*3.4. Quantitative Species Delimitation Methods*

The bPTP species delimitation method estimated the number of species to be between 708 and 889 (mean: 791, output delimitation with 853 clades), with an acceptance rate of 0.826 (Tables 3 and S4). MCMC generations merged at 249,739 and split at 250,261. The GMYC method, on the other hand, resulted in an estimation of 189 species, with a confidence interval of 145–237 (Tables 3 and S4).

Barcoding gap analysis using DNADIST via BioEdit resulted in an identity threshold value of 99.4% (distance threshold value = 0.006), retaining 94.6% of all within-species and excluding 95.4% of all between-species pairwise identities based on ad hoc delimitations. Most species delimited ad hoc were well delimited using this threshold value, such as the *Cora applanata*-*reticulifera* clade (Figure 7, File S11, Table S4). However, we detected several cases of ad hoc delimited species that may represent more than one taxon (e.g., *C. arachnoidea*, *C. davidia*, and *C. hawksworthiana*), as well as cases of diffuse species complexes where the threshold value did not fully discriminate between ad hoc-delimited species, including the *C. galapagoensis* clade and the *C. squamiformis*-*ciferrii* clade (Figure 7, File S11, Table S4). Overall, we found six cases of ad hoc-delimited species complexes to be potentially merged based on the threshold value, resulting in a possible reduction from 15 to six species. On the other hand, four cases would sugges<sup>t</sup> further splitting, resulting in a potential increase from four to nine species. Applying both corrections strictly to the initial number of 175 ad hoc-delimited species (plus one subspecies) would yield 171 species, a minor difference of only 2.3%.

ABGD on the subset of 716 *Cora* terminals returned stable results for relative gap width settings X = 1.0, 0.5, and 0.1. Setting X = 1.0 estimated between 147 and 231 species, corresponding to a barcode gap distance between 0.004 and 0.011 (Table 3). The three best-scoring ASAP scenarios (lowest score) estimated 161 species, with the following two scenarios ranging between 128 and 205 species, corresponding the threshold distances of 0.0086 (n = 3), 0.0130, and 0.0046, respectively (Table 3). The best-scoring scenario (161 species), therefore, appears to be a reasonable approximation.


**Table 3.** Species delimitation methods utilized and their respective results.

Extrapolation of these distance-based approaches to the entire taxon set (265 ad hocdelimited species) would result in estimates of 259 species (DNADIST), 223–350 species (ABGD), and (194–)244–310 species (ASAP).

**Figure 7.** Barcoding gap analysis using DNADIST in BioEdit. The different colors represent pairwise identity percentages (see legend). Highlighted clades show instances where the DNADIST and the ad hoc approaches inferred different species delimitations, potentially over- or underestimating species richness.
