Crem. peringueyi Emery, 1895

Quek, Swee-Peck, Pringle, Ernest L. & Heath, Alan, 2022, Chrysoritis Butler (Papilionoidea: Lycaenidae: Aphnaeinae) - Part I: Molecular phylogenetic analyses of a South African genus of myrmecophilous butterflies, Metamorphosis 33 (1), pp. 107-126 : 109-110

Crem. peringueyi Emery, 1895

Crematogaster peringueyi is understood to be a subjective synonym of Crem. capensis Mayr, 1862 (B. Blaimer & H.G. Robertson, pers. comm.); this view is as yet unpublished and so both names are currently valid and available. To avoid confusion, only the former name is used herein.

Unresolved cryptic species diversity is prevalent among ants. Even putatively well-known ant species like Lasius niger and Lasius alienus in Europe have turned out to be species complexes. Few of the ant species-level identifications listed above have been scrutinised by myrmecologists, and thus may be suspect ( Fiedler, 2021). For example, Crem. peringueyi and Crem. liengmei are widespread common species that could potentially contain cryptic species (see also Bickford et al., 2007) but they have yet to be studied in detail to determine if this could be the case here (B. Blaimer, pers. comm.).

Male patrolling terrain (MPT)

MPT is an informative trait in circumscribing Chrysoritis species. In some taxa, males congregate at specific topographic features (e.g., hilltop, gulley etc.), awaiting the arrival of newly eclosed virgin females. Likewise, virgin females will seek out particular terrain features to find their mate soon after eclosing. This phenomenon, possibly a form of lekking, is sometimes known as "hilltopping." Specificity in MPT maximises the chances of finding a mate of the same species and could serve as a prezygotic barrier to gene flow between species in sympatry. MPT specificity appears to be characteristic of thysbe clade species and has so far not been observed in species outside the thysbe clade. Males of C. dicksoni (outside the thysbe clade) show aggregating behaviour but it seems not to be associated with a consistent topographical feature (see HEA23a). In non- thysbe clade species (noting the exception of C. dicksoni ), most males and females congregate and mate near their host plants, and some species (e.g. C. zonarius ) prefer to stay very close to their host plant.

Analyses of molecular data

Phylogenetic analyses

Molecular data for population and phylogenetic analyses are from TEA20. The 406 Chrysoritis samples reported therein were found to contain a few samples that were sequenced twice for COI. After removing the duplicates, the total number of Chrysoritis samples was 399 (excluding 4 outgroup samples), of which all have sequences of COI (1220 base pairs [bp]), 97 have EF (1039 bp) and CAD (745 bp) sequences, and 98 have H3 (328 bp) sequences. We also discovered that the COI sequence for sample AH06M581 (identified as C. pan but appearing as part of C. stepheni) was likely the result of contamination and we deduced the correct sequence (see Note S1). The trees presented by TEA20 based on all four genes combined lacked well supported resolution in the thysbe clade. Examination of the nuclear gene trees ( CAD, EF and H3, unpublished, courtesy of G. Talavera) showed scant resolution within the thysbe clade. Thus we performed further analyses on the COI data alone (1220 nucleotides). Identical sequences were identified using Arlequin 3.5. ( Excoffier et al., 1992; Excoffier & Lischer, 2010) and removed prior to phylogenetic analyses, resulting in a sample set of 270. Maximum likelihood (ML) analyses were performed on the unpartitioned COI dataset using IQTree ( Nguyen et al., 2015; Minh et al., 2020) run on IQTree’s web server (Trifinopoulos et al., 2016) at https://www.hiv.lanl.gov/content/sequence/IQTREE/iqtre e.html using the “find best and apply” substitution model setting (ModelFinder, Kalyaanamoorthy et al., 2017, resulting in the TVM + F + R3 model selected using AIC, BIC and AICc), and branch support was obtained from 1000 repetitions of ultrafast bootstrap ( Hoang et al., 2018). ML analyses were also performed on the three nuclear genes separately using the same software and settings as for the COI data.

The aligned and combined nuclear gene dataset matrix is available for download at https://doi.org/10.6084/m9.figshare.19225203 and the COI dataset matrix at: https://doi.org/10.6084/m9.figshare.19225101.

Haplotype network construction

A statistical parsimony network (Templeton et al., 1992) of COI haplotypes (with duplicates removed as described above) from thysbe clade (sensu TEA20) was constructed using TCS 1.21 ( Clement et al., 2000), and the network visualised using the program tcsBU ( Murias dos Santos et al., 2016). The 334 samples within the thysbe clade collapsed into 200 unique haplotypes; however, some haplotypes differed only by missing data. A list of samples sharing identical haplotypes is provided in Table S1, available at: https://doi.org/10.6084/m9.figshare.19225038.

AMOVAs

To ascertain the degree to which genetic structure in the COI data of the thysbe clade can be explained by taxonomic designation versus geographic distribution, we performed analyses of molecular variance (AMOVAs) using Arlequin 3.5. ( Excoffier et al., 1992, Excoffier & Lischer, 2010). AMOVAs were run using four grouping schemes: 1) by species, 2) by subspecies, 3) by region, and 4) by locality group (comprising a cluster of localities in the same vicinity – see Table S2). In addition to the overall fixation index (ΦST), fixation indices for all pairwise comparisons were calculated to determine how differentiated a species or subspecies was from its sister taxon. Two samples were excluded from the AMOVAs: AH06M581 ( C. pan which had a contaminated COI sequence, see above) and AH12C011 (a brooksi x rileyi hybrid).

Genetic distances

In this study, the authors used COI data from TEA20 to generate a pairwise COI distance matrix of Chrysoritis samples. Genetic distance is not used to determine rank designations or species-level splits. This table is available as a.xlsx file at: https://doi.org/10.6084/m9.figshare.17241566.v1.