A comprehensive data set of this study is presented in the Supplementary Information (S).
The marine crabs, Percnon affine and Grapsus albolineatus, and the terrestrial crab, Geothelphusa albogilva, analyzed in this study were of similar size. Their fresh masses were 25.4 ± 10.6 g, 23.1 ± 9.6 g, and 22.5 ± 5.9 g, respectively (Fig. 2a, Table S1). The semi-terrestrial Orisarma intermedium was significantly smaller and lighter (13.1 ± 3.2 g) than the other species (one Way ANOVA, F(3.44) = 5.587, p = 0.0025).
The hepatosomatic indices (HSI, Fig. 2b, Table S2) were highest in the terrestrial species with 4.5 ± 1.3 in O. intermedium and 4.7 ± 1.3 in G. albogilva. The HSI of the marine P. affine was slightly lower (3.9 ± 0.8). G. albolineatus had the lowest HSI (3.0 ± 0.8). The HSI differed significantly between species (one Way ANOVA, F(3.44) = 6.822, p = 0.0007).
Total lipid content
The total lipid contents (TL in %DM) of the midgut glands were displayed on an ordinal scale, ranked from minimum to maximum values (Fig. 3). Lipid levels in the marine species ranged from 4 to 42%DM (average 22.3%) in P. affine and from 12 to 39%DM (average 21.7%) in G. albolineatus. Data of starved animals, displayed as open symbols, were distributed along the whole scale (Fig. 3a). Applying the sigmoidal model, maximum lipid values were projected to reach about 50%DM in either marine species. The lipid content of the semi-terrestrial O. intermedium ranged from 5 to 35%DM (average 16.3%) and that of the terrestrial G. albogilva from 3 to 25%DM (average 9.0%) (Fig. 3b). Similar to the other specimens, data of starved animals were distributed along the ascending series. No approximation towards a maximum lipid level could be deduced from the curve.
Triacylglycerols (TAG) were the only major storage lipid in all investigated species. The amount of TAG correlated with the total lipid content (Fig. 4).
Maximum values ranged from 70%TL in Grapsus albolineatus (Fig. 4a) to 91%TL in Geothelphusa albogilva (Fig. 4b). Wax ester and sterol ester (WE/SE) levels were low in all species (< 6.9%TL) except in Grapsus albolineatus, where WE/SE reached a maximum of 16.6%TL. The amount of wax esters showed no statistically significant correlation with the total lipid content. The amounts of free fatty acids (FFA) and sterols (ST) were negligible (Table 2). The shares of polar lipids (PL) were negatively correlated with TAG values.
Fatty acids and fatty alcohols
The FA compositions of the midgut glands of the four crab species differed from each other (Table 3). Major FA representing more than 10%TFA (%total fatty acid), were palmitic acid (16:0), arachidonic acid (20:4(n-6)), oleic acid (18:1(n-9)), and linoleic acid (18:2(n-6)). While 16:0 was the dominant FA in P. affine with 27.2%TFA, it was lower in G. albolineatus (19.0%TFA) and O. intermedium (13.7%TFA). A minimum of 10.8%TFA was present in G. albogilva. Percentages of 20:4(n-6) comprised a wide range among marine and terrestrial species: 13.3%TFA and 18.2%TFA for P. affine and G. albolineatus, respectively, and 13.5%TFA and 18.9%TFA for O. intermedium and G. albogilva, respectively. 18:1(n-9) was high in the semi-terrestrial O. intermedium (16.5%TFA) and the terrestrial G. albogilva (19.1%TFA). In contrast, the 18:1(n-9) values of the two marine species were much lower, accounting for 6.9%TFA in P. affine and 9.9%TFA in G. albolineatus. The same was true for 18:2(n-6): the marine species P. affine and G. albolineatus had low levels with 4.1 and 5.9%TFA, respectively, whereas O. intermedium and G. albogilva showed higher values of 10.1 and 11.2%TFA, respectively.
Among the other FA, palmitoleic acid (16:1(n-7)) was highest in P. affine with 4.3%TFA, while the other species showed lower values between 1.5 and 2.6%TFA. The opposite was detected for stearic acid (18:0): P. affine had lowest levels with 7.3%TFA and the other species ranged between 7.8 and 9.6%TFA. 20:5(n-3) was relatively high in P. affine with 9.8%TFA, but only comprised between 5.3%TFA and 7.8%TFA in the other species.
The potential food sources, algae, grasses, and leaf litter, contained high amounts of palmitic acid (16:0) (19.4 to 27.2%TFA, Table 3). Leaf litter was also rich in stearic acid (18:0) (13.5%TFA). All samples contained high amounts of linoleic acid (18:2(n-6)) and α-linolenic acid (18:3(n-3)), the latter showing a maximum of 47.2%TFA in the grasses.
All species had very low amounts of fatty alcohols, usually 14:0 and/or 16:0. Maximum values of 0.4%TL were present in G. albogilva. Apparently, fatty alcohols were minor components in these species and, hence, not considered in further analyses.
The Principal Component Analysis (PCA) of the FA compositions of the four crab species (midgut glands) and the three dietary items revealed a distinct separation of species with increasing lipid content. The first three PCs explained 81.3% of the variation. PC1 covered 45.9% of variation (Fig. 5a, b) and was primarily determined according to their Eigen vectors by the FA 20:4(n-6), 16:0, 18:3(n-3), 14:0, and 18:0 (Tables S3, S4). The individuals of each species lined up according to their lipid content, which was graphically indicated by the intensity of the symbol colors (Fig. 5a). In all crustacean species, the amount of storage lipids correlated positively with the amount of the saturated FA 16:0 and negatively with the FAs 20:4(n-6) and 18:0 (Fig. S1, Table S1). PC2 (23.3%) separated the four crustacean species and particularly the marine species from the semi-terrestrial and terrestrial species. PC2 was primarily determined by 18:2(n-6), 18:1(n-9), and 18:3(n-3). The separation along PC2 was most distinct in the lipid-rich individuals. Lipid-poor individuals of all species showed a more similar lipid composition and approached each other along PC1 and PC2 (Fig. 5a). PC3 covered another 12.1% of the variation and was primarily determined by 18:1(n-9), 18:3(n-3), and 16:2(n-4). It principally separated the crustaceans from the dietary plant material.
Dietary indices varied considerably among the four crab species. The index for the consumption of vascular plants IV was lowest in the two marine species P. affine (4.1 ± 0.9) and G. albolineatus, (5.9 ± 1.4) but significantly higher in the two terrestrial species O. intermedium (10.1 ± 2.8) and G. albogilva (11.2 ± 3.7) (Fig. 6a, Table S5).
The 16:0/IV index for marine/terrestrial diet decreased significantly from the marine to the terrestrial species (Fig. 6b, Table S5). P. affine showed the highest mean value (6.7 ± 3.1), followed by G. albolineatus (3.5 ± 2.0), O. intermedium (1.4 ± 0.6), and G. albogilva (1.0 ± 0.3). The 16:0/IV indices of O. intermedium and G. albogilva were not significantly different from each other.
The second marine/terrestrial diet index, the Σalgae FA/IV index, showed a similar pattern as the 16:0/IV index (Fig. 6c, Table S5). The two marine species, P. affine and G. albolineatus, had mean values of 8.4 ± 1.5 and 6.1 ± 1.2, respectively. They were significantly different from those of the two terrestrial species, O. intermedium (3.3 ± 1.8) and G. albogilva (3.0 ± 1.6).
Additional dietary indices of the four crab species are listed in Table 4. The carnivory index ICa of the two terrestrial species, O. intermedium and G. albogilva, was higher than that of the two marine species, P. affine and G. albolineatus. The Chlorophyta index ICh was lowest in the terrestrial G. albogilva. The index for Phaeophyceae consumption IP decreased from the marine towards the terrestrial species, whereas the index for Rhodophyta IR was quite similar among the four species. The Bacillariophyceae index IB was relatively high in P. affine, intermediate in O. intermedium and G. albolineatus, and low in G. albogilva.