The contribution of mitochondrial metagenomics to large-scale data mining and phylogenetic analysis of Coleoptera.

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 We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of 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.

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.

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.

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.

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.

Merging two eDNA metabarcoding approaches and citizen-science-based sampling to facilitate fish community monitoring along vast Sub-Saharan coastlines.

The coastline of Sub-Saharan Africa hosts highly diverse fish communities of great conservation value, which are also key resources for local livelihoods. However, many costal ecosystems are threatened by overexploitation and their conservation state is frequently unknown due to their vast spatial extent and limited monitoring budgets. Here, we evaluated the potential of citizen science-based eDNA surveys to alleviate such chronic data deficiencies and assessed fish communities in Mozambique using two 12S metabarcoding primer sets. Samples were either collected by scientific personnel or trained community members and results from the two metabarcoding primers were combined using a new data merging approach. Irrespective of the background of sampling personnel, a high average fish species richness was recorded (38 ± 20 OTUs per sample). Individual sections of the coastline largely differed in the occurrence of threatened and commercially important species, highlighting the need for regionally differentiated management strategies. A detailed comparison of the two applied primer sets revealed an important trade-off in primer choice with MiFish primers amplifying a higher number of species but Riaz primers performing better in the detection of threatened fish species. This trade-off could be partly resolved by applying our new data-merging approach, which was especially designed to increase the robustness of multiprimer assessments in regions with poor reference libraries. Overall, our study provides encouraging results but also highlights that eDNA-based monitoring will require further improvements of, for example, reference databases and local analytical infrastructure to facilitate routine applications in Sub-Saharan Africa.

Metagenomics: A viable tool for reconstructing herbivore diet.

Metagenomics can generate data on the diet of herbivores, without the need for primer selection and PCR enrichment steps as is necessary in metabarcoding. Metagenomic approaches to diet analysis have remained relatively unexplored, requiring validation of bioinformatic steps. Currently, no metagenomic herbivore diet studies have utilized both chloroplast and nuclear markers as reference sequences for plant identification, which would increase the number of reads that could be taxonomically informative. Here, we explore how in silico simulation of metagenomic data sets resembling sequences obtained from faecal samples can be used to validate taxonomic assignment. Using a known list of sequences to create simulated data sets, we derived reliable identification parameters for taxonomic assignments of sequences. We applied these parameters to characterize the diet of western capercaillies (Tetrao urogallus) located in Norway, and compared the results with metabarcoding trnL P6 loop data generated from the same samples. Both methods performed similarly in the number of plant taxa identified (metagenomics 42 taxa, metabarcoding 43 taxa), with no significant difference in species resolution (metagenomics 24%, metabarcoding 23%). We further observed that while metagenomics was strongly affected by the age of faecal samples, with fresh samples outperforming old samples, metabarcoding was not affected by sample age. On the other hand, metagenomics allowed us to simultaneously obtain the mitochondrial genome of the western capercaillies, thereby providing additional ecological information. Our study demonstrates the potential of utilizing metagenomics for diet reconstruction but also highlights key considerations as compared to metabarcoding for future utilization of this technique.

An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies.

BACKGROUND: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FINDINGS: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. CONCLUSIONS: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

Mitochondrial metagenomics: letting the genes out of the bottle.

'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.

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.

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.