Challenging the concept that eumelanin is the polymorphic brown banded pigment in Cepaea nemoralis

Abstract

The common grove snail Cepaea nemoralis displays a stable pigmentation polymorphism in its shell that has held the attention of scientists for decades. While the details of the molecular mechanisms that generate and maintain this diversity remain elusive, it has long been employed as a model system to address questions related to ecology, population genetics and evolution. In order to contribute to the ongoing efforts to identify the genes that generate this polymorphism we have tested the long-standing assumption that melanin is the pigment that comprises the dark-brown bands. Surprisingly, using a newly established analytical chemical method, we find no evidence that eumelanin is differentially distributed within the shells of C. nemoralis. Furthermore, genes known to be responsible for melanin deposition in other metazoans are not differentially expressed within the shell-forming mantle tissue of C. nemoralis. These results have implications for the continuing search for the supergene that generates the various pigmentation morphotypes.

Figure 1

Quantification of eu- and pheomelanin markers from colour-sorted shell fragments of C. nemoralis. (A) Shell fragments used for LC-UV-MS were sorted by colour. (B) Mass spectra for all four melanin oxidation markers as measured in a representative yellow background sample (III from panel A). (C) Representative UV chromatograms used for quantitation of melanin oxidation markers. (D) Eumelanin and pheomelanin oxidation products were quantitated by HPLC-UV with external calibration. Eumelanin (PDCA and PTCA) and pheomelanin (TDCA and TTCA) oxidation markers were quantified for each shell colour fraction and normalised to initial sample weight. n = 3 for each shell colour fraction with each replicate comprised of 8 shells.

Figure 2

qPCR analysis of normalised relative expression values (ddCt) of four melanin pathway genes. (A) RNA was extracted from pigmented and immediately adjacent non-pigmented mantle tissue (white boxed regions). (B) Expression levels were compared between dark-brown pigmented mantle tissue vs. non-pigmented mantle tissue, and between whole mantle vs. foot tissue. For each tissue n = 6. * indicates p ≤ 0.05, ** indicates p ≤ 0.01 as per a Mann-Whitney test.