences to test for species present in local samples. This approach simultaneously established the species richness, phylogenetic composition and community turnover at species and phylogenetic levels. We find a strong signature of vertical structuring in soil fauna that shows high local community differentiation between deep soil and superficial horizons at phylogenetic levels. Within the two vertical layers, turnover among regions was primarily at the tip (species) level and was stronger in the deep soil than leaf litter communities, pointing to layer-mediated drivers determining species diversification, spatial structure and evolutionary assembly of soil communities. This integrated phylogenetic framework opens the application of phylogenetic community ecology to the mesofauna of the soil, among the most diverse and least well-understood ecosystems, and will propel both theoretical and applied soil science.

Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species-level resolution is difficult to obtain. Next-generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 "barcode" and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (southern England). Operational taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, filtering, pair merging and clustering). Similar taxonomic profiles were obtained under a broad range of parameters. The SSU marker recovered Platyhelminthes and Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A reference set was created from all available barcode entries for Arthropoda in the BOLD database and clustered into OTUs. The River Kennet metabarcoding produced matches to 207 of these reference OTUs, five times the number of species recognized with morphological monitoring. The increase was due to the following: greater taxonomic resolution (e.g., splitting a single morphotaxon "Chironomidae" into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration-flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between "impacted" vs. "control" samples, detectable with each gene marker, for each major taxonomic group, and for meio- and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology.