Museum skins enable identification of introgression associated with cytonuclear discordance

Increased sampling of genomes and populations across closely related species has revealed that levels of genetic exchange during and after speciation are higher than previously thought. One obvious manifestation of such exchange is strong cytonuclear discordance, where the divergence in mitochondrial DNA (mtDNA) differs from that for nuclear genes more (or less) than expected from differences between mtDNA and nuclear DNA (nDNA) in population size and mutation rate. Given genome-scale datasets and coalescent modelling, we can now confidently identify cases of strong discordance and test specifically for historical or recent introgression as the cause. Using population sampling, combining exon capture data from historical museum specimens and recently collected tissues, we showcase how genomic tools can resolve complex evolutionary histories in the brachyotis group of rock-wallabies (Petrogale). In particular, applying population and phylogenomic approaches, we can assess the role of demographic processes in driving complex evolutionary patterns and assess the role of ancient introgression and hybridisation. We find that described species are well supported as monophyletic taxa for nDNA genes, but not for mtDNA, with cytonuclear discordance involving at least four operational taxonomic units (OTUs) across four species which diverged 183–278 kya. ABC modelling of nDNA gene trees supports introgression during or after speciation for some taxon pairs with cytonuclear discordance. Given substantial differences in body size between the species involved, this evidence for gene flow is surprising. Heterogenous patterns of introgression were identified but do not appear to be associated with chromosome differences between species. These and previous results suggest that dynamic past climates across the monsoonal tropics could have promoted reticulation among related species.

Methods
This dataset includes ~1000 nuclear loci from a custom-designed exon capture approach (SeqCap EZ Developer Library; Roche NimbleGen), designed from a yellow-footed rock-wallaby (Petrogale xanothpus) transcriptome. The raw sequencing data were processed following the workflow of Singhal (2013), and scripts for this pipeline are available from 10.5061/dryad.7c99f (see Supplementary Materials and Methods here for more detail). The dataset includes samples from both modern and historical specimens, and these were processed in different pipelines. Modern samples were assembled de novo from the cleaned sequencing reads following the workflow described in Bragg et al. (2016). The historical samples were assembled using custom scripts https://github.com/CGRL-QB3-UCBerkeley/denovoTargetCapturePhylogenomics) and published methods (Bi et al. 2012; Portik et al. 2016; refer to Supplementary Materials and Methods here for more detail). Data was finally aligned and filtered using the Eaphy (v1.2; Blom 2015) pipeline. 

Mitogenomes were assembled following the methods of Hahn et al. (2013) using a docker version for MITObim (https://github.com/chrishah/MITObim). Data were generated in one of two approaches: (1) mapped to the Osphranter robustus mitochondrial genome (Janke et al. 1997), or (2) reconstructed from mitochondrial ND2, COI and Cytb seeds from previously sequenced individuals (Potter et al. 2012, 2014). Final mitochondrial genome assemblies were then aligned using Geneious Prime as well as mafft (Katoh et al. 2002) and edited to protein-coding regions of the genome (~11,500 bp).