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).