Phylogenetic Analyses and Species Delimitation
CO1 sequences were aligned by hand using the reading frame for reference in BioEdit v 7.2.5 (Hall, 1999) as there were no indels across sequences. Each sequence was evaluated for evidence of amplification of a nuclear mitochondrial insertion (NUMT) (Lopez, Yuhki, Masuda, Modi, & O’Brien, 1994) based on the criteria outlined by Zhang and Hewitt (1996). Sequences suspected of being NUMTs were removed from future analysis. Maximum likelihood and Bayesian phylogenetic analyses were performed to identify specimens destructively sampled using IQTree v 1.6.10 (Nguyen, Schmidt, von Haeseler, & Minh, 2014) and MrBayes v 3.2.7 (Ronquist et al., 2012) on the CIPRES Science Gateway v 3.3 (Miller, Pfeiffer, & Schwartz, 2010). Afrocampsis sp. (Hymenoptera: Braconidae: Helconinae) and Johannica gemellata(Coleoptera: Chrysomelidae: Chrysomelinae) were used as outgroups for the Eadya and host beetle phylogenies, respectively. Confidently identified voucher sequences of both parasitoids and beetles from Peixoto et al. (2018) and Nahrung et al. (2020) were included in each analysis to confirm the species of parasitoids and beetle hosts that were destructively sampled. In addition, four Eadya specimens collected in Tasmania and sent to the first author for identification were included (RDR129 - RDR132). One specimen (RDR237) was removed from the wasp dataset prior to phylogenetic analyses as a nucleotide BLAST (Altschul, Gish, Miller, Myers, & Lipman, 1990) search (megablast under default settings) of the nr nucleotide NCBI database indicated greatest similarity to unrelated species of Maxfischeria (Braconidae: Maxfischeriinae) (Boring, Sharanowski, & Sharkey, 2011). Two specimens were removed from the host dataset prior to phylogenetic analyses. RDR274 was most similar to species of Eadya based on a BLAST search (same as above), indicting the host sample was contaminated by the parasitoid. RDR301 contained a stop codon in the coding sequence, indicating a NUMT was potentially amplified.
The best fitting model of evolution was determined for each alignment based on Bayesian Information Criterion (BIC) using ModelFinder (Kalyaanamoorthy, Minh, Wong, von Haeseler, & Jermiin, 2017) within IQTree v 1.6.10 on the CIPRES Science Gateway v 3.3. For the wasp (Eadya ) dataset, the top performing model was K3Pu (BIC = 5986.3377) with parameters for empirical base frequencies (+F) and a gamma distribution of rate heterogeneity (+G). As this model is not supported within MrBayes, HKY+F+G (BIC = 5988.4978) was used as its BIC score was within Δ5 of the top performing model. For the beetle dataset, the top performing model was HKY+F+G (BIC = 6554.0634). The maximum likelihood analyses were performed with 1000 ultrafast bootstraps (Hoang, Chernomor, Von Haeseler, Minh, & Vinh, 2017), while the Bayesian analyses consisted of two independent runs with four chains each, for a total of 15,000,000 generations, sampling every 1000 generations with a 25% burn-in applied. Convergence of the two independent runs was evaluated using the average standard deviation of split frequencies, the potential scale reduction factor, and minimum estimated sample size output by MrBayes. Additionally, parasitoid and beetle Bayesian phylogenies were constructed using a dataset supplemented with Tasmanian specimens from Peixoto et al. (2018) for use in the Procrustes Approach to Cophylogeny (PACo) analysis outlined below. These phylogenies were constructed using the same methods listed above, using a HKY+F+G (BIC = 7587.3901) for the parasitoid dataset and HKY+F+G with a parameter for invariant sites (+I) for the beetle dataset (BIC = 8949.7961; Δ5.8 from top model). The intra- and inter-specific genetic distances were calculated for each clade recovered in the phylogenetic analyses using MEGA v 7.0 (Kumar, Stecher, & Tamura, 2016) with the Kimura 2-parameter model of molecular evolution (Kimura, 1980). The maximum likelihood and Bayesian trees were visualized using FigTree v 1.4.3 (Rambaut, 2012), and edited using Adobe Illustrator Creative Cloud (Adobe Systems Inc.). Alignment files can be found on Figshare (www.figshare.com DOI: 10.6084/m9.figshare.17105768).