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Complex communities of fungi are regularly characterised by deep sequencing with standard barcode markers, especially the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus. Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations or for early detection of pathogens and invasive species, but reliance on a single short sequence fragment may be problematic due to various technological and analytical obstacles. Here we conducted a side-by-side comparison of fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens, using ITS and LSU. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type). Identification against the respective reference databases using various classifiers revealed similar higher-level taxonomic composition, but decreasing resolution towards lower levels, especially at the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, a phylogenetic analysis (focused on the epidemiologically important Sordariomycetes) placed OTUs relative to reference sequences spanning both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. The analysis of congruence in both markers also suggested the biologically most defensible threshold values for OTU delimitation (98% for ITS, 99% for LSU). Studies of complex fungal communities using the canonical ITS metabarcode marker require corroboration across additional loci, and benefit from phylogenetic analyses and their greater precision compared to conventional taxonomic classifiers, even in the face of incomplete and partially identified reference databases.

Dytiscidae is the most diverse family of beetles in which both adults and larvae are aquatic, with examples of extreme morphological and ecological adaptations. Despite continuous attention from systematists and ecologists, existing phylogenetic hypotheses remain unsatisfactory because of limited taxon sampling or low node support. Here we provide a phylogenetic tree inferred from four gene fragments (cox1, rrnL, H3 and SSU, � 4000 aligned base pairs), including 222 species in 116 of 174 known genera and 25 of 26 tribes. We aligned ribosomal genes prior to tree building with parsimony and Bayesian methods using three approaches: progressive pair-wise alignment with refinement, progressive alignment modeling the evolution of indels, and deletion of hypervariable sites. Results were generally congruent across alignment and tree inference methods. Basal relationships were not well defined, although we identified 28 well supported lineages corresponding to recognized tribes or groups of genera, among which the most prominent novel results were the polyphyly of Dytiscinae; the grouping of Pachydrini with Bidessini, Peschetius with Methlini and Coptotomus within Copelatinae; the monophyly of all Australian Hydroporini (Necterosoma group), and their relationship with the Graptodytes and Deronectes groups plus Hygrotini. We found support for a clade formed by Hydroporinae plus Laccophilini, and their sister relationship with Cybistrini and Copelatinae. The tree provided a framework for the analysis of species diversification in Dytiscidae. We found a positive correlation between the number of species in a lineage and the age of the crown group as estimated through a molecular clock approach, but the correlation with the stem age was non-significant. Imbalances between sister clades were significant for several nodes, but the residuals of the regression of species numbers with the crown age of the group identified only Bidessini and the Coptotomus + Agaporomorphus clade as lineages with, respectively, above and below expected levels of species diversity. � The Willi Hennig Society 2008.

Disentangling the relative role of environmental filtering and spatial processes in driving metacommunity structure across mountainous regions remains challenging, as the way we quantify spatial connectivity in topographically and environmentally heterogeneous landscapes can influence our perception of which process predominates. More empirical data sets are required to account for taxon- and context-dependency, but relevant research in understudied areas is often compromised by the taxonomic impediment. Here we used haplotype-level community DNA metabarcoding, enabled by stringent filtering of amplicon sequence variants (ASVs), to characterize metacommunity structure of soil microarthropod assemblages across a mosaic of five forest habitats on the Troodos mountain range in Cyprus. We found similar β diversity patterns at ASV and species (OTU, operational taxonomic unit) levels, which pointed to a primary role of habitat filtering resulting in the existence of largely distinct metacommunities linked to different forest types. Within-habitat turnover was correlated to topoclimatic heterogeneity, again emphasizing the role of environmental filtering. However, when integrating landscape matrix information for the highly fragmented Quercus alnifolia habitat, we also detected a major role of spatial isolation determined by patch connectivity, indicating that stochastic and niche-based processes synergistically govern community assembly. Alpha diversity patterns varied between ASV and OTU levels, with OTU richness decreasing with elevation and ASV richness following a longitudinal gradient, potentially reflecting a decline of genetic diversity eastwards due to historical pressures. Our study demonstrates the utility of haplotype-level community metabarcoding for characterizing metacommunity structure of complex assemblages and improving our understanding of biodiversity dynamics across mountainous landscapes worldwide.

Climate change is predicted to progressively shift habitat characteristics that will alter the distribution and eco-physiological responses of organisms. High-elevation stream insects without extensive dispersal are expected to be highly vulnerable if they are unable to track predicted shifts. Understanding dispersal is therefore crucial to implement practical solutions in conservation. The evolutionary history of the Palaearctic genus Annitella (Trichoptera), the population dynamics and habitat distribution (present and future) of two endemic species (A. esparraguera and A. iglesiasi) confined to Baetic Mountains (SE-Iberian Peninsula) were assessed to gain fundamental insights into species responses to climate warming and to identify populations at risk. Diversification of Annitella was dated to the Pleistocene and was likely associated with southern and extra-Mediterranean refugia located across Europe. The two endemic species belong to distant lineages that preferred similar cold headwater pristine habitats. The range of A. esparraguera was larger than that of A. iglesiasi. Both species showed low genetic diversity in cox1, but only A. esparraguera exhibit locally unique haplotypes, indicating limited gene flow. For A. esparraguera, modelled future habitat suitability showed 88.4% range contraction by 2050 (RCP scenario 8.5) and a displacement of 41.5% of the current potential distribution to higher elevations. Populations of A. esparraguera are predicted to be lost because of the reduction of optimal habitat and limited propensity for tracking future suitable conditions. Beyond the preservation of their current habitat, their conservation might require proactive measures (translocations). Similar predictions may apply to other Mediterranean endemic headwater specialist restricted to isolated high-elevation streams.

The high-level classification of Chrysomelidae (leaf beetles) currently recognizes 12 or 13 well-established subfamilies, but the phylogenetic relationships among them remain ambiguous. Full mitochondrial genomes were newly generated for 27 taxa and combined with existing GenBank data to provide a dataset of 108 mitochondrial genomes covering all subfamilies. Phylogenetic analysis under maximum likelihood and Bayesian inference recovered the monophyly of all subfamilies, except that Timarcha was split from Chrysomelinae in some analyses. Three previously recognized major clades of Chrysomelidae were broadly supported: the ‘chrysomeline’ clade consisting of (Chrysomelinae (Galerucinae + Alticinae)); the ‘sagrine’ clade with internal relationships of ((Bruchinae + Sagrinae) + (Criocerinae + Donaciinae)), and the ‘eumolpine’ clade comprising (Spilopyrinae (Cassidinae (Eumolpinae (Cryptocephalinae + Lamprosomatinae)))). Relationships among these clades differed between data treatments and phylogenetic algorithms, and were complicated by two additional deep lineages, Timarcha and Synetinae. Various topological tests favoured the PhyloBayes software as the preferred inference method, resulting in the arrangement of (chrysomelines (eumolpines + sagrines)), with Timarcha placed as sister to the chrysomeline clade and Synetinae as a deep lineage splitting near the base. Whereas mitogenomes provide a solid framework for the phylogeny of Chrysomelidae, the basal relationships do not agree with the topology of existing molecular studies and remain one of the most difficult problems of Chrysomelidae phylogenetics., This research was supported by grants from the National Science Foundation of China (nos 31772496, 31301900 and 3181101902), and the President's International Fellowship Initiative of the Chinese Academy to APV. APV's research on Coleoptera phylogenetics was funded by the Leverhulme Trust (F/00696/P and IAF‐2018‐038) and mitogenome sequencing was supported by the Natural History Museum Biodiversity Initiative.

The gut microbiome plays an important role in a host’s development and adaption to its dietary niche. In this study, a group of bamboo-feeding insects are used to explore the potential role of the gut microbiota in the convergent adaptation to extreme diet specialization. Specifically, using a 16S rRNA marker and an Illumina sequencing platform, we profiled the microbial communities of 76 gut samples collected from nine bamboo-feeding insects, including both hemimetabolous (Orthoptera and Hemiptera) and holometabolous (Coleoptera and Lepidoptera) species, which are specialized in three distinct dietary niches: bamboo leaf, shoot, and sap. The gut microbiota of these insects were dominated by Proteobacteria, Firmicutes, and Bacteroidetes and were clustered into solid (leaf and shoot) and liquid (sap) dietary niches. The gut bacterial communities of insects feeding on solid diet overlapped significantly, even though these insects belong to phylogenetically distant lineages representing different orders. In addition, the presence of cellulolytic bacterial communities within the gut microbiota allows bamboo-feeding insects to adapt to a highly specialized, fiber-rich diet. Although both phylogeny and diet can impact the structure and composition of gut microbiomes, phylogeny is the primary driving force underlying the convergent adaptation to a highly specialized diet, especially when the related insect species harbor similar gut microbiomes and share the same dietary niche over evolutionary timescales. These combined findings lay the foundation for future research on how convergent feeding strategies impact the interplays between hosts and their gut microbiomes and how the gut microbiota may facilitate convergent evolution in phylogenetically distant species in adaptation to the shared diet.

Metabarcoding of DNA extracted from community samples of whole organisms (whole organism community DNA, wocDNA) is increasingly being applied to terrestrial, marine and freshwater metazoan communities to provide rapid, accurate and high resolution data for novel molecular ecology research. The growth of this field has been accompanied by considerable development that builds on microbial metabarcoding methods to develop appropriate and efficient sampling and laboratory protocols for whole organism metazoan communities. However, considerably less attention has focused on ensuring bioinformatic methods are adapted and applied comprehensively in wocDNA metabarcoding. In this study we examined over 600 papers and identified 111 studies that performed COI metabarcoding of wocDNA. We then systematically reviewed the bioinformatic methods employed by these papers to identify the state-of-the-art. Our results show that the increasing use of wocDNA COI metabarcoding for metazoan diversity is characterised by a clear absence of bioinformatic harmonisation, and the temporal trends show little change in this situation. The reviewed literature showed (i) high heterogeneity across pipelines, tasks and tools used, (ii) limited or no adaptation of bioinformatic procedures to the nature of the COI fragment, and (iii) a worrying underreporting of tasks, software and parameters. Based upon these findings we propose a set of recommendations that we think the wocDNA metabarcoding community should consider to ensure that bioinformatic methods are appropriate, comprehensive and comparable. We believe that adhering to these recommendations will improve the long-term integrative potential of wocDNA COI metabarcoding for biodiversity science.

Metabarcoding has revolutionized the study of ecological communities, but PCR bias hampers quantitative analyses, as required in studies of trophic interactions. Direct DNA shotgun sequencing can avoid the amplification step when unassembled reads are mapped to a reference database, enabling identification and quantification of prey items.Two feeding bioassays tested the precision and accuracy of quantitative assessments with the coccinellids Harmonia axyridis and Hippodamia convergens, the chrysopid Chrysoperla externa, and the aphid Myzus persicae. Guts were dissected and the total DNA extracted was directly sent to sequence by Illumina HiSeq2500 (insert 350 bp, PE 250). Predator gut content reads were blasted against an arthropod mitochondrial DNA reference database for taxonomic assignment and the matches curated for false positive prey identification using a series of bioinformatics pipelines, mostly in R. Taxonomic assignment through KrakenUnique, which is based on the counts of unique k-mers, was compared.In a Prey Quantity bioassay, the number of prey reads was correlated to the amount of prey consumed and the elapsed time since consumption. In a Direct and Indirect Predation bioassay, prey was detectable in the predator 6 h after feeding (direct predation) and the prey’s prior prey was detectable 3 h after feeding (indirect predation). Detection of indirect predation was related to the species-specific decay rates of the two predators, but not to the order of predation.We demonstrate that degraded prey DNA was quantifiable across trophic levels with high accuracy (98.4% positive predictive value) and taxonomic resolution.

In recent years, soluble amyloid beta oligomers (AßO) have emerged as key elements in the cascade leading to synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). Previous work from our lab has shown that synaptic zinc released during excitatory neurotransmission increases the formation and accumulation of AßO at synaptic sites, and that the AßO-zinc interaction accelerates oligomer formation. Other recent research has shown that sequestration of synaptic zinc by AßO disrupts synaptic function, a significant finding as zinc has been demonstrated to modulate seizure activity and signaling pathways, has a high affinity for AßO and accumulates in Aß plaques in AD brain. We sought to investigate markers of seizure activity in zinc transporter ZnT3 knockout (ZnT3KO) mice, which lack synaptic zinc, and the effects of treatment with an anti-seizure drug on the cognitive impairment demonstrated by aged ZnT3KO mice. Hippocampus tissue collected from age-matched cohorts of wild type and ZnT3KO were assayed for markers of seizure activity, finding age-dependent alterations in levels of these markers consistent with seizure activity in ZnT3KO. To study the effects of seizure activity on cognition, memory was assessed after acute treatment with an anti-seizure drug, finding no significant improvement in six month old ZnT3KO.We also investigated alterations in neurotrophic signaling pathways in acute hippocampal slices from ZnT3KO mice. Neurotrophic protein expression and phosphorylation were assessed, finding reduction in basal and activity-dependent levels of Erk1/2 and p-Erk1/2, reduction in AKT, age-dependent reduction in BDNF, and reduction in activity-dependent BDNF mRNA expression. Pharmacological treatments of acute hippocampus sections were performed to investigate the effects of Zn2+ on activation of Erk1/2 through NMDA receptors and receptor subunits NR2A and NR2B, finding that Zn2+ inhibits NR2B-mediated activation of Erk1/2. Neurodegeneration was assessed through histochemistry of age-matched hippocampus sections, demonstrating an increase in neurodegeneration in aged ZnT3KO.Collectively, the results discussed in this dissertation support the hypothesis that dysregulation of synaptic zinc results in excessive excitatory neuronal activity and neurodegenerative alterations in signaling pathways. Consequently, therapeutics targeting maintenance of zinc homeostasis and reduction of AßO interference with zinc neurotransmission may prove beneficial in managing neuronal hyperactivity and neurodegenerative changes in AD.

For organisms to survive and prosper in a harsh environment, particularly under rapid climate change, poses tremendous challenges. Recent studies have highlighted the continued loss of megafauna in terrestrial ecosystems and the subsequent surge of small mammals, such as rodents, bats, lagomorphs, and insectivores. However, the ecological partitioning of these animals will likely lead to large variation in their responses to environmental change. In the present study, we investigated the evolutionary history and genetic adaptations of white-bellied rats (Niviventer Marshall, 1976), which are widespread in the natural terrestrial ecosystems in Asia but also known as important zoonotic pathogen vectors and transmitters. The southeastern Qinghai-Tibet Plateau was inferred as the origin center of this genus, with parallel diversification in temperate and tropical niches. Demographic history analyses from mitochondrial and nuclear sequences of Niviventer demonstrated population size increases and range expansion for species in Southeast Asia, and habitat generalists elsewhere. Unexpectedly, population increases were seen in N. eha, which inhabits the highest elevation among Niviventer species. Genome scans of nuclear exons revealed that among the congeneric species, N. eha has the largest number of positively selected genes. Protein functions of these genes are mainly related to olfaction, taste, and tumor suppression. Extensive genetic modification presents a major strategy in response to global changes in these alpine species.

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully account for the complex nature of concomitant sequences and the highly variable DNA contribution of specimens in a metabarcoding sample. We propose, as a complement to denoising, the metabarcoding Multidimensional Abundance Threshold Evaluation (metaMATE) framework, a novel approach that allows comprehensive examination of multiple dimensions of abundance filtering and the evaluation of the prevalence of unwanted concomitant sequences in denoised metabarcoding datasets. metaMATE requires a denoised set of ASVs as input, and designates a subset of ASVs as being either authentic (mitochondrial DNA haplotypes) or nonauthentic ASVs (NUMTs and erroneous sequences) by comparison to external reference data and by analysing nucleotide substitution patterns. metaMATE (i) facilitates the application of read abundance filtering strategies, which are structured with regard to sequence library and phylogeny and applied for a range of increasing abundance threshold values, and (ii) evaluates their performance by quantifying the prevalence of nonauthentic ASVs and the collateral effects on the removal of authentic ASVs. The output from metaMATE facilitates decision-making about required filtering stringency and can be used to improve the reliability of intraspecific genetic information derived from metabarcode data. The framework is implemented in the metaMATE software (available at )., C. A. was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) (CGL2015‐74178‐JIN) and Fundación CajaCanarias/Obra social “la Caixa.” B. C. E. was supported by the project CGL2017‐85718‐P (AEI, Spain/FEDER, EU). P. A., T. J. C., B. C. E. and A. P. V. were supported by the iBioGen project funded by the H2020 European Research Council, Grant/Award Number: 810729. We extend our gratitude to the regional governments of Andalucía and Canarias (Spain) for facilitating collecting of samples, to Jesús Arribas for assistance with field sampling, and Carlos Martínez for the mathematical advice.

Soil arthropod communities are highly diverse and critical for ecosystem functioning. However, our knowledge of spatial structure and the underlying processes of community assembly are scarce, hampered by limited empirical data on species diversity and turnover. We implement a high‐throughput sequencing approach to generate comparative data for thousands of arthropods at three hierarchical levels: genetic, species and supra‐specific lineages. A joint analysis of the spatial arrangement across these levels can reveal the predominant processes driving the variation in biological assemblages at the local scale. This multihierarchical approach was performed using haplotype‐level COI metabarcoding of entire communities of mites, springtails and beetles from three Iberian mountain regions. Tens of thousands of specimens were extracted from deep and superficial soil layers and produced comparative phylogeographic data for >1,000 codistributed species and nearly 3,000 haplotypes. Local assemblage composition differed greatly between grasslands and forests and, within each habitat, showed strong spatial structure and high endemicity. Distance decay was high at all levels, even at the scale of a few kilometres or less. The local distance decay patterns were self‐similar for the haplotypes and higher hierarchical entities, and this fractal structure was similar in all regions, suggesting that uniform processes of limited dispersal determine local‐scale community assembly. Our results from whole‐community metabarcoding provide insight into how dispersal limitations constrain mesofauna community structure within local spatial settings over evolutionary timescales. If generalized across wider areas, the high turnover and endemicity in the soil locally may indicate extremely high richness globally, challenging our current estimations of total arthropod diversity on Earth., This research was funded by Newton International Program, UK, to PA and the NHM Biodiversity Initiative to APV. PA was supported by postdoctoral grants from the Royal Society (Newton International Program, UK) and the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) within the Juan de la Cierva Formación Program. CA was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Spain; CGL2015‐74178‐JIN MINECO/FEDER, UE).

Host specificity of parasitoids may be measured by various specialization indices to assess the variation of interaction strength among species and the structure of the wider interaction network. However, the conclusions from analyses at the species and network levels may differ, which remains poorly explored. In addition, the recovery of cryptic species of hosts and parasitoids with molecular data may affect the structure of inferred interaction links. We quantified host specificity of hymenopteran parasitoids (family Encyrtidae) on armored scale insects (Hemiptera: Diaspididae) from a wide geographic sampling range across the Chinese Mainland based on both morphological and molecular species delimitation. Mitochondrial COI and nuclear 28S markers detected high cryptic species diversity in the encyrtids and to a lesser degree in the diaspidids, which divided generalist morphospecies into complexes of specialists and generalists. One‐to‐one reciprocal host–parasite links were increased in the molecular data set, but different quantitative species‐level indices produced contrasting estimates of specificity from various one‐to‐multiple and multiple‐to‐multiple host–parasite links. Network indices calculated from DNA‐based species, compared to morphology‐based species definitions, showed lower connectance and generality, but greater specialization and compartmentalization of the interaction network. We conclude that a high degree of cryptic species in host–parasitoid systems refines the true network structure and may cause us overestimating the stability of these interaction webs.

Rodents are the major remaining mammals in many terrestrial ecosystems after the historical loss of megafauna and large-bodied taxa. Niviventer confucianus is a dominant habitat generalist in natural forests in most of China. It is also recorded as an important vector of diverse zoonotic diseases. Here, three mitochondrial and one nuclear DNA fragments were sequenced from samples covering most of the species range to study intraspecific genetic diversification and demographic history. Molecular voucher specimens of N. confucianus revealed that its assumed distribution range has been overestimated because of the hitherto unrecognized separation from parapatric species. Phylogenetic inferences recognized three geographically delimited intraspecific lineages that diverged at approximately 1.28 and 0.68 Mya. Hengduan Mountains, the east margin of Qinghai Tibetan Plateau, and the mountains surrounding Sichuan Basin were recognized as the major geographical barriers. Demographic analysis revealed dramatic population growth in southwest, central, and northern China in the late Pleistocene, but only slight growth in Yunnan/Tibet. The population boom apparently coincided with the reduction of predation and competition from the loss of megafauna in the late Pleistocene. Distributional ranges were inferred to be fairly stable through the late Quaternary glacial-interglacial climatic oscillations, possibly enabled by the species’ seed hoarding behavior and wide climatic tolerance. The demographic history of N. confucianus suggests that these rodents directly profited from the loss of megafauna, while their most recent increases potentially led to the proliferation of zoonotic disease by this species.

A phylogenetic tree at the species level is still far off for highly diverse insect orders, including the Coleoptera, but the taxonomic breadth of public sequence databases is growing. In addition, new types of data may contribute to increasing taxon coverage, such as metagenomic shotgun sequencing for assembly of mitogenomes from bulk specimen samples. The current study explores the application of these techniques for large-scale efforts to build the tree of Coleoptera. We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of >3000 bp. These sequences were combined into a single dataset together with all mitochondrial data available at GenBank, in addition to nuclear markers widely used in molecular phylogenetics. The resulting matrix of nearly 16,000 species with two or more loci produced trees (RAxML) showing overall congruence with the Linnaean taxonomy at hierarchical levels from suborders to genera. We tested the role of full-length mitogenomes in stabilizing the tree from GenBank data, as mitogenomes might link terminals with non-overlapping gene representation. However, the mitogenome data were only partly useful in this respect, presumably because of the purely automated approach to assembly and gene delimitation, but improvements in future may be possible by using multiple assemblers and manual curation. In conclusion, the combination of data mining and metagenomic sequencing of bulk samples provided the largest phylogenetic tree of Coleoptera to date, which represents a summary of existing phylogenetic knowledge and a defensible tree of great utility, in particular for studies at the intra-familial level, despite some shortcomings for resolving basal nodes., This work was funded by the Biodiversity Initiative of the NHM and NERC grant NE/M021955. PA received funding through a Newton Fellowship of the Royal Society.

Plant invasions often act as ecosystem 'simplifiers' to simplify diversity and community structure of soil biota. However, inconsistent relationships between plant invasion and soil fauna have been found and few studies have addressed how soil fauna communities change upon plant invasions across taxa and feeding guilds. Here, we investigated the effects of moso bamboo (Phyllostachys edulis) invasion in subtropical China on soil mesofauna communities using novel high-throughput sequencing (HTS). Specifically, we analyzed the spatio-temporal dynamics of fauna diversity and feeding guilds in the litter and soil layers for three stages of moso bamboo invasion, i.e., uninvaded (secondary broadleaved forest), moderately invaded (mixed bamboo forest) and completely invaded (P. edulis forest). Overall, we found that the completely invaded bamboo forest decreased species richness and diversity of total fauna, herbivores, and microbivores consistently across different soil layers, but less so detritivores and predators. Although we did not find any interaction effects of bamboo invasion and soil layers on soil fauna diversity indices, significant interaction effects were found on the community composition, for total fauna and their feeding guilds. Specifically, the detrimental effects of bamboo invasion on the trophic structure of soil fauna communities were more profound in the litter layer than in the soil layer, suggesting that a litter layer with more diverse taxa does not mean higher resistance to plant invasion in maintaining the soil food web structure. Taken together, our findings suggest that different responses within fauna feeding guilds to plant invasion were pervasive, and a deeper soil layer may better alleviate the negative effects of pant invasion on fauna community structure. These shifts in soil biodiversity may further degrade ecosystem functioning.

The beetle suborder Adephaga consists of several aquatic ('Hydradephaga') and terrestrial ('Geadephaga') families whose relationships remain poorly known. In particular, the position of Cicindelidae (tiger beetles) appears problematic, as recent studies have found them either within the Hydradephaga based on mitogenomes, or together with several unlikely relatives in Geadephaga based on 18S rRNA genes. We newly sequenced nine mitogenomes of representatives of Cicindelidae and three ground beetles (Carabidae), and conducted phylogenetic analyses together with 29 existing mitogenomes of Adephaga. Our results support a basal split of Geadephaga and Hydradephaga, and reveal Cicindelidae, together with Trachypachidae, as sister to all other Geadephaga, supporting their status as Family. We show that alternative arrangements of basal adephagan relationships coincide with increased rates of evolutionary change and with nucleotide compositional bias, but these confounding factors were overcome by the CAT-Poisson model of PhyloBayes. The mitogenome + 18S rRNA combined matrix supports the same topology only after removal of the hypervariable expansion segments. Densely sampled mitogenomes, analyzed with site heterogeneous mixture models, support a plausible hypothesis of basal relationships in the Adephaga.

We present a mtDNA gene tree of tarantula spiders (Araneae: Mygalomorphae: Theraphosidae) based on the mitochondrial 16S-tRNA (leu)-ND1 gene region as a promising initial molecular hypothesis to clarify the taxonomy of the largest subfamily, Theraphosinae. Many species of this New World subfamily are traded widely as exotic pets, yet few scientific studies on them exist, and the robustness of many supposed taxonomic groupings is debatable. Yet the validity of taxon names and knowledge of their distinctiveness is vital for trade regulation, most notably for the Neotropical genus Brachypelma Simon 1891, which is listed under CITES (Appendix II, see online supplemental material, which is available from the article's Taylor & Francis Online page at https://doi.org/10.1080/14772000.2017.1346719). The use of molecular markers for tarantula taxonomy has been limited until recently, with most previous studies relying on morphological methods. Our findings, from newly collected molecular data, have several nomencl...

High-throughput DNA methods hold great promise for phylogenetic analysis of lineages that are difficult to study with conventional molecular and morphological approaches. The mites (Acari), and in particular the highly diverse soil-dwelling lineages, are among the least known branches of the metazoan Tree-of-Life. We extracted numerous minute mites from soils in an area of mixed forest and grassland in southern Iberia. Selected specimens representing the full morphological diversity were shotgun sequenced in bulk, followed by genome assembly of short reads from the mixture, which produced >100 mitochondrial genomes representing diverse acarine lineages. Phylogenetic analyses in combination with taxonomically limited mitogenomes available publicly resulted in plausible trees defining basal relationships of the Acari. Several critical nodes were supported by ancestral-state reconstructions of mitochondrial gene rearrangements. Molecular calibration placed the minimum age for the common ancestor of the superorder Acariformes, which includes most soil-dwelling mites, to the Cambrian–Ordovician (likely within 455–552 Ma), whereas the origin of the superorder Parasitiformes was placed later in the Carboniferous-Permian. Most family-level taxa within the Acariformes were dated to the Jurassic and Triassic. The ancient origin of Acariformes and the early diversification of major extant lineages linked to the soil are consistent with a pioneering role for mites in building the earliest terrestrial ecosystems., This research was funded by the NHM Biodiversity Initiative and the CGL2015-66192-R and CGL2015-74178-JIN projects. PA was supported by the Royal Society UK (Newton International Program) and the Spanish Ministry of Economy and Competitiveness (Juan de la Cierva Formación); BL by the NHM Biodiversity Initiative and French Labex Agro-CeMEB-NUMEV

High-throughput DNA methods hold great promise for the study of the hyperdiverse arthropod fauna of the soil. We used the mitochondrial metagenomic approach to generate 39 mitochondrial genomes from adult and larval specimens of Coleoptera collected from soil samples. The mitogenomes correspond to species from the families Carabidae (6), Chrysomelidae (1), Curculionidae (9), Dermestidae (1), Elateridae (1), Latridiidae (1), Scarabaeidae (3), Silvanidae (1), Staphylinidae (12), and Tenebrionidae (4). All the mitogenomes followed the putative ancestral gene order for Coleoptera. We provide the first available mitogenome for 30 genera of Coleoptera, including endogean representatives of the genera Torneuma, Coiffaitiella, Otiorhynchus, Oligotyphlopsis, and Typhlocharis., The study was funded by the NHM Biodiversity Initiative (UK) and the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad) under a project with the number CGL2015-74178-JIN.

Improved taxonomic methods are needed to quantify declining populations of insect pollinators. This study devises a high-throughput DNA barcoding protocol for a regional fauna (United Kingdom) of bees (Apiformes), consisting of reference library construction, a proof-of-concept monitoring scheme, and the deep barcoding of individuals to assess potential artefacts and organismal associations. A reference database of Cytochrome Oxidase subunit 1 (cox1) sequences including 92.4% of 278 bee species known from the UK showed high congruence with morphological taxon concepts, but molecular species delimitations resulted in numerous split and (fewer) lumped entities within the Linnaean species. Double tagging permitted deep illumina sequencing of 762 separate individuals of bees from a UK-wide survey. Extracting the target barcode from the amplicon mix required a new protocol employing read abundance and phylogenetic position, which revealed 180 molecular entities of Apiformes identifiable to species. An additional 72 entities were ascribed to mitochondrial pseudogenes based on patterns of read abundance and phylogenetic relatedness to the reference set. Clustering of reads revealed a range of secondary Operational Taxonomic Units (OTUs) in almost all samples, resulting from traces of insect species caught in the same traps, organisms associated with the insects including a known mite parasite of bees, and the common detection of human DNA, besides evidence for low-level cross-contamination in pan traps and laboratory steps. Custom scripts were generated to conduct critical steps of the bioinformatics protocol. The resources built here will greatly aid DNA-based monitoring to inform management and conservation policies for the protection of pollinators.

Several species of swallowtail butterflies (genus Papilio) are Batesian mimics that express multiple mimetic female forms, while the males are monomorphic and nonmimetic. The evolution of such sex-limited mimicry may involve sexual dimorphism arising first and mimicry subsequently. Such a stepwise scenario through a nonmimetic, sexually dimorphic stage has been proposed for two closely related sexually dimorphic species: Papilio phorcas, a nonmimetic species with two female forms, and Papilio dardanus, a female-limited polymorphic mimetic species. Their close relationship indicates that female-limited polymorphism could be a shared derived character of the two species. Here, we present a phylogenomic analysis of the dardanus group using 3964 nuclear loci and whole mitochondrial genomes, showing that they are not sister species and thus that the sexually dimorphic state has arisen independently in the two species. Nonhomology of the female polymorphism in both species is supported by population genetic analysis of engrailed, the presumed mimicry switch locus in P. dardanus. McDonald-Kreitman tests performed on SNPs in engrailed showed the signature of balancing selection in a polymorphic population of P. dardanus, but not in monomorphic populations, nor in the nonmimetic P. phorcas. Hence, the wing polymorphism does not balance polymorphisms in engrailed in P. phorcas. Equally, unlike in P. dardanus, none of the SNPs in P. phorcas engrailed were associated with either female morph. We conclude that sexual dimorphism due to female polymorphism evolved independently in both species from monomorphic, nonmimetic states. While sexual selection may drive male-female dimorphism in nonmimetic species, in mimetic Papilios, natural selection for protection from predators in females is an alternative route to sexual dimorphism.

Phylogenetic relationships of dung beetles in the tribe Onthophagini, including the species-rich, cosmopolitan genus Onthophagus, were inferred using whole mitochondrial genomes. Data were generated by shotgun sequencing of mixed genomic DNA from >100 individuals on 50% of an Illumina MiSeq flow cell. Genome assembly of the mixed reads produced contigs of 74 (nearly) complete mitogenomes. The final dataset included representatives of Onthophagus from all biogeographic regions, closely related genera of Onthophagini, and the related tribes Onitini and Oniticellini. The analysis defined four major clades of Onthophagini, which was paraphyletic for Oniticellini, with Onitini as sister group to all others. Several (sub)genera considered as members of Onthophagus in the older literature formed separate deep lineages. All New World species of Onthophagus formed a monophyletic group, and the Australian taxa are confined to a single or two closely related clades, one of which forms the sister group of the New World species. Dating the tree by constraining the basal splits with existing calibrations of Scarabaeoidea suggests an origin of Onthophagini sensu lato in the Eocene and a rapid spread from an African ancestral stock into the Oriental region, and secondarily to Australia and the Americas at about 20-24 Mya. The successful assembly of mitogenomes and the well-supported tree obtained from these sequences demonstrates the power of shotgun sequencing from total genomic DNA of species pools as an efficient tool in genus-level phylogenetics.

Hymenopterans - one of the 'big four' of insect orders - includes the sawflies, wasps, ants and bees. Two phylogenomic studies now resolve hymenopteran phylogeny and provide scenarios for the evolution of major lineages and key lifestyle transitions.

Polymorphic Batesian mimics exhibit multiple protective morphs that each mimic a different noxious model. Here, we study the genomic transitions leading to the evolution of different mimetic wing patterns in the polymorphic Mocker Swallowtail Papilio dardanus. We generated a draft genome (231 Mb over 30 chromosomes) and re-sequenced individuals of three morphs. Genome-wide single nucleotide polymorphism (SNP) analysis revealed elevated linkage disequilibrium and divergence between morphs in the regulatory region of engrailed, a developmental gene previously implicated in the mimicry switch . The diverged region exhibits a discrete chromosomal inversion (of 40 kb) relative to the ancestral orientation that is associated with the cenea morph, but not with the bottom-recessive hippocoonides morph or with non-mimetic allopatric populations. The functional role of this inversion in the expression of the novel phenotype is currently unknown, but by preventing recombination, it allows the stable inheritance of divergent alleles enabling geographic spread and local coexistence of multiple adaptive morphs.

Plastoceridae Crowson, 1972, Drilidae Blanchard, 1845 and Omalisidae Lacordaire, 1857 (Elateroidea) are families of the Coleoptera with obscure phylogenetic relationships and modified morphology showing neotenic traits such as soft bodies, reduced wing cases and larviform females. We shotgun sequenced genomes of Plastocerus, Drilus and Omalisus and incorporated them into data matrices of 66 and 4202 single-copy nuclear genes representing Elateroidea. Phylogenetic analyses indicate their terminal positions within the broadly defined well-sclerotized and fully metamorphosed Elateridae and thus Omalisidae should now be considered as Omalisinae stat. nov. in Elateridae Leach, 1815. The results support multiple independent origins of incomplete metamorphosis in Elateridae and indicate the parallel evolution of morphological and ecological traits. Unlike other neotenic elateroids derived from the supposedly pre-adapted aposematically coloured and unpalatable soft-bodied elateroids, such as fireflies (Lampyridae) and net-winged beetles (Lycidae), omalisids and drilids evolved from well-sclerotized click beetles. These findings suggest sudden morphological shifts through incomplete metamorphosis, with important implications for macroevolution, including reduced speciation rate and high extinction risk in unstable habitats. Precise phylogenetic placement is necessary for studies of the molecular mechanisms of ontogenetic shifts leading to profoundly changed morphology.

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., Ministerio de Economía y Competitividad. Grant Number: CGL2015‐74178‐JIN.

The insect order Hymenoptera presents marvelous morphological and ecological diversity. Higher-level hymenopteran relationships remain controversial, even after recent phylogenomic analyses, as their taxon sampling was limited. To shed light on the origin and diversification of Hymenoptera, in particular the poorly studied Parasitica, we undertook phylogenetic analyses of 40 newly and 43 previously sequenced mitochondrial genomes representing all major clades of Hymenoptera. Various Bayesian inferences using different data partitions and phylogenetic methods recovered similar phylogenetic trees with strong statistical support for almost all nodes. Novel findings of the mitogenomic phylogeny mainly affected the three infraorders Ichneumonomorpha, Proctotrupomorpha and Evaniomorpha, the latter of which was split into three clades. Basal relationships of Parasitica recovered Stephanoidea + (Gasteruptiidae + Aulacidae) as the sister group to Ichneumonomorpha + (Trigonalyoidea + Megalyroidea). This entire clade is sister to Proctotrupomorpha, and Ceraphronoidea + Evaniidae is sister to Aculeata (stinging wasps). Our divergence time analysis indicates that major hymenopteran lineages originated in the Mesozoic. The radiation of early apocritans may have been triggered by the Triassic–Jurassic mass extinction; all extant families were present by the Cretaceous.

[Background] Rhinorhipidae Lawrence, 1988 is an enigmatic beetle family represented by a single species, Rhinorhipus tamborinensis Lawrence, 1988, from Australia, with poorly established affinities near the superfamily Elateroidea (click beetles, soldier beetles and fireflies) or the more inclusive series (infraorder) Elateriformia. Its evolutionary position may inform the basal relationships of the suborder Polyphaga, the largest clade of Coleoptera. [Results] We analyzed four densely sampled DNA datasets of major coleopteran lineages for mitogenomes, rRNA genes and single copy nuclear genes. Additionally, genome sequencing was used for incorporation of R. tamborinensis into a set of 4220 orthologs for 24 terminals representing 12 polyphagan superfamilies. Topologies differed to various degrees, but all consistently refute the proposed placement of Rhinorhipidae in Elateroidea and instead indicate either sister relationships with other Elateriformia, frequently together with Nosodendridae, another divergent small family hitherto placed in Derodontoidea, or in an isolated position among the deepest lineages of Polyphaga. The phylogenomic analyses recovered Rhinorhipus in a sister position to all other Elateriformia composed of five superfamilies. Therefore, we erect the new superfamily Rhinorhipoidea Lawrence, 1988, stat. Nov., with the type-family Rhinorhipidae. The origins of the Rhinorhipidae were dated to the Upper Triassic/Lower Jurassic at the very early phase of polyphagan diversification. [Conclusions] Thus, Rhinorhipidae adds another example to several recently recognized ancient relict lineages which are interspersed within contemporaneous hugely species-rich lineages of Coleoptera., The study was funded by the IGA and GACR projects (PrF2018, 18-14942S to L.B., D.K., M.Mo. and Ma.B.), Leverhulme Trust (F/00696/P to A.P.V. and L.B.) and the NHM Biodiversity Initiative.

© 2018 by the authors., Establishing well-supported monophyletic groups is a key requirement for producing a natural classification that reflects evolutionary descent. In a phylogenetic framework this is best achieved through dense taxon sampling and the analysis of a robust character dataset, combined with statistical testing of topological hypotheses. This study assesses the monophyly of tribes and subfamilies within the diverse ‘broad-nosed weevils’ (Curculionidae: Entiminae, Cyclominae and Hyperinae) through analysis of single-locus sequence data for mitochondrial cox1 and rrnL genes, in combination with a ‘backbone’ of complete and near-complete mitochondrial genome sequences. Maximum likelihood phylogenetic analyses incorporating topological constraints for various higher-taxa were statistically tested using the AU, SH, and KH tests, which indicated that three tribes within Entiminae, as presently classified, are not monophyletic. Moderate and high bootstrap support was also consistent with two entimine tribes (Peritelini and Cylydrorhinini) being each recovered as monophyletic in an unconstrained analysis. Furthermore, one genus of cyclomine weevils (Aphela) is recovered outside the clade of ‘broad-nosed weevils’, although its taxonomic placement remains uncertain. It is apparent that the present approach may be hampered by limited taxon sampling in the ‘backbone’ dataset, rendering it difficult for divergent taxa to robustly match to their closest lineages. However, with improved taxon sampling of the mitogenome tree, the general approach can be a useful taxonomic tool for weevils., The authors are indebted to Karen Siu-Ting (NUI Maynooth) for her assistance with some analyses. This work was supported by a Natural Environment Research Council CASE PhD studentship (NE/G01194X/1) to C.P.D.T.G., the University of East Anglia, and The Natural History Museum., Peerreviewed

With efficient sequencing techniques, full mitochondrial genomes are rapidly replacing other widely used markers, such as the nuclear rRNA genes, for phylogenetic analysis but their power to resolve deep levels of the tree remains controversial. We studied phylogenetic relationships of leaf beetles (Chrysomelidae) in the tribes Galerucini and Alticini (root worms and flea beetles) based on full mitochondrial genomes (103 newly sequenced), and compared their performance to the widely sequenced nuclear rRNA genes (full 18S, partial 28S). Our results show that: (i) the mitogenome is phylogenetically informative from subtribe to family level, and the per-nucleotide contribution to nodal support is higher than that of rRNA genes, (ii) the Galerucini and Alticini are reciprocally monophyletic sister groups, if the classification is adjusted to accommodate several ‘problematic genera’ that do not fit the dichotomy of lineages based on the presence (Alticini) or absence (Galerucini) of the jumping apparatus, and (iii) the phylogenetic results suggest a new classification system of Galerucini with eight subtribes: Oidina, Galerucina, Hylaspina, Metacyclina, Luperina, Aulacophorina, Diabroticina and Monoleptina.

The evolution of animal life strategies is among the main themes of current evolutionary biology. Checkered beetles, soft-winged flower beetles and their allies (superfamily Cleroidea), exhibit well-known aposematic colour patterns, particularly in the family Cleridae, which participate in mimicry complexes mostly with unpalatable beetles, ants and velvet ants representing a Mullerian–Batesian continuum. Many cleroids also exhibit attenuated hardening of cuticular layers resulting in a soft-bodied appearance. Here, a molecular phylogenetic analysis of the entire Cleroidea was performed using sequences of two nuclear and two mitochondrial loci of ~4 kb total length. Inferred phylogenies were used to reconstruct ancestral colour patterns and involvement in mimicry complexes. The hypothesis of a soft-bodied ancestor of Cleridae and allies was tested. The phylogenetic analyses corroborated the expanded Cleroidea concept including Byturidae and Biphyllidae formerly classified as Cucujoidea. Character state optimization showed cryptic coloration was the ancestral state in Cleroidea, from which aposematic coloration originated several times in distant cleroid lineages. Within Cleridae, mimicry also arose from an ancestor that was cryptic, and multiple lineages that mimicked unpalatable beetles (Chrysomelidae, Meloidae, Lycidae) and stinging Hymenoptera evolved. Aposematic coloration was acquired in all major clerid lineages including Thanerocleridae, which are either the sister group of Chaetosomatidae or Cleridae. These findings suggest that mimetic traits in the clerid clade evolved at various times, possibly soon after the origin of soft-bodiedness. The adaptive value of aposematism in cleroids is likely to be enhanced in soft-bodied species, as this trait provides limited means of protection against predators, and therefore may promote the acquisition of aposematic and mimetic coloration in various ecological situations.

Aim Neotropical highland streams have shown diminished ecosystem functioning after amphibian extirpation infected by the chytrid fungus Batrachochytrium dendrobatidis. The loss of amphibians could affect communities of aquatic insects co-occurring in these streams in various ways. We examined patterns of species and genetic diversity of these communities and their evolutionary history along the chytrid expansion gradient to elucidate potential community responses. Location Six streams over a 320-km transect in Panama affected by chytrid expansion from west to east for up to 14 years, and two apparently chytrid-free streams in the east. Methods Patterns of α- and β-diversity were investigated at three hierarchical levels: genus, species and haplotypes. Genus identification was based on morphology, and putative species were inferred by grouping the DNA barcodes (749 cox1 sequences) with the GMYC method on all collected individuals of Ephemeroptera, Trichoptera, Coleoptera and Plecoptera. Results A total of 96 genera in 43 families (9 orders) of insects were encountered. Genus-level α-diversity was higher in the easternmost streams, possibly due to a separate biogeographical history, whereas β-diversity was constant along the chytrid expansion gradient. Community DNA barcoding resulted in 426 cox1 haplotypes and 154 putative species, most of them limited to single sites. High β-diversity along the gradient at both species and haplotype levels argues against community homogenization by migration in the wake of amphibian declines. In contrast, phylo-β-diversity was low, indicating community similarity at deep levels. Main conclusions Aquatic insect communities in this region are influenced by long-term limited dispersion that generated high endemicity. The pattern persists mostly unperturbed after disease-driven amphibian declines; hence, if indeed insects fill the niches vacated by tadpoles, they would originate from local communities rather than immigration. Given the unique evolutionary history and physical isolation of local assemblages, the ecosystem deterioration carries the risk of losing unique diversity.

Polymerase chain reaction approaches for DNA sequencing from dried collection specimens of insects usually suffer from contamination, short fragment length, and limited phylogenetic power. As an alternative, we use shotgun metagenomic sequencing from mixed pools of DNA, from which mitochondrial sequences are filtered in silico. We extracted DNA from a single leg of 35 species of British butterflies that had been stored in the Natural History Museum for between 9 and 35 years. Illumina MiSeq (1/3 of flow cell) produced mitogenome sequences of > 10 kb for ten species, of which six were (almost) complete. In addition, full or partial barcode sequences for 31 species were recovered, which, in almost all cases, showed perfect (100%) identity to sequences available in the BOLD database under the same taxon name. The mean length of sequence reads was 167 bp (putative mitochondrial reads) and 153 bp (all reads). Assembly of contigs > 10 kb from these short reads was apparently error free, except in the case of two closely-related species of Pieris that formed chimeras. Contiguous sequences are difficult to obtain from degraded DNA with Sanger technology but the Illumina short reads are a natural fit for dry-collection specimens. The obvious possibility for scaling up this protocol provides exciting prospects for the large-scale indexing of natural history collections using partial or full mitochondrial genomes.

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.

Aim Large-scale DNA barcoding allows the simultaneous assessment of variation in community composition at species level and below. We here propose that the patterns emerging across multiple hierarchical levels can be used to discern the effects of neutral and non-neutral macroecological processes, which otherwise have proven difficult to separate. Location Iberian Peninsula. Methods We performed cox1 barcoding on 20 complete assemblages of leaf beetles for 4533 individuals of 203 species. The neutrality of cox1 sequence evolution was tested using Tajima's test. Haplotypes (n = 2020) were grouped into nested n-step networks of up to five intraspecific hierarchical levels. We then assessed whether the spatial variation in assemblage composition at all hierarchical levels from haplotype to species was self-similar (fractal) and predictable from level to level. Results Tajima's test on a subset of widely sampled species (n = 136) was consistent with neutral evolution in 83% of the species, but only 3% of cases exhibited balancing selection. Multiple hierarchical levels representing haplotype genealogies of various ages showed a similar rate of distance decay of assemblage similarity. In addition, we found strong log-log correlations between hierarchical level (lineage age) and number of lineages, lineage range size and assemblage similarity. Similarity at the species level was strongly correlated to similarity at the haplotype level for the whole assemblage (r2 = 0.75) or for within-species haplotype similarity (mean r2 = 0.17, SE = 0.03). Main conclusions These findings suggest great regularities in the pattern of assemblage variation at all lineage ages that are best explained by the enduring action of stochastic (neutral) processes of mutation and dispersal. The multi-hierarchical analysis therefore bridges predictions of the neutral theory of molecular evolution and the neutral theory of biodiversity. Neutral processes thus emerge as a unifying principle of ecology and evolution, which has deep implications in biodiversity assessment and conservation.

The biodiversity of mixed-species samples of arthropods can be characterized by shotgun sequencing of bulk genomic DNA and subsequent bioinformatics assembly of mitochondrial genomes. Here, we tested the power of mitochondrial metagenomics by conducting Illumina sequencing on mixtures of >2600 individuals of leaf beetles (Chrysomelidae) from 10 communities. Patterns of species richness, community dissimilarity and biomass were assessed from matches of reads against three reference databases, including (i) a custom set of mitogenomes generated for 156 species (89% of species in the study); (ii) mitogenomes obtained by the de novo assembly of sequence reads from the real-world communities; and (iii) a custom set of DNA barcode (cox1-5') sequences. Species detection against the custom-built reference genomes was very high (>90%). False presences were rare against mitogenomes but slightly higher against the barcode references. False absences were mainly due to the incompleteness of the reference databases and, thus, more prevalent in the de novo data set. Biomass (abundance × body length) and read numbers were strongly correlated, demonstrating the potential of mitochondrial metagenomics for studies of species abundance. A phylogenetic tree from the mitogenomes showed high congruence with known relationships in Chrysomelidae. Patterns of taxonomic and phylogenetic dissimilarity between sites were highly consistent with data from morphological identifications. The power of mitochondrial metagenomics results from the possibility of rapid assembly of mitogenomes from mixtures of specimens and the use of read counts for accurate estimates of key parameters of biodiversity directly from community samples.

Tiger beetles are an important insect group in Sri Lanka due to their high species richness and endemicity. The present study investigated the distribution of tiger beetle species populations in Sri Lanka and history of species populations. Distribution was studied by conducting extensive field work that recorded the locations, habitat types and habitat preferences of tiger beetle species. DNA sequence data of three mitochondrial genes were used to study the history of species populations. The genetic indices: haplotype diversity, nucleotide diversity and Tajima’s D statistic were computed using DNA sequence data. The study revealed the distribution of ten tiger beetle species of Sri Lanka and their habitat types. According to genetic indices the tiger beetles of Sri Lanka are single species populations that consist of subpopulations distributed in locations that are not separated by major geographical barriers. There is a high probability that all species are experiencing population expansion after either habitat displacement by other tiger beetle species, expansion of suitable new habitats and natural disasters.Ceylon Journal of Science (Bio. Sci.) 43 (2): 47-63, 2014

International audience; DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR-based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross-reactions with the predator and related species genomes. Here, we use PCR-free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding , multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h À1 respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h À1 , respectively). However, the facultative aphid symbionts Hamil-tonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1–2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.

Dispersal is a critical factor determining the spatial scale of speciation, which is constrained by the ecological characteristics and distribution of a species’ habitat and the intrinsic traits of species. Endogean taxa are strongly affected by the unique qualities of the below‐ground environment and its effect on dispersal, and contrasting reports indicate either high dispersal capabilities favoured by small body size and mediated by passive mechanisms, or low dispersal due to restricted movement and confinement inside the soil. We studied a species‐rich endogean ground beetle lineage, Typhlocharina, including three genera and more than 60 species, as a model for the evolutionary biology of dispersal and speciation in the deep soil. A time‐calibrated molecular phylogeny generated from >400 individuals was used to delimit candidate species, to study the accumulation of lineages through space and time by species–area–age relationships and to determine the geographical structure of the diversification using the relationship between phylogenetic and geographic distances across the phylogeny. Our results indicated a small spatial scale of speciation in Typhlocharina and low dispersal capacity combined with sporadic long distance, presumably passive dispersal events that fuelled the speciation process. Analysis of lineage growth within Typhlocharina revealed a richness plateau correlated with the range of distribution of lineages, suggesting a long‐term species richness equilibrium mediated by density dependence through limits of habitat availability. The interplay of area‐ and age‐dependent processes ruling the lineage diversification in Typhlocharina may serve as a general model for the evolution of high species diversity in endogean mesofauna., This study was partially supported by the NERC Grant NE/M021955. CA was partly founded by The European Commission (MSCA‐IF‐2015‐705639) and The Spanish “Ministerio de Economía y Competitividad” (CGL2010‐16944) during the development of this study.

Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels. This article is protected by copyright. All rights reserved.

Whilst cartographers of the 19th century endeavoured to chart the last unknown lands, the great challenge for biologists in the 21st century is to fill the gaps on the biodiversity map of the Earth. And one of the largest gaps concerns the biodiversity of soils, a terra incognita right under our feet. The study of soil biodiversity, and particularly the complex communities of small invertebrates, has suffered from a severe ‘taxonomic impediment’ (Decaëns 2010) leading to great uncertainties about total species richness, phylogenetic diversity, geographical structure, temporal dynamics of soil organisms, and consequently about their role on ecosystem function (Bardgett & van der Putten 2014). However, the revolution in high‐throughput sequencing is now revealing the hidden biodiversity of the soil with unprecedented detail (e.g. Arribas et al. 2016). In a noteworthy from the Cover article in this issue of Molecular Ecology, Cicconardi et al. (2017) apply these new tools to study soil communities of Collembola in three distant oceanic islands of volcanic origin, obtaining a striking result: only 38 of 70 species (54%) are exclusively found in a single island, with the remaining shared among islands or with other distant regions, suggesting a massive recent introduction of soil species, whose impact is entirely unknown.

Wild Mediterranean fruit fly specimens collected from various regions worldwide were screened for the glycine to alanine (Gly->Ala) point mutation (G328A) in the acetylcholinesterase enzyme, presumably causing resistance to organophosphates. We found that the single nucleotide polymorphism (SNP) responsible for this amino acid change is located at the beginning of exon 6 of the Ccace2 gene. The identification of the exact location of the SNP permitted PCR primer design around this site and direct sequencing of the corresponding genomic region. We detected the resistance allele in natural Mediterranean fruit fly populations from Brazil and Spain, but not from other sites in four continents. The known treatment history of sites suggests that the resistance buildup is linked to organophosphate application in the field. The PCR-based detection provides a screening method useful for monitoring Mediterranean fruit fly insecticide resistance in local populations and improving pest management strategies accordingly.

Tiger beetles have been observed in many riverine ecosystems of Sri Lanka. However, current locations, species, distribution of species, habitat preferences and possible interactions between species are unknown. The present study intends to investigate these details and provide information that can be used in further studies. Tiger beetles are sampled from 15 riverine locations and examined for identification, body weight and body length. The riverine locations are analysed for locational, climatic and soil parameters and microhabitat details are recorded. Statistical analysis using One-Way Analysis of Variance and Tukey’s pair comparison method of Minitab 16.0 statistical software package is conducted to compare the body sizes of species. Further, a statistical comparison between the climatic and soil parameters of the locations of Cylindera (Ifasina) labioaenea and that of other species are carried out. The study reveals five tiger beetle species Cylindera (Ifasina) labioaenea Horn, Cylindera (Ifasina) willeyi Horn, Cylindera (Ifasina) waterhousei Horn, Calomera cardoni Fleutiaux, Calomera angulata Fabricius, from the riverine ecosystems of Sri Lanka. Cylindera labioaenea is the most common species; C. willeyi and C. waterhousei are endemic to Sri Lanka. Cylindera labioaenea, C. willeyi and C. waterhousei are small, while Calomera cardoni and Calomera angulata are medium in size. Cylindera labioaenea is significantly smaller than C. willeyi and C. waterhousei, and resides in locations with significantly higher air temperatures, solar radiations and significantly lower relative humidity than the other two species. An optimal temperature range for the riverine tiger beetles is suggested and their preferences to soil moisture, soil temperature, soil colour and soil salinity are discussed. The occurrence of C. labioaenea as a single species population while the fact that other species co-exist may be due to a defensive strategy.

Bark and ambrosia beetles (Curculionidae: Scolytinae) are known for their symbioses with fungi and play a key role in the dispersal of phytopathogens. The scolytine community of eight pine stands along a latitudinal gradient in the UK was surveyed and beetle-associated fungal communities (mycobiota) were assessed using ITS2 metabarcoding (304 specimens, 12 species). Distribution patterns among 2,257 detected fungal Operational Taxonomic Units (OTUs) revealed that beetle species identity was an important predictor of mycobiotic richness and composition, while the effects of environmental and spatial variables were negligible. Network-based specificity analysis suggested that a relatively small subset of OTUs (75 in total) exhibit an affinity for a subset of beetle species and that these include many Microascales and Saccharomycetes. Notably though, of the OTUs belonging to the family Ophiostomataceae, relatively few display host specificity. Our results add to the complex picture of host-associated fungal communities and suggest that host range limits are unlikely to restrict the spread of economically important phytopathogens.

The species representation of public databases is growing rapidly and permits increasingly detailed phylogenetic inferences. We present a supermatrix based on all gene sequences of Coleoptera available in Genbank for two nuclear (18S and 28S rRNA) and two mitochondrial (rrnL and cox1) genes. After filtering for unique species names and the addition of ˜2000 unpublished sequences for cox1 and 18S rRNA, the resulting data matrix included 8441 species-level terminals and 6600 aligned nucleotide positions. The concatenated matrix represents the equivalent of 2.17% of the 390 000 described species of Coleoptera and includes 152 beetle families. The remaining 29 families constitute small lineages with ˜250 known species in total. Taxonomic coverage remains low for several major lineages, including Buprestidae (0.16% of described species), Staphylinidae (1.03%), Tenebrionidae (0.90%) and Cerambycidae (0.58%). The current taxon sampling was strongly biased towards the Northern Hemisphere. Phylogenetic trees obtained from the supermatrix were in very good agreement with the Linnaean classification, in particular at the family level, but lower for the subfamily and lowest for the genus level. The topology supports the basal split of Derodontidae and Scirtoidea from the remaining Polyphaga, and the broad paraphyly of Cucujoidea. The data extraction pipeline and detailed tree provide a framework for placement of any new sequences, including environmental samples, into a DNA-based classification system of Coleoptera.

Body size is an important feature of an animal that is linked with its life history, morphology, physiology and ecology. Understanding the association between body size and habitat type of an animal and the underlying causes for this are important for determining their distribution. The present study examines the association between body size and habitat types of tiger beetles in Sri Lanka and the environmental correlations for these associations. Morphometric parameters of tiger beetles and environmental parameters (air temperature, solar radiation, relative humidity, wind speed and, the colour, temperature, moisture content, pH and salinity of soil) in each habitat were recorded. Ten tiger beetle species were found from 37 locations which included coastal, riverine, urban and reservoir habitats. Species with larger body and mandible sizes prefer coastal and reservoir habitats with high wind speed, low soil moisture and high soil pH, whereas species with smaller body and mandible sizes prefer riverine habitats with low wind speed, high soil moisture and low soil pH. Species with smaller body size may also prefer urban habitats which may be due to similarity of environmental conditions that prevails in these two sites. These findings will assist in predicting the distribution of tiger beetles within various habitat types of Sri Lanka.DOI: http://dx.doi.org/10.4038/cjsbs.v42i1.5898 Ceylon Journal of Science (Bio. Sci.) 42 (1): 41-53, 2013