Rhytiphora saundersii. Photo Jiri Lochman

Ignition Grant Round 7 (July 2017)

• Lauren Ashman, ANU PhD student
• David Rowell, ANU
• Adam Slipinski, CSIRO
• Andreas Zwick, CSIRO

Rhytiphora is the largest Australian genus of longhorn beetles (Cerambycidae: Lamiinae), with a large Australian radiation comprising about 200 species. It is the most morphologically diverse clade of longhorn beetles, mostly medium-sized and often colourful.

The genus is also known from a few species in New Guinea and SE Asia, their likely origin. Lamiinae appear to have arrived in Australia relatively late (as compared to the other Australian subfamilies Prioninae and Cerambycinae), where they diversified enormously, which is reflected in the unusually high diversity of species and genera. Aridification of the continent and availability of new plant communities (including their hosts Eucalyptus and Acacias) were probably major factors for diversification of this clade. 

The current concept of the genus (encompassing many former genera) by Slipinski and Escalona (2013) is supported by a single common trait: variously developed setose areas on the male abdomen, probably dispersing pheromones of unknown function. This trait is a putative apomorphy for Rhytiphora but has not been rigorously tested. There is currently no molecular phylogeny for Rhytiphora, and few molecular studies on Lamiinae in general, despite the fact that cerambycid beetles are a recognised pest of timber and some crop species. 

To study species-rich taxa like Rhytiphora it is essential to utilise the great diversity of samples that has been accumulated and taxonomically sorted in collections over many decades. The DNA of these older samples is typically poorly preserved, but can still be sequenced with a Whole Genome Shotgun (WGS) approach, which is rather costly and commonly targets only genes with high copy numbers, e.g. ribosomal and mitochondrial genes.

Molecular Inversion Probes (MIPs; Hardenbol et al. 2003; Niedzicka et al. 2016) provide an interesting alternative approach that can target any part of the genome (i.e. nuclear single copy genes suitable for phylogenetics). It is cost-effective for organisms with large genomes (like Rhytiphora) and should be highly suitable for fragmented, low quantity DNA; however, it has not been used for collection genomics so far. MIPs do not require DNA library preparation and contain sequences at each end that are complementary to conserved regions flanking the short target sequences.
 
We aim to use Rhytiphora as a test case to trial MIPs with old collection specimens and compare this approach against WGS sequencing of the same samples. We have already generated 105Gbp of low-coverage WGS sequence data from 24 dry museum specimens (dating back to 1966), which will be sufficient to assemble whole mitochondrial genomes, ribosomal genes and some fragments of the nuclear genome.

We will use these genome fragment data from divergent Rhytiphora species, as well as published cerambycid genomes and transcriptomes, to design MIPs that will target ~100 variable loci of the nuclear and mitochondrial genome of Rhytiphora. We will then compare the results obtained from MIPs and WGS sequencing of the same 24 specimens to evaluate the performance of these methods.

The results of this pilot study will inform our decision on which approach we will use for sequencing the bulk of Rhytiphora specimens in our larger phylogeographic study of the genus.

Hardenbol, P., J. Banér, M. Jain, M. Nilsson, E.A. Namsaraev, G.A. Karlin-Neumann, H. Fakhrai-Rad, M. Ronaghi, T.D. Willis, U. Landegren and R.W. Davis (2003). Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol 21: 673-678

Niedzicka, M., A. Fijarczyk, K. Dudek, M. Stuglik and W. Babik (2016). Molecular inversion probes for targeted resequencing in non-model organisms. Sci Rep 6: 24051