We here applied DNA metabarcoding approach and stable isotope analyses to explore the trophic niche differentiation of genetically different but geographically proximate populations of marine iguanas. We demonstrate for the first time that the macroalgal diet differs between populations of A. c. mertensi and A. c. godzilla on the island of San Cristóbal, which could be one factor contributing to the maintenance of genetic divergence between these geographically proximate marine iguana subspecies.
We found a diverse assemblage of green, red and brown macroalgae in the iguanas’ foraging grounds on San Cristóbal. As confirmed by the isotope analyses, red and green algae were found to be important food resources for marine iguana populations (except for A. c. mertensi at La Loberìa, Fig. 3). On the other hand, our DNA metabarcoding analysis indicated much higher preference towards red algae in terms of detected macroalgal taxa from the feces samples. In addition, although the primer design included sequences from brown algae, the metabarcoding data did not include any OTUs from this group. In contrast, the stable isotope data suggested that brown algae are a part of the marine iguanas’ diet (Fig. 3). These inconsistencies may be caused by seasonal variance in foraging preferences, which has previously been described to be an important factor affecting the diet patterns of marine herbivores [e.g. 10]. For example, Shepherd and Hawkes  reported that during the hot season (December-May), the proportional cover of the green alga Ulva sp. was much lower compared to red algae (e.g. Gelidium sp.). As the availability of the food items (e.g. seasonality) may determine the foraging patterns of marine iguanas , the low detection rate of green algae in our metabarcoding data might reflect its low abundance during our sampling period on the hot season (December). Moreover, December is generally considered to be the breeding season for marine iguanas (also in San Cristóbal, personal observations) and it has been suggested that the quality of the food has a positive relationship with the timing of reproduction . Thus, during the hot season, there is a strong preference for red algae, which is associated with their better digestibility, protein and energy content (higher quality) compared to green and brown algae [11–13]. Nevertheless, green algae (especially Ulva spp.) have also been reported to represent an important part of marine iguanas’ diet [14, 15]. However, the latter studies were conducted during cool season (June-November), where the abundance of Ulva is high, therefore potentially contributing much stronger to the overall diet. When there is a lack of both, red and green algae, brown algae may be used as a substitute ’emergency’ food . Therefore, these incongruences between metabarcoding data and SIA reflect the different timescale assessed by these two methods, where metabarcoding captures a short-term snapshot of ingested food items, while stable isotopes indicate the assimilated diets over a longer time period and could include seasonal resource use shifts.
The OTU-level community analyses in our metabarcoding data set revealed a clear diet partitioning between A. c. mertensi and A. c. godzilla (Fig. 1B); i.e. the similarity of the macroalgal OTU composition is higher in geographically proximate locations. Interestingly, a similar pattern was found for gut microbial communities in marine iguanas . This may indicate the potential of selective foraging for the algae from which the extraction of nutrients via specialized microbiota could be more efficient. However, besides the above mentioned seasonal effects on the availability and quality of food items, fine scale differences between habitats may be an additional factor determining the composition of local macroalgal communities . As there was little overlap between stable isotope values between sampling sites on the main island, our data indicate that individuals use mostly local foraging grounds during at least the previous season and may not move far along the coastline. Thus, whether preference for different red algae of A. c. mertensi and A. c. godzilla during parts of the year reflects selective foraging behavior or is driven by patterns of local algal diversity (i.e. available food), remains to be clarified.
In previously studied populations of marine iguanas, the majority of individuals forage in the intertidal, i.e., on exposed rocks, during low tides. Only 5%, mostly large-bodied individuals, dive beneath the sea surface to graze on offshore algal beds . From our field observations, we do not expect substantial differences in intertidal vs. subtidal feeding at our study site as an adaptive response to different spatial distribution of algae. Instead, we interpret the dietary differences among sites as a result of either different resource availability, or different food preferences. These differences in the trophic niche of marine iguanas on San Cristóbal island have important implications. Strong genetic differentiation between populations of A. c. mertensi and A. c. godzilla are paralleled by significant differences in their diet, thus proving a first hint to a concurring ecological divergence in these two subspecies. Additionally, limited movement behavior may additionally enhance the separation and add to the observed fine scale genetic differentiation.