Building the tree of life: Reconstructing the evolution of a recent and megadiverse branch (Calyptrates : Diptera)

Ph.DDOCTOR OF PHILOSOPH

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 via masstrapping, many of which will be new species, could be incorporated routinely in phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures via mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from ~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 are induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, whilst the ecological sample expands 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

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications

Evaluation of a diatom eDNA-based technique for assessing water quality variations in tropical lakes and reservoirs

Freshwater bodies, both lotic (canals, rivers) and lentic (lakes and reservoirs), in the tropics have only rarely been subjected to intense monitoring regimes owing to limited expertise and resources, despite tropical habitats being among the most dynamic and threatened globally. Diatoms are an important part of assessing aquatic environmental changes in parts of the world where monitoring is well-established because of their sensitivity to a range of indicators of water quality. In such cases, diatoms have traditionally been identified based on morphology. Morphological information on tropical diatom taxa and the expertise to assemble it are sparse. However, DNA-based assessment is less reliant on taxonomic expertise and has the potential to be more cost-efficient. The application of DNA-based technology could thus expedite the incorporation of diatoms into routine monitoring in the tropics. Here we evaluate an environmental DNA (eDNA) metabarcoding workflow for diatom identifications based on 109 water samples from six freshwater reservoirs in Singapore, a highly urbanised part of tropical Southeast Asia. The six reservoirs span a range of sizes and catchment settings. We generate baseline information for lentic systems by targeting planktonic assemblages and including two commonly used barcodes, rbcL and 18S, to assess detection differences. Congruence with morphological identifications is generally low and each technique reveals distinct species lists, but overall congruence between methods improves when comparisons are at the genus level. We discuss the probable methodological and analytical causes for this incongruence and conclude that both approaches be used concurrently for generating reference databases of relatively poorly-known diatom diversity in tropical freshwater habitats. More promisingly, we find that eDNA datasets were sufficient to reveal both spatial and temporal patterns in diatom assemblages across the six study sites. We also show that regardless of the detection method used, diatom assemblages correlated with similar environmental variables. Results highlight the potential feasibility of eDNA-based diatom monitoring of freshwater habitats in Southeast Asia and in the tropics more broadly

A phylogenomic analysis of Culicomorpha (Diptera) resolves the relationships among the eight constituent families

Culicomorpha is a particularly species‐rich clade within Diptera (true flies) that comprises c. 10% of the described diversity, including many medically important flies. Morphological studies – even when all life stages are included – yield relationships different from those derived from molecular data, notably with regard to the position of Chironomidae. Congruence amongst molecular studies has been weak due to limitations in gene‐ and family‐level taxon coverage. Here we use a whole‐transcriptome shotgun phylogenomic approach to clarify the relationships among all families of Culicomorpha. The dataset comprised 30 species (27 ingroup) and 364 888 amino acid residues for 1233 single‐copy protein‐encoding genes. Likelihood and parsimony analyses produce robust and highly congruent phylogenetic trees, with only one node in conflict. The superfamily Culicoidea is well supported and comprises Dixidae + [Corethrellidae + (Chaoboridae + Culicidae)]. As suggested previously, Chironomoidea is not monophyletic. The well supported Thaumaleidae + Simuliidae is sister group to Culicoidea, with the weakly supported Chironomidae + Ceratopogonidae probably being the sister group of all remaining Culicomorpha. We used random addition concatenation analysis (RADICAL) and four‐cluster likelihood mapping (FcLM) to assess the strengths of nodal support. The sister‐group relationship between Chironomidae + Ceratopogonidae is consistent with the FcLM results but support for this relationship emerges only when 1150 of the 1233 loci are analysed. We discuss briefly nodes that remain poorly supported even with thousands of genes and mention problems with vouchering in transcriptomic studies.We acknowledge grant support from NUS SEABIG (R-154-000-648-646 and R-509154-000-648-733)

Monophyletic blowflies revealed by phylogenomics

Abstract Background Blowflies are ubiquitous insects, often shiny and metallic, and the larvae of many species provide important ecosystem services (e.g., recycling carrion) and are used in forensics and debridement therapy. Yet, the taxon has repeatedly been recovered to be para- or polyphyletic, and the lack of a well-corroborated phylogeny has prevented a robust classification. Results We here resolve the relationships between the different blowfly subclades by including all recognized subfamilies in a phylogenomic analysis using 2221 single-copy nuclear protein-coding genes of Diptera. Maximum likelihood (ML), maximum parsimony (MP), and coalescent-based phylogeny reconstructions all support the same relationships for the full data set. Based on this backbone phylogeny, blowflies are redefined as the most inclusive monophylum within the superfamily Oestroidea not containing Mesembrinellidae, Mystacinobiidae, Oestridae, Polleniidae, Sarcophagidae, Tachinidae, and Ulurumyiidae. The constituent subfamilies are re-classified as Ameniinae (including the Helicoboscinae, syn. nov.), Bengaliinae, Calliphorinae (including Aphyssurinae, syn. nov., Melanomyinae, syn. nov., and Toxotarsinae, syn. nov.), Chrysomyinae, Luciliinae, Phumosiinae, Rhiniinae stat. rev., and Rhinophorinae stat. rev. Metallic coloration in the adult is shown to be widespread but does not emerge as the most likely ground plan feature. Conclusions Our study provides the first phylogeny of oestroid calyptrates including all blowfly subfamilies. This allows settling a long-lasting controversy in Diptera by redefining blowflies as a well-supported monophylum, and blowfly classification is adjusted accordingly. The archetypical blowfly trait of carrion-feeding maggots most likely evolved twice, and the metallic color may not belong to the blowfly ground plan

Next-Generation Sequencing Identification Tools for Nee Soon freshwater swamp forest, Singapore

10.26492/gbs70(suppl.1).2018-08The Gardens's Bulletin Singapore70Supplement 1155-174ISSN 0374-785

ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

10.1186/s12915-021-01141-xBMC Biology19121