Alex Crampton-Platt

Building the Coleoptera tree-of-life for >8000 species: composition of public DNA data and fit with Linnaean classification

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 After filtering for unique species names and the addition of ˜2000 unpublished sequences for cox1 and 18S rRNA, the resulting data matrix included 8441 species‐level terminals and 6600 aligned nucleotide positions.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.

Bulk De Novo Mitogenome Assembly from Pooled Total DNA Elucidates the Phylogeny of Weevils (Coleoptera: Curculionoidea)

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverse animal groups. However, the relative difficulty and high cost associated with obtaining de novo full mitogenomes have frequently led to conspicuously low taxon sampling in ensuing studies. Here, we report the successful use of an economical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-generation sequencing of a single library made from pooled total DNA extracts of numerous target species. To avoid the use of separate indexed libraries for each specimen, and an associated increase in cost, we incorporate standard polymerase chain reaction-based “bait” sequences to identify the assembled mitogenomes. The method was applied to study the higher level phylogenetic relationships in the weevils (Coleoptera: Curculionoidea), producing 92 newly assembled mitogenomes obtained in a single Illumina MiSeq run. The analysis supported a separate origin of wood-boring behavior by the subfamilies Scolytinae, Platypodinae, and Cossoninae. This finding contradicts morphological hypotheses proposing a close relationship between the first two of these but is congruent with previous molecular studies, reinforcing the utility of mitogenomes in phylogeny reconstruction. Our methodology provides a technically simple procedure for generating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for which bait sequences are the only required prior genome knowledge.

Soup to tree: the phylogeny of beetles inferred by mitochondrial metagenomics of a Bornean rainforest sample

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA “superbarcodes” for testing hypotheses regarding global patterns of diversity.

Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages

Summary 1. 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. 2. 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 0 ) sequences. 3. 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 9 body length) and read numbers were strongly correlated, demonstrating the potential of mitochondrial metagenomics for studies of species abundance. 4. 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. 5. 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.

Phylogenetic community ecology of soil biodiversity using mitochondrial metagenomics

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.

Mitochondrial metagenomics: letting the genes out of the bottle

Abstract‘Mitochondrial metagenomics’ (MMG) is a methodology for shotgun sequencing of total DNA from specimen mixtures and subsequent bioinformatic extraction of mitochondrial sequences. The approach can be applied to phylogenetic analysis of taxonomically selected taxa, as an economical alternative to mitogenome sequencing from individual species, or to environmental samples of mixed specimens, such as from mass trapping of invertebrates. The routine generation of mitochondrial genome sequences has great potential both for systematics and community phylogenetics. Mapping of reads from low-coverage shotgun sequencing of environmental samples also makes it possible to obtain data on spatial and temporal turnover in whole-community phylogenetic and species composition, even in complex ecosystems where species-level taxonomy and biodiversity patterns are poorly known. In addition, read mapping can produce information on species biomass, and potentially allows quantification of within-species genetic variation. The success of MMG relies on the formation of numerous mitochondrial genome contigs, achievable with standard genome assemblers, but various challenges for the efficiency of assembly remain, particularly in the face of variable relative species abundance and intra-specific genetic variation. Nevertheless, several studies have demonstrated the power of mitogenomes from MMG for accurate phylogenetic placement, evolutionary analysis of species traits, biodiversity discovery and the establishment of species distribution patterns; it offers a promising avenue for unifying the ecological and evolutionary understanding of species diversity.

Metagenome skimming of insect specimen pools: potential for comparative genomics

Metagenomic analyses are challenging in metazoans, but high-copy number and repeat regions can be assembled from low-coverage sequencing by “genome skimming,” which is applied here as a new way of characterizing metagenomes obtained in an ecological or taxonomic context. Illumina shotgun sequencing on two pools of Coleoptera (beetles) of approximately 200 species each were assembled into tens of thousands of scaffolds. Repeated low-coverage sequencing recovered similar scaffold sets consistently, although approximately 70% of scaffolds could not be identified against existing genome databases. Identifiable scaffolds included mitochondrial DNA, conserved sequences with hits to expressed sequence tag and protein databases, and known repeat elements of high and low complexity, including numerous copies of rRNA and histone genes. Assemblies of histones captured a diversity of gene order and primary sequence in Coleoptera. Scaffolds with similarity to multiple sites in available coleopteran genome sequences for Dendroctonus and Tribolium revealed high specificity of scaffolds to either of these genomes, in particular for high-copy number repeats. Numerous “clusters” of scaffolds mapped to the same genomic site revealed intra- and/or intergenomic variation within a metagenome pool. In addition to effect of taxonomic composition of the metagenomes, the number of mapped scaffolds also revealed structural differences between the two reference genomes, although the significance of this striking finding remains unclear. Finally, apparently exogenous sequences were recovered, including potential food plants, fungal pathogens, and bacterial symbionts. The “metagenome skimming” approach is useful for capturing the genomic diversity of poorly studied, species-rich lineages and opens new prospects in environmental genomics.

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents.

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks.

Shotgun mitogenomics across body size classes in a local assemblage of tropical Diptera: Phylogeny, species diversity and mitochondrial abundance spectrum

Mitochondrial genomes can be assembled readily from shotgun‐sequenced DNA mixtures of mass‐trapped arthropods (“mitochondrial metagenomics”), speeding up the taxonomic characterization. Bulk sequencing was conducted on some 800 individuals of Diptera obtained by canopy fogging of a single tree in Borneo dominated by small (<1.5 mm) individuals. Specimens were split into five body size classes for DNA extraction, to equalize read numbers across specimens and to study how body size, a key ecological trait, interacts with species and phylogenetic diversity. Genome assembly produced 304 orthologous mitochondrial contigs presumed to each represent a different species. The small‐bodied fraction was the by far most species‐rich (187 contigs). Identification of contigs was through phylogenetic analysis together with 56 reference mitogenomes, which placed most of the Bornean community into seven clades of small‐bodied species, indicating phylogenetic conservation of body size. Mapping of shotgun reads against the mitogenomes showed wide ranges of read abundances within each size class. Ranked read abundance plots were largely log‐linear, indicating a uniformly filled abundance spectrum, especially for small‐bodied species. Small‐bodied species differed greatly from other size classes in neutral metacommunity parameters, exhibiting greater levels of immigration, besides greater total community size. We suggest that the established uses of mitochondrial metagenomics for analysis of species and phylogenetic diversity can be extended to parameterize recent theories of community ecology and biodiversity, and by focusing on the number mitochondria, rather than individuals, a new theoretical framework for analysis of mitochondrial abundance spectra can be developed that incorporates metabolic activity approximated by the count of mitochondria.

Lessons from genome skimming of arthropod-preserving ethanol

Field‐collected specimens of invertebrates are regularly killed and preserved in ethanol, prior to DNA extraction from the specimens, while the ethanol fraction is usually discarded. However, DNA may be released from the specimens into the ethanol, which can potentially be exploited to study species diversity in the sample without the need for DNA extraction from tissue. We used shallow shotgun sequencing of the total DNA to characterize the preservative ethanol from two pools of insects (from a freshwater habitat and terrestrial habitat) to evaluate the efficiency of DNA transfer from the specimens to the ethanol. In parallel, the specimens themselves were subjected to bulk DNA extraction and shotgun sequencing, followed by assembly of mitochondrial genomes for 39 of 40 species in the two pools. Shotgun sequencing from the ethanol fraction and read‐matching to the mitogenomes detected ~40% of the arthropod species in the ethanol, confirming the transfer of DNA whose quantity was correlated to the biomass of specimens. The comparison of diversity profiles of microbiota in specimen and ethanol samples showed that ‘closed association’ (internal tissue) bacterial species tend to be more abundant in DNA extracted from the specimens, while ‘open association’ symbionts were enriched in the preservative fluid. The vomiting reflex of many insects also ensures that gut content is released into the ethanol, which provides easy access to DNA from prey items. Shotgun sequencing of DNA from preservative ethanol provides novel opportunities for characterizing the functional or ecological components of an ecosystem and their trophic interactions.