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.

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The authors thank L. Roman Carrasco for his advice on statistics and the National Parks Board, Singapore for the permit (number NP/RP 15-088) under which the specimens were collected.


This study was funded by a Singapore Ministry of Education Academic Research Fund (MOE AcRF) Tier 1 Grant (R154-000-660-112) and by the National Research Foundation, Prime Minister’s Office, Singapore under its Marine Science R&D Programme (MSRDP-P03).

Author information




I.Z.W.C. conceived and designed the experiment, performed image and data analysis and wrote the manuscript, including the preparation of the figures and tables. J.J.M.C. and D.H. conceived and designed the experiment, collected the specimens and performed genetic and morphological analyses and contributed to the final draft of the manuscript. P.A.T. conceived and designed the experiment and contributed to the final draft of the manuscript.

Corresponding author

Correspondence to Ian Z. W. Chan.

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The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed by the authors.

Sampling and field studies

The specimens were collected under a permit issued by the National Parks Board, Singapore (number NP/RP 15-088).

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The datasets and scripts used for all analyses are included in the “Electronic supplementary material” of the article.

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Chan, I.Z.W., Chang, J.J.M., Huang, D. et al. Colour pattern measurements successfully differentiate two cryptic Onchidiidae Rafinesque, 1815 species. Mar Biodiv 49, 1743–1750 (2019).

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  • Peronia
  • Sensory ecology
  • Integrative taxonomy
  • Pattern quantification