In this investigation we present the most comprehensive data on the microbiota composition of sea turtles to date, and demonstrate a clear link between microbiota composition and sea turtle phylogeny. Furthermore, this is one of the only studies in wild animals to have obtained samples from all extant species of an entire clade of the evolutionary tree. This enabled us to perform a detailed evaluation of the phylogenetic signal that exists between host and microbiota. We showed that microbiota composition differs among all sea turtle species, but in all species, the predominant bacterial phylum in nesting turtles was Proteobacteria. In an investigation into the microbiota of juvenile green turtles from Florida, Proteobacteria was also the major phylum dominating samples . In contrast, the microbiota of juvenile green turtles from coastal areas of Brazil are co-dominated by the phyla Bacteroidetes and Firmicutes, with the investigators speculating that Proteobacteria increased in abundance in three individual turtles in this investigation in response to anthropogenic factors . Similarly, in a study conducted on green turtles from the Great Barrier Reef, Firmicutes was the most common phylum isolated from healthy individuals, but in sick turtles Proteobacteria was the dominate phylum . There are also reports of Firmicutes dominating the microbiota of stranded loggerhead turtles [18, 37], but these investigations are confounded because samples were collected from sick individuals, and in many cases there were delays between when the turtle was rescued and when samples were collected. Given that both captivity [58-62] and health  have both been shown to affect the microbiota of individuals, these results should be interpreted with caution as they are unlikely to represent the normal gut microbiota. In our investigation, all turtles sampled were wild and apparently healthy, and some had been sourced from extremely remote locations with no human habitation (e.g. Rosemary Island and Tiwi Islands), so we think that the likelihood of anthropogenic or other factors influencing our results is low. Furthermore, given that Proteobacteria was overwhelmingly the predominate phylum in all sea turtle species, and the strong phylogenetic relationship between microbiota and sea turtle species, we think that in nesting animals (i.e. animals with prolonged periods of inappetence), any microbiota in which Proteobacteria is not the most abundant phylum represents an atypical gut flora.
In comparison to other taxa, there are few studies on the microbiota of wild reptiles, and the relative abundance of specific bacterial phyla in herpetofauna appears to vary greatly. For example, Firmicutes was the dominant phylum identified in anoles (Anolis sp.) , Galápagos tortoises (Chelonoidis nigra) , gopher tortoises (Gopherus polyphemus) , green iguanas (Iguana iguana) , Galápagos land iguanas (Conolophus subcristatus) , marine iguanas (Amblyrhynchus cristatus) , and the montane iguana species Liolaemus parvus, Liolaemus ruibali, and Phymaturus williamsi . Such findings have led some researchers to believe that reptile microbiotas resemble that of mammals [21, 32], but in a wide-ranging investigation into squamate microbiotas, in which individuals representing 22 squamate families were sampled, Proteobacteria was the predominate phylum, and supports a hypothesis that the reptile gut microbiota is similar to that of birds . However, in the single reptile study conducted in the species most closely related to birds, the archosaurian American alligator (Alligator mississippiensis), gut microbiota was overwhelmingly dominated by the phylum Fusobacteria . Furthermore, the results of our investigation are similar to those seen in fish, in which the predominate phyla are Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria . These discrepancies highlight the difficulties associated with making any assumptions on microbial assembly between taxa based solely on compositional data without incorporating any phylogenetic techniques in the analysis.
Unlike their microbiotas, the natural histories of sea turtles differ widely among the seven extant species. Leatherbacks are largely oceanic-pelagic throughout their life-history, and have the most specialised diet, feeding almost entirely on an array of dense gelatinous zooplankton . Their large size means that they are more cold-adapted than other species, allowing them to traverse through the boreal waters that act as a barrier to other warm-water adapted turtles . Green turtles have an oceanic-neritic developmental pattern , are found in tropical and temperate waters worldwide , and consume a variety of seagrass, marine algae, and invertebrates [49, 70]. Like green turtles, loggerheads also have an oceanic-neritic developmental pattern and prefer temperate to tropical waters . However, unlike green turtles, they are largely carnivorous, feeding on a wide array of prey items including Hydrozoa, Bivalvia, Cephalopodia, Porifera, Scyphoza, Bryozoa, Gastropoda, Polychaeta, Maxillopoda, Malacostraca, Insecta, Holothuroidea, Echinoidea, Anthozoa, Actinopterygii, and occasional plant material . Evidence suggests that hawksbills also have an oceanic-neritic developmental pattern , with a preference for tropical waters . Although primarily carnivorous, the composition of prey items varies among populations of hawksbills, with some having a preference for sponges, while others feed predominately on corals . The flatback turtle has a completely neritic life history , residing within the tropical waters of the Australian continental shelf . Detailed investigations into their diet are lacking, but it is presumed that they are carnivorous . The olive ridley turtle is predominately oceanic for the duration of its life , and is found in both temperate and tropical waters, but most feeding probably occurs in warm water and they are likely omnivorous [49, 68]. Finally, Kemp’s ridleys have an oceanic-neritic developmental pattern , have a preference for tropical waters, and as adults are primarily carnivorous ingesting a range of molluscs, fish, jellyfish and gastropods . Given the vast array of niche occupancy and dietary preferences of sea turtles, some with significant overlap between species, it is likely that the similarities seen in relative abundance of the major bacterial phyla are driven by their shared evolutionary history rather than dietary and ecological factors. The similarities seen between gut microbiotas in nesting female sea turtles is remarkable, since there is nearly 100 million years of evolution separating the most ancient species, the leatherback, and the most modern species, the Kemp’s ridley . Such conservation of community similarity may be an indication that specific combinations of bacterial phyla are fundamental to sea turtle normal function, but this remains to be demonstrated in any taxa.
Based on the results of our study, it appears that gut microbial composition and sea turtle phylogeny are intrinsically linked, and it is likely that a process of co-evolution exists between host and microbial community composition. Microbes have been identified as a key driver of vertebrate evolution , and more recent research has focused on the role that phylogeny may play in convergence of microbiotas in some species . Investigations in primates suggest that evolutionary trends in host physiology are more important than dietary niche in determining gastrointestinal microbiota , which is supported by this research. For example, previous investigations into sea turtle microbiotas have speculated that the high proportion of Firmicutes found in some green turtle samples may be due to this bacterial phylum’s ability to break down plant-derived polysaccharides  and thus facilitate digestion of a cellulose-rich food such as the seagrass species that form the primary diet item of green turtles. However, we found that green turtles, along with loggerheads, had the lowest relative abundance of Firmicutes of all the sea turtle species, suggesting that this phylum may not be as important for cellulose digestion in herbivorous reptiles as previously reported. However, it should be remembered that since the animals sampled in our investigation were not feeding, it is possible that there may have been a shift away from phyla important for digestive function and thus they were underrepresented relative to what would be expected in feeding animals. Nevertheless, there are many examples in vertebrates, both terrestrial and aquatic, and from disparate branches of the evolutionary tree, where Firmicutes are lower in abundance than other phyla in herbivorous species [73-81]. Therefore, in the absence of specific functional testing, caution should be applied when making assumptions on functionality of the microbiota within a species, especially where phylogeny has not been considered as a component of the analysis. The results of this analysis show that extant sea turtle microbiotas have changed very little over the course of nearly 70 million years of evolution, despite the phenotypic changes that have occurred in their hosts, and this may be an indication that certain combinations of microbes are fundamental to specific aspects of all sea turtle physiology, regardless of differences in natural history between species.
We showed that the bacterial phylum SR1 was strongly linked to sea turtle phylogeny. The candidate phylum SR1 (Absconditabacteria), includes ubiquitous organisms found in marine and terrestrial high-temperature environments, fresh-water lakes, subsurface aquifers, and animals [82, 83]. There are no cultured representatives of SR1, with all current knowledge on their presence and diversity obtained from genomic sequencing [84, 85]. They have a predilection for sulphur-rich and oxygen-limited environments, suggesting a potential microaerophilic, sulphur-based metabolism, and in general, environmental and animal-derived SR1 species appear to cluster differently [83, 86]. SR1 is routinely found in a range of vertebrates, but is most commonly associated with H2S-related malodour and periodontal disease in humans [87, 88]. How SR1 is involved in sea turtle gastrointestinal function remains unknown at this stage. The role of the microbiota in shaping vertebrate phylogeny should be a focus for future investigations, and an effort must be made to sample as diverse an array of species as possible, spanning multiple clades of the evolutionary tree, so that these relationships can be further explored.
Using results obtained from extant turtles, we reconstructed possible microbiotas of extinct sea turtle species. This analysis showed that the composition of sea turtle microbiotas has not changed greatly over time, with only relatively minor fluctuations in the relative abundance of specific phyla responsible for observed differences. In contrast, the human microbiota has diverged rapidly from our closest relatives , and among populations of humans, continues to rapidly evolve . Sea turtles are among the most ancient species on Earth, with the leatherback existing in its current form for nearly 70 million years , while modern humans first appeared around 350,000 to 260,000 years ago . These seemingly marked differences in rate of evolution of microbiotas, may be reflective of broad differences in rates of evolution between taxa, and warrants further investigation.
A feature of our study was that we were restricted to sampling nesting females. This was because living males are difficult to obtain, and this can only be done in-water. Although this means that our analysis and interpretation is made on a particular subset of animals of the same sex, life stage and reproductive state for each species, it removes potentially confounding variation in the composition of microbiota associated with these traits and thus we think it strengthens our phylogenetic comparisons. It is likely that all of the females sampled in this investigation had not eaten for an extended period, as sea turtles may undergo long periods of fasting, particularly during breeding and migration [51, 92]. Periods of inappetence have been shown to affect the microbiotas of a range of vertebrates including humans , fish , bears , alligators , mice , penguins , and the Burmese python , however such changes are not consistent across all taxa, with fasting having negligible effects on the microbiota of geckoes . How this may have influenced our results is unknown, but it may explain the differences in our results and those of other green turtles captured on the Great Barrier Reef , and future investigations should focus on obtaining samples from a range of age classes, sexes, and physiological states. Although we were not able to determine if fasting in nesting turtles affected microbial diversity, some authors propose that fasting samples represent the core microbial OTUs, with other OTUs fluctuating in number in response to post-prandial physiological changes [17, 32]. If, as hypothesised, diet has little effect on this core microbiota, then this strengthens the results of our phylogenetic analysis because our results have not been confounded by transient microbial species that might be associated with dietary variation.