Colour pattern measurements successfully differentiate two cryptic Onchidiidae Rafinesque, 1815 species

Cryptic species, by definition, appear very similar to each other. In the absence of obvious external morphological differences, quantitative measurements of fine-scale colour pattern differences may be used to distinguish between cryptic species. To demonstrate how this is accomplished, 30 specimens each of two cryptic onchidiid sea slug species in Singapore were collected and identified by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit I gene. Sequences displayed a clear barcode gap: intraspecific distances (0–0.4%) and interspecific distances (4.8–5.5%) were distinct. To quantify colour patterns, eight pattern properties on the animals’ dorsal surface were measured using the PAT-GEOM software. Linear discriminant analysis and classification tree analysis were able to classify specimens with 80% and 81.7% accuracy respectively, and both identified proportion cover and randomness as the most important properties for differentiating the two species. Agreement between the genetic and pattern data is demonstrated by a significant correlation between the pairwise genetic and pattern distance matrices, as well as the significantly greater interspecific than intraspecific distances in both datasets. These results demonstrate that fine-scale pattern differences can be used to differentiate Peronia cryptic species. This approach has potential applications for a range of disciplines, including behaviour and ecology, and as an additional line of evidence for integrative taxonomy.