Marine mammals harbor unique microbiotas shaped by and yet distinct from the sea

Abstract

Marine mammals play crucial ecological roles in the oceans, but little is known about their microbiotas. Here we study the bacterial communities in 337 samples from 5 body sites in 48 healthy dolphins and 18 healthy sea lions, as well as those of adjacent seawater and other hosts. The bacterial taxonomic compositions are distinct from those of other mammals, dietary fish and seawater, are highly diverse and vary according to body site and host species. Dolphins harbour 30 bacterial phyla, with 25 of them in the mouth, several abundant but poorly characterized Tenericutes species in gastric fluid and a surprisingly paucity of Bacteroidetes in distal gut. About 70% of near-full length bacterial 16S ribosomal RNA sequences from dolphins are unique. Host habitat, diet and phylogeny all contribute to variation in marine mammal distal gut microbiota composition. Our findings help elucidate the factors structuring marine mammal microbiotas and may enhance monitoring of marine mammal health.

Figure 1. Bacterial phyla in specimens from 38 dolphins and 18 sea lions.

Only one time point per animal and only specimens with ⩾372 pyrosequencing reads are shown (n=199, average number of reads per specimen was 3119.4). Four other single time point specimens and all 26 extraction controls yielded <50 reads. Specimens are shown in the same order as listed in Supplementary Table 1. The relative proportions of each phylum within each specimen and in the total data set are shown sorted on the average abundance in the combined data set (most abundant at the bottom). A total of 51 phyla were found. Only the 20 most abundant phyla are shown; the remaining 31 as well as all unclassifiable sequences were grouped together in ‘other phyla'; these are shown in Supplementary Fig. 1. Of the 20 phyla shown here, 19 were found in specimens obtained from the marine mammals; phylum SAR406 was only found in seawater samples. The horizontal lines at the top show the specimen source. ‘M' and ‘W' indicate specimens obtained from MMP and wild dolphins, respectively. No gastric or respiratory specimens were obtained from the wild dolphins, and in several cases different MMP dolphins were used for the respiratory sampling than for the oral/gastric/rectal sampling. Column ‘All' on the right displays the average relative phyla abundance in all specimens. Blow, blowhole; Gast, gastric; F&S, fish and squid.

Figure 2. Gain in phylogenetic diversity according to specimen type.

Phylogenetic diversity (PD) gain refers to the cumulative branch length in a phylogenetic tree that is exclusive to a particular specimen, as samples are added in a user-defined order. Calculations were done on the PS data set. Two different orders are shown. In a, the dolphin oral specimens were added first, followed by dolphin gastric, rectal and respiratory samples, sea lion specimens, fish and squid, and seawater samples. Panel b shows the animal samples in reversed order. Only one time point per animal was included. Of the marine mammal specimens, the dolphin oral specimens show the most phylogenetic gain, even when the sea lion specimens are considered first.

Figure 3. Habitat-specificity of bacterial groups found in this study.

(a) Relative OTU abundance in the pyrosequencing data set. Specimens with ⩾372 reads (n=199; single time point per animal; same specimens and order as in Fig. 1) are shown in columns, and OTUs with ⩾20 reads (n=236) are shown in rows. The OTUs are clustered using an NMDS sorting based on Bray–Curtis distance, while the specimens (shown in columns) are sorted per specimen group. Relative abundance is shown in grey scale (white, absent; black, high abundance). (b) Venn diagram showing sharing of the 4,137 OTUs found in a rarefied pyrosequencing data set of 18 dolphins, 18 sea lions and 18 water specimens. Each data set was rarefied to 69,377 reads to match the smallest data set (from the water samples). Green, dolphins (1,452 OTUs total, includes oral, gastric, and rectal specimens from MMP and wild dolphins); red, sea lions (659 OTUs oral, gastric, rectal); blue, seawater (2,212 OTUs). The overlap between the circles is not to scale. (c) Quantitative PCR on four specimen types. Results are presented as average gene copy number per millilitre for seawater and gastric fluid, or per swab for oral and rectal specimens, for each of four different qPCR tests. Each individual qPCR was done in triplicate, error bars indicate s.d.. The numbers above the bars indicate the number of specimens positive for each qPCR test.

Figure 4. Relationships among bacterial communities from different host species and habitats.

Only samples with ⩾372 reads were included (n=199; one specimen per body site per animal). (a) NMDS Bray–Curtis ordination of PS-analysed specimens from dolphins and sea lions included in this study. Colours indicate the different specimen types and sources. Shapes display the location of the animal during sampling (San Diego or an undisclosed alternate, distant location for the MMP animals and Sarasota Bay for the wild, free-living dolphins). (b) Distance Threshold Network analysis of marine mammal-associated bacterial communities. The network displays binary relationships between specimens and OTUs using a Bray–Curtis distance and a Fruchterman–Reingold layout. Each data point represents the bacterial community from an individual specimen. Two specimens are considered ‘connected' if the distance between them is less than a user-defined threshold, here, 0.6. The thickness of the connecting edge is related to the distance between two specimens. OTU abundances were proportionally transformed.

Figure 5. Oral and rectal bacterial communities associated with bottlenose dolphins and sea lions.

Oral (a) and rectal (b) bacterial communities associated with bottlenose dolphins and sea lions. NMDS Bray–Curtis ordination of PS reads in which colour shows specimen type and data point shape depicts location (San Diego or alternate location for the MMP animals, Sarasota Bay for the wild animals). Only one time point per animal is shown. A bootstrap test in which the wild/MMP status of the dolphins was resampled showed that the average Bray–Curtis distance within oral specimens from MMP and within wild dolphins was significantly lower than the average distance between oral specimens from MMP and wild dolphins (P<0.001). However, average Bray–Curtis distances of dolphin rectal communities were not statistically different within or between location groups (P=0.11).

Figure 6. Relative stability of dolphin-associated microbial communities.

(a) Oral communities, (b) gastric communities and (c) rectal communities. NMDS Bray–Curtis ordination of PS reads for each body site individually for the seven dolphins sampled monthly for 5 or 6 months. Six of these were sampled 3 years later as well (open circles). For each of the three body sites, intra-individual specimens were significantly more similar than interindividual specimens during the monthly sampling period (P<0.001, Mantel test). This heightened intra-individual similarity decayed or disappeared entirely at three years, although conclusions are limited by the small number of animals.

Figure 7. Comparison of distal gut communities obtained from different mammalian host species.

(a) NMDS Bray–Curtis ordination of FL sequences from distal gut bacterial communities in 68 terrestrial and marine mammalian species, coloured according to host habitat. For these data sets, subsets of six individuals per host species (dolphins, sea lions and manatees) were chosen to provide a more balanced representation of marine mammal orders and diets. Specimens ‘100' (dugong24) and specimens starting with ‘Mt' (manatees, this study) were derived from herbivorous marine mammals (Order: Sirenia). Panels b,c show the same data as in a, coloured according to host diet (b) or mammalian phylogeny (Order) (c). See Supplementary Data 4 for more details on the individual data points and a key to the numbering shown in a. Supplementary Table 2 lists the most closely related communities to those from average dolphin and sea lion distal gut microbiotas.

Figure 8. CCA of mammalian distal gut communities.

Canonical Correspondence Analysis (CCA) plot showing the ordination of gut microbiotas from mammalian species (same as shown in Fig. 7, with subsets of six animals each for dolphins, sea lions and manatees) and bacterial taxa. Text in black displays the centroids of the three determinants that were tested, that is, habitat (aquatic and terrestrial), diet (carnivore, omnivore and herbivore) and host group (Carnivora, Cetartiodactyla, Primates and Other).