Table 2. Main protein sequences identified in the double-buttons from Havnø, Hornstaad-Hörnle IA and Peştera Ungurească and their presence/absence in the analysed set of reference freshwater and marine shells (black dots).

Numbers indicate total number of peptide sequences identified and the cell colour is proportional to the coverage of the sequence itself. Threshold values for peptide and protein identification: false discovery rate (protein FDR) = 0.5%, protein score −10lgP ≥ 40, unique peptides ≥ 2, de novo sequences scores (ALC%) ≥ 50. Asterisks (*) indicate proteins identified only when using less stringent parametres: protein score −10lgP ≥ 20; unique peptides ≥ 1. Note that molecular sequence databases for molluscan species are incomplete and biased towards well-studied model organisms. The peptide sequences recovered in our study were identified using sequence homologies with proteins originally described from Hyriopsis cumingii, Crassostrea sp., Pinctada sp., Mytilus sp. and several others. As a result of database insufficiency, the bioinformatic search of these ‘shellomes’ could not identify the exact taxon of our samples, but provided a strong indication of the fact that the closest taxon to that of the ornaments (and of the freshwater reference shells) is the pearl-producing triangle sail mussel Hyriopsis cumingii (Unionoida).

Proteins present in database from		Identified proteins	Double-buttons							Freshwater				Marine
Order	Genus		HavA	HavB	HavC	HorA	HorB	HorC	PesB	U.p	U.c	M.m	P.a	Mo.M	O.e
Unionoida	Hyriopsis	Hic74 [Hyriopsis cumingii]	132	158	260		6	11	21	●	●	●	●
		Hic52 nacreous layer matrix protein [Hyriopsis cumingii]	1*	1*	2*					●	●	●	●
		Silkmapin (isoforms: nasilin 1 and nasilin 2) [Hyriopsis cumingii]	1*	3	3		5			●	●	●*	●*
Ostreida	Pinctada	MSI60-related protein [Pinctada fucata]	6		27			12		●	●		●	●
		Insoluble matrix protein [Pinctada fucata]	4		33					●	●		●
	Crassostrea	Glycine-rich cell wall structural protein-like [Crassostrea virginica/gigas]	17	11	12				14		●
		Glycine-rich protein 23-like [Crassostrea virginica]	8	11	6					●		●
		Antifreeze protein Maxi-like [Crassostrea virginica]		4	4								●
Mytilida	Bathymodiolus	MSI60-related protein partial [Bathymodiolus platifrons]		6	11					●			●	●
	Mytilus	Precollagen D [Mytilus edulis]	16	26	23				9	●	●	●
		Nongradient byssal precursor [Mytilus edulis]	10	10					25		●
Other	Other	Predicted: transcription factor hamlet-like partial [Octopus bimaculoides]	5	6	11					●		●
		Hypothetical protein OCBIM_22008720 mg partial [Octopus bimaculoides]	6	11
							Coverage		≥55%	≥35%	≥15%	≥10%	≥1%	Presence ●