Weevil phylogenomics using hybrid enrichment
Massively parallel sequencing (aka next generation sequence [NGS]) enables rapid access to genomic-scale DNA data at a relatively low cost. Phylogenomics is a flourishing field that capitalizes on the power of NGS and uses hundreds or even thousands of loci, often amounting to millions of base pairs (bp), to reconstruct phylogenies. Methods for obtaining loci are many, varying in mainly marker design and library preparation.
I am taking a target enrichment approach to capture and sequence >500 loci or >1000,000 bp to reconstruct a phylogeny of weevils at the family or subfamily level. These loci are derived from seven previously published weevil transcriptomes. The program SISRS is used to determine homologous loci across these transcriptomes. The sequence data are provided to a company called MYcroarray, which designs and produces biotinylated RNA baits (MYbaits), used for hybridizing to and capturing target loci.
I have obtained a MYbaits kit for capturing 500 loci. At the initial stage I will work on 24 weevil samples. I am collaborating with Dr. Rachel Schwartz (author of 'SISRS') on this project. Dr. Schwartz has done the heavy lifting of selecting markers using SISRS. I will be performing library preparation (using MYbaits) and the subsequent analyses after sequencing. The sequencing will be done on a Illumina Miseq. The sequence reads will be assembled and homologous loci determined with SISRS.
I will be using the resulting phylogeny to test familial and to some extent subfamilial relationships within weevils. Some contested or unresolved issues include, but are not limited to, the monophyly of Anthribidae, if Nemonychidae together with Anthribidae or the latter (or part of it) alone is the sister to the remainders of Curculionoidea, and the phylogenetic relationships within some families (e.g., Attelabidae).
Diversity and evolution of bacterial symbionts in weevils by 16s sequencing
Bacterial symbionts play diverse and critical roles in insects, such as provisioning nutrients, promoting temperature tolerance, conferring parasite resistance, manipulating reproduction, overcoming host defense, and most recently discovered, even in helping producing aggregation pheromones. Advances in high-throughput sequencing technologies are increasing the capacity to study endosymbiosis by orders of magnitude. We have thus entered a new age primed to unravel the fascinating phenomena of endosymbiosis in the most diverse eukaryotic organisms, the insects.
My project is addressing this challenge with a focus on beetles of the weevil superfamily Curculionoidea (aka weevils). Existing knowledge of weevil endosymbionts is scattered, but offers promising insights. At least three unique lineages of possibly obligate endosymbionts ("Candidatus Nardonella", "Candidatus Curculioniphilus" and SOPE [S. oryzae principal endosymbiont]) have been found in various weevils. Facultative endosymbionts – e.g., Wolbachia, Rickettsia, Sodalis and Spiroplasma – have also been recorded.
Previous surveys of bacterial symbionts in weevils had one common limitation--they had very sparse taxonomic sampling. In this project I am sampling four families or 17 subfamilies of weevils, which would be largest taxonomic sample gathered to survey the diversity of symbionts in weevils.
Experiments were completed and procedures as follows: (1) weevil specimens were dissected and gut contents recovered; (2) samples were subjected to bead-beating (see advantages) and then DNA was isolated with the Qiagen DNeasy Blood & Tissue kit; (3) the 16s gene (V4-V5 region, positions 515-909) was amplified using PCR with barcoded primers (for sample-indexing); (4) PCR products were gel-purified using the Freeze 'N Squeeze spin columns (Bio-Rad); (5) PCR products were quantified on a Nanodrop and normalized using the SequalPrep™ Normalization Plate Kit (ThermoFisher Scientific); (6) normalized PCR products were pooled into a single sample, which was processed for library preparation; and finally (7) sequenced on an Illumina Miseq (paired end) platform.
We have performed data exploration and preliminary analyses based on the 11 million + assembled sequence reads. Some insights we gained from the analyses are (1) "Ca. Nardonella", "Ca. Curculioniphilus" and SOPE, previously found only from weevils, were found in all weevil specimens examined in the current study, indicating their conservation in weevils; (2) these bacteria were also detected in other related weevils (Chrysomeloidea, Tenebrinoidea and Cleroidea), and hence may be more widespread than previously thought; (3) a single weevil generated several 16s sequences that belonged to the same bacterial lineage (e.g., "Ca. Nardonella") but were found to be polyphyletic, indicating horizontal transfer or duplication and selective extinction; and (4) other 'generic' symbionts such as Wolbachia, Rickettsia, and Spiroplsma were also found across nearly all samples.
Current analyses are testing co-evolutionary scenarios, examining for possible horizontal transfer or duplication of symbionts, and comparing diversity across samples.
Biogeography of the Neotropical Exophthalmus genus complex (Entiminae)
The Exophthalmus genus complex (Curculionidae: Entiminae) contains a large radiation (>90 species) in the Caribbean region and about another 50 species on the Neotropical mainland. The Caribbean species show a high degree of single-island endemicity, with more than 90% of the species found from only a single island. This project aims to reconstruct a molecular phylogeny of the Exophthalmus genus complex and investigate the historical biogeography of this group.
I have assembled a data set consisting of ~65 species belonging to the Exophthalmus genus complex. More than 10 field trips were conducted in various Caribbean islands, several Central American countries and some South American localities. I participated in one such trip to Cuba in 2013.
... ... (TBC)
I am currently revising a manuscript previously submitted to the Journal of Biogeography.