High diversity and unique composition of gut microbiomes in pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales

Abstract

Mammals host diverse bacterial and archaeal symbiont communities (i.e. microbiomes) that play important roles in digestive and immune system functioning, yet cetacean microbiomes remain largely unexplored, in part due to sample collection difficulties. Here, fecal samples from stranded pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales were used to characterize the gut microbiomes of two closely-related species with similar diets. 16S rRNA gene sequencing revealed diverse microbial communities in kogiid whales dominated by Firmicutes and Bacteroidetes. Core symbiont taxa were affiliated with phylogenetic lineages capable of fermentative metabolism and sulfate respiration, indicating potential symbiont contributions to energy acquisition during prey digestion. The diversity and phylum-level composition of kogiid microbiomes differed from those previously reported in toothed whales, which exhibited low diversity communities dominated by Proteobacteria and Actinobacteria. Community structure analyses revealed distinct gut microbiomes in K. breviceps and K. sima, driven by differential relative abundances of shared taxa, and unique microbiomes in kogiid hosts compared to other toothed and baleen whales, driven by differences in symbiont membership. These results provide insight into the diversity, composition and structure of kogiid gut microbiomes and indicate that host identity plays an important role in structuring cetacean microbiomes, even at fine-scale taxonomic levels.

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SCieNtifiC REPORTs | 7: 7205 | DOI:10.1038/s41598-017-07425-z
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High diversity and unique
composition of gut microbiomes in
pygmy (Kogia breviceps) and dwarf
(K. sima) sperm whales
Patrick M. Erwin , Ryan G. Rhodes, Kevin B. Kiser, Tiany F. Keenan-Bateman,
William A. McLellan & D. Ann Pabst
Mammals host diverse bacterial and archaeal symbiont communities (i.e. microbiomes) that play
important roles in digestive and immune system functioning, yet cetacean microbiomes remain largely
unexplored, in part due to sample collection diculties. Here, fecal samples from stranded pygmy
(Kogia breviceps) and dwarf (K. sima) sperm whales were used to characterize the gut microbiomes of
two closely-related species with similar diets. 16S rRNA gene sequencing revealed diverse microbial
communities in kogiid whales dominated by Firmicutes and Bacteroidetes. Core symbiont taxa were
aliated with phylogenetic lineages capable of fermentative metabolism and sulfate respiration,
indicating potential symbiont contributions to energy acquisition during prey digestion. The diversity
and phylum-level composition of kogiid microbiomes diered from those previously reported in toothed
whales, which exhibited low diversity communities dominated by Proteobacteria and Actinobacteria.
Community structure analyses revealed distinct gut microbiomes in K. breviceps and K. sima, driven by
dierential relative abundances of shared taxa, and unique microbiomes in kogiid hosts compared to
other toothed and baleen whales, driven by dierences in symbiont membership. These results provide
insight into the diversity, composition and structure of kogiid gut microbiomes and indicate that host
identity plays an important role in structuring cetacean microbiomes, even at ne-scale taxonomic levels.
Microorganisms form symbiotic relationships with nearly all animal taxa, from basal invertebrate phyla (e.g.
sponges)1 to humans2, and play important roles in the biology, ecology and evolution of animal life3. e struc-
tural and functional diversity of microbial communities (“microbiomes”) have been particularly well-studied in
mammals and shown to be a key determinant of health and disease4. Indeed, host-microbe interactions in the
mammalian gastrointestinal tract contribute signicantly to host metabolism and pathogen defense. e gut
microbiome provides essential nutrients (e.g. vitamins)5 and aids in the digestion of complex macromolecules
to deliver energy sources otherwise unavailable to animal hosts2. Furthermore, these microbes are crucial to the
development and regulation of the immune system6 and contribute to pathogen defense through colonization
resistance, competing for colonization sites and secreting substances that inhibit pathogen growth7. e study
of the complex bacterial and archaeal taxa that comprise mammalian gut microbiomes can thus provide insight
into host health and homeostasis, and is particularly timely for species threatened by anthropogenic disturbances,
such as marine mammals.
Marine mammals are emblematic and ecologically signicant members of coastal and pelagic ecosystems8 that
are represented in three mammalian orders (Carnivora, Cetartiodactyla and Sirenia), united by lifestyle rather
than evolutionary history. e cumulative eects of direct (e.g. hunting) and indirect (e.g. habitat exploitation)
anthropogenic impacts have led to worldwide declines in marine mammal populations, including extinction
events9, and have resulted in over 20% of extant species being classied as threatened or endangered10. Mass
mortality events from viral pathogens and secondary opportunistic infections11, 12 have prompted investigations
of infectious diseases, their impacts of cetacean populations and their zoonotic implications13. In addition to
the study of pathogenic bacteria and viruses, recent work has also focused on determining the composition and
Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington,
Wilmington, NC, 28409, USA. Correspondence and requests for materials should be addressed to P.M.E. (email:
erwinp@uncw.edu)
Received: 14 February 2017
Accepted: 28 June 2017
Published: xx xx xxxx
OPEN

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function of resident microbial communities inhabiting marine mammals. To date, most studies have focused on
mammals in the orders Sirenia (e.g. dugongs14, 15, manatees16) and Carnivora (e.g. seals17–19, sea lions20), while
cetaceans remain largely unexplored (whales, dolphins and porpoises, Order Cetartiodactyla)21, due in part to
diculties in in situ sample collection and the opportunistic nature of stranding events22. e emerging eld of
cetacean microbiology has largely focused on gut microbiome composition20, 23, 24, with recent metagenomic
surveys characterizing the putative functionality of these symbiotic communities25. Preliminary trends in the
structure of cetacean gut microbiomes include divergence between cetacean-associated and free-living micro-
bial communities20, a high degree of host-specicity, and lower diversity microbiomes in toothed whale species
compared to baleen whales25. Broader host taxon sampling is required to conrm these trends and apply this
knowledge towards conservation and rehabilitation eorts for threatened and endangered cetacean species26.
In this study, we characterized the gut microbiomes of two closely-related species in the cetacean genus Kogia,
K. breviceps (pygmy sperm whale) and K. sima (dwarf sperm whale), which exhibit similar gross morphology
and ecological niches in open ocean habitats27, 28. Both species have a shark-like appearance, characterized by a
blunt rostrum (or pointed snout), underslung jaw and cervical pigmentation pattern that resembles a gill slit28.
Historically, the shared morphological characteristics between these species have confounded taxonomic analy-
ses of the genus Kogia. Current species-level resolution of kogiid whales recognizes two species, based on mor-
phology and molecular evidence29–31, although at-sea identication remains challenging. In general, K. breviceps
exhibits greater body size than K. sima, with distinct dierences in dorsal n location, dorsal n height and exter-
nal “false gill slit” pigmentation between the two species28, 31, 32. It has been suggested that K. breviceps may dive
more deeply and be found further oshore than K. sima32, but these species display a high degree of dietary over-
lap that indicates very similar foraging ecologies27. Both species exhibit a worldwide distribution in tropical and
temperate o-shore waters and predominantly feed on cephalopods during extended, deep dives. Observations
at-sea of kogiid whales have been rare, due to their inconspicuous surface behavior and extended dive times, yet
K. breviceps and K. sima are among the most commonly stranded cetaceans along the southeastern U.S.33, 34. Here,
we examined fecal samples of stranded individuals of K. breviceps and K. sima to study kogiid gut microbiomes in
a comparative context controlling for broad dierences in diet and phylogenetic history. Our objectives were to
provide the rst characterization of kogiid gut microbiomes and to determine their host-specicity, by comparing
microbiome diversity and structure between kogiid species and across other toothed (odontocete) and baleen
(mysticete) whale hosts. We hypothesized that kogiid gut microbiomes would exhibit: (1) similar richness and
diversity compared to other toothed whale species, (2) lower richness and diversity compared to baleen whale
species, and (3) distinct community structure compared to other toothed and baleen whale species.
Results
Composition of cetacean gut microbiomes. A total of 1,720 symbiont OTUs were recovered from
K. breviceps (n = 1,368) and K. sima (n = 890), representing 11 bacterial phyla and one archaeal lineage (Fig.1,
TableS1). e gut microbiomes of K. breviceps and K. sima were dominated by the bacterial phyla Firmicutes
and Bacteroidetes, together accounting for >68% of the total gut microbial communities in each kogiid host.
Actinobacteria, Proteobacteria, Synergistetes and Verrucomicrobia were also common members of kogiid gut
microbiomes (Fig.1), with six additional rare phyla (<1% relative abundance) detected in both K. breviceps and
K. sima (TableS1). Notably, some dierentiation of gut microbiomes in K. breviceps and K. sima was observed
at the phylum level. In total, ve phyla exhibited dierential abundances between hosts, including signicantly
(P < 0.05) higher relative abundances of Bacteroidetes, Verrucomicrobia and Lentisphaerae in K. breviceps, and
Actinobacteria and Cyanobacteria in K. sima (TableS1).
Comparative analysis with other toothed and baleen whale species revealed that the dominance of the bacterial
phyla Firmicutes and Bacteroidetes in kogiid microbiomes was more characteristic of baleen whales than those of
other toothed whales (Fig.1). e vast majority (84 to 91%) of the gut microbiomes in baleen whale hosts (hump-
back whales, right whales, and sei whales) were comprised of Firmicutes and Bacteroidetes, whereas non-kogiid
toothed whale hosts were dominated by Proteobacteria (60% in bottlenose dolphins) and Actinobacteria (66% in
beluga whales, Fig.1). Despite these shared dominant phyla, several bacterial phyla distinguished kogiid micro-
biomes from those in baleen whales: Actinobacteria and Synergistetes were common in kogiid hosts (6–15 and
2–4%, respectively) while rare in baleen hosts (<2 and <0.2%, respectively), while Spirochaetes were common
members of baleen whale microbiomes (3–8%) and extremely rare in kogiid hosts (<0.03%).
Diversity of cetacean gut microbiomes. Gut microbiomes in kogiid whales exhibited high OTU-level
diversity, averaging 432 (±7 SE) and 416 (±18 SE) symbiont OTUs per host in K. breviceps and K. sima, respec-
tively. No signicant dierences in richness (S), diversity (1/D), evenness (E1/D) and dominance (d) were observed
in gut microbial communities between kogiid hosts (post hoc Tukey’s HSD tests, Fig.2), indicating similar
richness and evenness of gut microbiomes in these closely related species. Broader comparisons of diversity
including previously characterized cetacean gut microbiomes revealed that kogiid whales hosted microbial com-
munities with signicantly (P < 0.05) higher diversity than those occurring in other toothed whales (bottlenose
dolphins = 52 ± 18 OTUs, beluga whales = 84 ± 8, post hoc Tukey’s HSD tests, Fig.2). Similarly, a single OTU
dominated the microbial communities of bottlenose dolphins (67%) and beluga whales (74%), while the most
abundant OTU accounted for less than a quarter of the microbiome in K. breviceps (23%) and K. sima (15%). In
contrast, kogiid gut microbiomes did not dier signicantly in richness, diversity and dominance compared to
the gut microbial communities inhabiting three baleen whale species (post hoc Tukey’s HSD tests, Fig.2).
Community structure of cetacean gut microbiomes. Despite similarities in community diversity, the
gut microbiomes in K. breviceps and K. sima exhibited signicant dierences in community structure according
to both OTU-based and phylogenetic beta-diversity metrics (PERMANOVA, P < 0.01, Table1), indicating that

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distinct microbial communities inhabit each host species. ese dierences were visible in cluster plots based on
microbiome similarity, which grouped kogiid individuals by host species (Fig.3). Importantly, these structural
dierences were driven by dierences in the relative abundance of shared symbiont taxa, as analyses based on
symbiont membership revealed no signicant dierences (PERMANOVA, P > 0.11) in microbiome community
structure between kogiid hosts for either beta-diversity metric (Table1, Fig.S1) and no host-specic clustering of
microbiomes in cluster plots (Fig.S1). Indeed, the 538 OTUs shared between K. breviceps and K. sima accounted
for >99% of the recovered sequences, while the unique OTUs in each hosts’ microbiome (n = 830 in K. breviceps,
n = 352 in K. sima) were extremely rare (in total, representing <1% of all sequences). Signicant dierences in
dispersion were detected between the microbiomes of K. breviceps and K. sima (PERMDISP, P < 0.01, TableS2),
indicating greater intra-specic variability in microbial communities among K. sima individuals. Again, these
dierences were driven by variability in the relative abundance of shared symbionts, as analyses based on symbi-
ont membership revealed no signicant dierences in dispersion (PERMDISP, P > 0.05, TableS2). No signicant
dierences in the structure of kogiid gut microbiomes were detected based on sex (ANOSIM, P > 0.18) or carcass
condition (P > 0.36, TableS3).
e high degree of host-specicity exhibited by kogiid gut microbiomes was also observed across more dis-
tantly related host species, including other toothed whales and baleen whales, as cluster plots based on microbial
community similarity grouped all individuals by host species (Fig.3). Host species accounted for the majority
(69%) of variation observed between samples and signicant dierences between hosts were observed for most
pairwise comparisons (Table1), with exceptions generally involving pairwise comparisons with low statistical
power (i.e. hosts with low replication, T. truncatus and D. leucas). Dierences between the gut microbiomes of
these more distantly related cetacean hosts were driven by symbiont membership, rather than relative abun-
dance of shared taxa alone, as analyses of presence-absence data revealed a higher proportion of variation (83%)
explained by host species and more distinct clustering of host species in cluster plots (Fig.S1).
Dominant symbiont OTUs in kogiid gut microbiomes. Core microbiomes in kogiid whales were iden-
tied as symbiont OTUs detected in all samples of each host species, thereby excluding potentially transient mem-
bers of the recovered communities. Core microbiomes were comprised of 147 and 155 OTUs in K. breviceps and
K. sima, respectively, and represented a small fraction of overall symbiont diversity (10.7% in K. breviceps, 17.4%
in K. sima). However, core OTUs also represented dominant symbiont taxa and accounted for the vast majority of
overall symbiont communities (96.2 to 97.4%). A high degree of overlap in symbiont membership was observed
when comparing the core communities in K. breviceps and K. sima. e shared component of core microbiomes
Figure 1. Phylum-level composition of gut microbiomes in Kogia breviceps and K. sima compared with
previous data from other toothed and baleen whales25. Relative abundances of common microbial phyla are
shown in color, with rare phyla (other) in black (Fusobacteria, Euryarchaeota, Lentisphaerae, Cyanobacteria,
Planctomycetes, Fibrobacteres, Deferribacteres, TM7, TM6, Acidobacteria, Elusimicrobia, Armatimonadetes,
ermi, and ermotogae).

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in these two kogiid hosts was comprised of 115 OTUs that accounted for most of the sequence reads recovered
from K. breviceps (95.6%) and K. sima (94.0%, TableS4).
e 25 most common OTUs in kogiid microbiomes were examined in further detail to compare their rel-
ative abundances between kogiid hosts and presence in the microbiomes of other toothed and baleen whales
(Table2). All 25 OTUs represented core members of both kogiid species microbiomes, yet exhibited dierent rel-
ative abundances in each host. Together, these 25 OTUs contributed to 66.3% of the variation observed between
the microbiomes of K. breviceps and K. sima, with 14 OTUs (56%) exhibiting signicantly dierent abundances
between kogiid hosts (Table2). Eighteen (72.0%) of these OTUs were unique to kogiid microbiomes (i.e., not
detected in the other toothed and baleen hosts included herein) and 15 OTUs (60%) exhibited greater than 3%
sequence divergence from any previously characterized sequence (GenBank database), likely representing novel
bacterial species. Combined with the unresolved taxonomic status of some dominant OTUs, these results indicate
that several novel taxa comprise kogiid microbiomes and are most closely related to symbionts identied in gut
communities of other mammalian hosts (Table2). For example, the most abundant OTU in both kogiid hosts
(OTU1) was unique to kogiid microbiomes, unclassied below the phylum level (Bacteroidetes) and exhibited
over 9% sequence divergence from the most closely related bacterium, a symbiont characterized from elephant
(Elephas maximus) feces (Table2).
e few symbiont OTUs shared between gut communities of kogiid and other cetacean hosts were generally
in very low abundance in one or both hosts, with notable exceptions. OTU2 (Firmicutes, Peptostreptococcaceae)
was a dominant member of the microbiomes in both kogiid hosts (10.2% in K. breviceps, 6.7% in K. sima) as
well as bottlenose dolphins (18.5%). Mycobacterium arupense (Actinobacteria, OTU4) and Oscillospira sp.
(Firmicutes, OTU8) were common in K. breviceps (3.8 and 2.6%, respectively) and K. sima (1.3 and 1.0%), with
the former symbiont a dominant member of beluga whale microbiomes (65.3%) and the latter symbiont a domi-
nant member of humpback whale microbiomes (14.5%).
Discussion
Characterization of gut microbiomes in pygmy (K. breviceps) and dwarf (K. sima) sperm whales revealed diverse
and host-specic microbial communities inhabiting kogiid whales. Gut microbiomes in K. breviceps and K.
sima exhibited similar levels of richness and evenness, along with high overlap in symbiont membership, yet
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K. breviceps
K. sima
M. novaeangliae
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D. leucas
T. truncatus
K. breviceps
K. sima
M. novaeangliae
E. glacialis
B. borealis
seicepStsoHseicepStsoH
To othed whales Baleen whales Toothed whales Baleen whales
Figure 2. Diversity metrics of kogiid gut microbiomes compared with previous data from other toothed
(gray bars) and baleen (black bars) whales25. Dierent letters above bars denote signicantly dierent means
among host species (P < 0.05). No signicant dierences in evenness occurred between host species (n.s. = not
signicant). Error bars represent ±1 standard deviation.

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were dierentiated by the relative abundance of shared symbiont taxa. As a result, distinct microbial community
structure was detected between kogiid species, as well as, among kogiid microbial communities and previously
characterized gut microbiomes of other toothed and baleen whales. Notably, these latter dierences were driven
by dierences in community membership and several dominant kogiid-associated OTUs exhibited high levels
of dierentiation from any described bacteria, indicating the presence of unique symbiont taxa in these hosts.
Kogiid gut microbiomes exhibited unique structure and composition and a high-degree of host-specicity, thus
highlighting the importance of host identity in structuring cetacean microbiomes.
e diversity of gut microbial communities in kogiid whales was unexpectedly high, as previous investiga-
tions of toothed whale gut microbiomes revealed low levels of phylum- and OTU-level diversity. Symbiont OTU
richness in K. breviceps (432 ± 7) and K. sima (416 ± 18) was nearly ve times higher than the gut microbiomes of
bottlenose dolphins (T. truncatus 52 ± 8) and beluga whales (D. leucas 84 ± 18) included in comparative analyses
Pairwise Comparison
Bray-Curtis Similarity UniFrac Distance
Rel. Abund. Presence-Abs. Weighted Unweighted
t P t P t P t P
K. breviceps - K. sima 2.405 0.001*1.350 0.119 2.906 0.001*1.187 0.145
K. breviceps - D. leucas 6.531 0.001*15.29 0.001*5.702 0.001*2.228 0.003*
K. breviceps - E. glacialis 10.405 0.001*46.88 0.001*5.006 0.001*3.085 0.001*
K. breviceps - M. novaeangliae 6.895 0.001*25.44 0.001*3.681 0.002*2.304 0.002*
K. breviceps - T. truncatus 4.045 0.001*8.223 0.001*3.287 0.001*2.143 0.003*
K. sima - D. leucas 3.509 0.005*9.296 0.001*2.844 0.010*1.856 0.033
K. sima - M. novaeangliae 3.854 0.001*15.827 0.001*1.927 0.025 1.900 0.025
K. sima - E. glacialis 6.071 0.001*30.62 0.001*3.014 0.001*2.447 0.001*
K. sima - T. truncatus 2.270 0.022 4.927 0.002*1.704 0.065 1.770 0.034
E. glacialis - D. leucas 5.831 0.001*11.558 0.001*3.877 0.001*2.061 0.005*
E. glacialis - M. novaeangliae 3.440 0.001*7.181 0.001*1.609 0.079 1.625 0.029
E. glacialis - T. truncatus 3.495 0.004*6.253 0.001*2.633 0.002*1.968 0.007*
M. novaeangliae - D. leucas 3.788 0.004*6.231 0.002*2.337 0.032 1.553 0.098
M. novaeangliae - T. truncatus 1.964 0.070 3.122 0.014*1.530 0.136 1.445 0.174
T. truncatus - D. leucas 1.770 0.141 2.321 0.075 1.121 0.379 1.108 0.386
Table 1. Pairwise statistical comparisons of microbial community structure (PERMANOVA) across cetacean
hosts, based on OTU-dependent (Bray Curtis) and OTU-independent (UniFrac) metrics of relative abundance
(Rel. Abund., Weighted) and presence-absence (Presence-Abs., Unweighted) data. Asterisks (*) indicate
signicant dierences following B-Y corrections.
Eubalaenaglacialis F16
Eubalaenaglacialis F9
Eubalaenaglacialis F2
Eubalaenaglacialis F5
Eubalaenaglacialis F8
Eubalaenaglacialis F11
Eubalaenaglacialis F12
Balaenoptera borealis JS1
MegapteranovaeangliaeJS10
MegapteranovaeangliaeJS11
MegapteranovaeangliaeJS9
Tursiops truncatusJS13
Tursiops truncatusJS14
Delphinapterus le ucas JS16
Delphinapterus le ucas JS17
KogiasimaK13
KogiasimaK11
KogiasimaK10
KogiasimaK12
Kogiabreviceps K02
Kogiabreviceps K07
Kogiabreviceps K06
Kogiabreviceps K09
Kogiabreviceps K04
Kogiabreviceps K08
Kogiabreviceps K01
Kogiabreviceps K03
Kogiabreviceps K05
Microbiome Similarity (Bray-Curtis, %)
Pygmy
Sperm Whales
Dwarf
Sperm Whales
Beluga Whales
Bottlenose Dolphins
Humpback
Whales
Right Whales
SeiWhale
Toothed Whales Baleen Whales
020406080100
Figure 3. Similarity of gut microbiomes in Kogia breviceps and K. sima compared with previous data from
other toothed and baleen whales25 based on relative abundance OTU data. Shaded boxes delineate toothed
whale species (light gray) and baleen whale species (dark gray).

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herein. In contrast to the kogiid colonic content sampled from stranded wild individuals, fecal samples from
bottlenose dolphins and beluga whales were collected following defecation by captive individuals25. However,
similarly low diversity communities have been reported in other toothed whale species, including rectal swabs
of wild (36 ± 2) and captive (41 ± 8) bottlenose dolphins20, striped dolphins (Stenella coeruleoalba, 75 OTUs)23
and colonic contents of the Yangtze nless porpoise (Neophocaena phocaenoides asiaeorientalis, 30 OTUs)24, indi-
cating that dierent sampling methods and host status (wild vs. captive) alone do not account for the observed
dierences in microbiome richness between kogiids and other toothed whales. As such, kogiid whales appear
to represent a more fertile microbial habitat compared to other toothed whale species, possibly related to the
unique colonic sac that enlarges the large intestine of kogiid whales (e.g. 50 L of feces were extracted from a
295 cm individual K. breviceps)35. e presence of this colonic feature, and its potential to sequester feces for
extended periods of time, are also hypothesized to contribute to the detectability of the neurotoxin domoic acid in
kogiids36. Otherwise, kogiid gastrointestinal tracts exhibit similar morphology to those of most other odontocetes
(including Physeter macrocephalus) and mysticetes, with a multi-chambered stomach consisting of single fore-,
main and pyloric chambers37. Interestingly, the low diversity of gut microbiomes in non-kogiid toothed whales
contrasts with other body sites of these animals, where high diversity microbial communities have been reported.
For example, the oral microbiome of bottlenose dolphins displayed twice the phylum-level and four times the
OTU-level diversity compared to their gut microbiomes20. Whether the observed dierences in microbiome
diversity between kogiid and other toothed whales translate into dierences in community functionality and
stability (e.g. via functional redundancy) are important questions for understanding host-symbiont interactions
and will require additional metagenomic and cultivation-based studies of whale microbiomes.
Kogiid whale gut microbiomes were dominated by symbiont taxa aliated with the phyla Firmicutes and
Bacteroidetes, two common lineages in mammalian gut communities. Members of the phylum Firmicutes were
rare in some non-kogiid toothed whale hosts, for example, representing <2% of gut communities in beluga
whales. In bottlenose dolphins, high variability was observed in the relative abundance of Firmicutes symbi-
onts (<1 to 55%), though recent work indicates that Firmicutes are indeed dominant members of the bottle-
nose dolphin gut microbiome20, 38. In contrast, Bacteroidetes were greatly reduced (<2%) in gut microbiomes
of both non-kogiid toothed whales investigated herein, consistent with previous studies20, 38, while comprising
a large fraction of symbiont communities in K. breviceps (31%) and K. sima (13%). Bacteroidetes are common
members of herbivorous14–16 and carnivorous18, 39, 40 marine mammal microbiomes and are well known for their
ability to degrade high molecular weight organic compounds, including complex polysaccharides41. Accordingly,
OTU Phylum (lowest taxonomy) K. breviceps K. sima % Contrib. Unique BLAST Match Source (Identity, Acc. No.)
*000001 Bacteroidetes (unclassied) 22.98 ± 2.12 9.72 ± 3.66 10.87 Ye s Elephant Feces (90.6, EU471682)
*000007 Firmicutes (o_Clostridiales) 3.33 ± 1.29 7.09 ± 1.28 4.14 Ye s Dolphin Rectum (96.0, JQ204280)
*000010 Firmicutes (f_Mogibacteriaceae) 5.07 ± 0.46 1.64 ± 0.95 1.97 Ye s Sea Lion Rectum (94.0, JQ207359)
*000018 Synergistetes (f_Synergistaceae) 3.78 ± 0.43 0.14 ± 0.08 2.94 Ye s Zebra Feces (92.4, EU470284)
*000021 Bacteroidetes (o_Bacteroidales) 3.22 ± 0.62 0.10 ± 0.04 2.52 Ye s Bovine Colon (90.5, JX096352)
*000008 Firmicutes (g_Oscillospira) 2.55 ± 0.45 0.97 ± 0.34 1.34 No Equine Manure (100, AY212772)
*000026 Proteobacteria (Campylobacter f etus) 2.62 ± 1.41 0.04 ± 0.00 2.08 Ye s Human Feces (99.2, CP015575)
*000030 Verrucomicrobia (f_RFP12) 2.26 ± 0.53 0.11 ± 0.07 1.73 Yes Sediment (92.6, GU453511)
*000020 Actinobacteria (f_Coriobacteriaceae) 0.25 ± 0.03 4.11 ± 1.85 3.1 Ye s Bovine Rumen (91.7, KT172105)
*000022 Actinobacteria (g_Adlercreutzia) 0.40 ± 0.05 3.63 ± 1.08 2.59 Ye s Swine Feces (93.3, KP102484)
*000039 Firmicutes (f_Ruminococcaceae) 1.57 ± 0.52 0.24 ± 0.06 1.07 Yes Chicken Feces (96.0, JQ248085)
*000038 Firmicutes (g_Butyrivibrio) 1.52 ± 0.20 0.29 ± 0.04 0.99 Yes Sea Lion Rectum (96.0, JQ208575)
*000047 Bacteria (unclassied) 1.40 ± 0.52 0.05 ± 0.01 1.1 Ye s Anaerobic Digester (90.1, KF631052)
*000058 Firmicutes (o_Clostridiales) 1.17 ± 0.81 0.05 ± 0.01 0.92 Ye s Swine Feces (96.5, KP107340)
000002 Firmicutes (f_Peptostreptococcaceae) 10.20 ± 2.29 6.67 ± 3.35 6.17 No Dolphin Rectum (98.8, JQ202598)
000005 Firmicutes (Clostridium perfringens) 1.65 ± 0.86 8.63 ± 5.37 6.79 No Human Feces (100, KX674026)
000004 Actinobacteria (Mycobacterium arupense) 3.83 ± 1.27 1.32 ± 0.56 2.7 No Porpoise Feces (100, JN792395)
000013 Bacteria (unclassied) 1.32 ± 0.43 3.81 ± 1.86 2.6 Ye s Dolphin Rectum (98.8, JQ203364)
000025 Bacteroidetes (unclassied) 1.92 ± 0.78 1.57 ± 1.42 1.89 Ye s Bovine Rumen (92.1, AB616513)
000016 Firmicutes (g_Oscillospira) 1.45 ± 0.21 2.38 ± 1.47 1.51 Ye s Human Feces (100, HQ808319)
000034 Bacteroidetes (unclassied) 1.52 ± 0.40 0.39 ± 0.21 1.02 Ye s Bovine Rumen (90.2, GQ327094)
000033 Proteobacteria (g_Citrobacter) 0.10 ± 0.04 3.29 ± 3.23 2.63 No Human Feces (100, CP016762)
000037 Firmicutes (f_Mogibacteriaceae) 0.78 ± 0.06 1.63 ± 0.82 0.92 Ye s Sea Lion Rectum (96.4, JQ207359)
000029 Firmicutes (Clostridium perfringens) 0.08 ± 0.03 2.60 ± 2.10 2.06 No Dolphin Rectum (100, JQ202064)
000049 Firmicutes (Faecalibacterium prausnitz ii) 0.94 ± 0.24 0.61 ± 0.42 0.68 No Sea l Colon (99.2, GQ867580)
Table 2. Common OTUs in the gut microbiomes of Kogia breviceps and K. sima, showing phylum- and lowest-
level taxonomy, relative abundances, and percentage contributions to dissimilarity between hosts (SIMPER
analysis). Unique OTUs were those not detected in other whale species (comparative analysis herein). BLAST
matches show the source and identity (%) of the closest known relative for each OTU. Asterisks (*) denote
dierentially abundant OTUs between kogiid hosts.

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these symbionts are thought to play a key role in the digestive health of mammals and their absence in many
toothed whale species may reect alternative nutrient acquisition strategies. Importantly, Bacteroidetes have been
reported as common (even dominant) in other body sites of toothed whale hosts, including the oral, upper gastric
and respiratory microbiomes of bottlenose dolphins20, thus their absence in the lower gut may indicate environ-
mental selection against these taxa in this particular microhabitat of these hosts.
e structural determinants of gut microbiome composition in marine mammals include age, diet and phy-
logenetic position17, 20, 25, similar to studies of terrestrial counterparts42, 43. In this study, the similar foraging niches
and dietary overlap between K. breviceps and K. sima27 allowed for the investigation of cetacean microbiomes in
a comparative context controlling for broad dierences in diet and isolating host-specic factors. Our results
suggest that host identity plays an important role in structuring cetacean microbiomes, even at ne-scale taxo-
nomic levels, as distinct microbiome structure was observed between closely-related kogiid hosts. In contrast,
host sex and carcass condition showed no signicant eects on microbiome structure in kogiid whales. Evidence
to date indicates that sex is not an important structuring factor for marine mammal microbiomes, as past studies
have similarly reported no signicant eect of sex on gut microbiomes of bottlenose dolphins20, leopard seals
(Hydrurga leptonyx)17, manatees (Trichechus manatus)16 and dugongs (Dugong dugon)14. An exception to this
trend occurs in elephant seals (Mirounga leonina) which, in contrast to the aforementioned species, exhibit pro-
nounced sexual size dimorphism hypothesized to drive observed dierences in gut microbiomes between males
and females17. Future studies of gut microbial communities in cetacean species that display drastic sexual size
dimorphism (e.g. the true sperm whale, Physeter macrocephalus) will yield further insight into the impacts of
sex-specic variations in body size and foraging behavior on gut microbiome structure. Importantly, no eect of
carcass condition was recovered herein. ese results are consistent with recent studies of postmortem mammal
microbiomes (e.g. murine models)44 showing that endogenous gut bacteria continue to dominate gastrointesti-
nal communities until advanced stages of decomposition, specically intestinal rupture and the exposure of the
abdominal cavity to oxygen. e postmortem stability of gut-associated communities indicates that stranded
whales in early stages of decomposition can be used to provide accurate representations of cetacean gut microbi-
omes in wild ranging animals.
e taxonomic composition of kogiid gut microbiomes also oers insight into the putative functionality of
gut-associated bacteria, as several core symbiont taxa were classied into bacterial lineages with known physio-
logical capabilities. Facultative and obligate anaerobes with fermentative metabolism were common, including
members of the families Ruminococcaceae (Faecalibacterium, Oscillospira), Enterobacteriaceae (Citrobacter)
and Lachnospiraceae (Buytrivibrio). Microbial fermentation transforms undigested dietary components into
end-products readily metabolized by mammal hosts, namely short-chain fatty acids (SCFA). Similar to rumi-
nant mammals, cetacean gastrointestinal tracts include a non-glandular forestomach that likely functions as a
fermentation vessel, based on cultivation studies of forestomach bacteria45, SCFA proling of this chamber46 and
metagenomic characterization of carbon metabolism in whales25. e taxonomic data herein, combined with
past evidence of fermentation in sperm whales (based on bile acid composition)47, indicates that fermentative
metabolism occurs in the gastrointestinal tract of K. breviceps and K. sima and potentially contributes to energy
acquisition during prey digestion. Other core members of kogiid gut microbiomes were aliated with sulfate
reducing bacteria, including Desulfovibrio (Desulfovibrionaceae) and Desulfomonile (Syntrophaceae), which may
persist in the anaerobic habitat of whale intestines by deriving energy from the respiration of sulfate present in
small volumes of seawater ingested with prey. Finally, several putative pathogens were detected in K. breviceps
and K. sima, including Campylobacter fetus, Clostridium perfringens and Mycobacterium arupense. e clinical
relevance of these bacteria is unclear, although their role as opportunistic pathogens in other hosts48, taxonomic
distinction from pathogens reported for toothed whale species49 and prevalence in diverse cetacean hosts50 sug-
gest low virulence and potentially a greater threat to immunocompromised individuals.
In summary, this study characterized the structure and taxonomic composition of gut microbiomes of kogiid
whales, a critical rst step in understanding the role of cetaceans as microbial habitats and the contributions of
symbiont communities to host metabolism and health. e selective pressures that contribute to the establish-
ment and maintenance of host-specic microbiomes in kogiid whales may include unique microhabitats within
each host, functional contributions of specic microbial guilds to host tness, or a combination of both phenom-
ena. Future studies targeting how these diverse, host-specic microbiomes form (e.g. comparative analyses across
cetacean life stages) and their physiological characteristics (e.g. metagenomic and culture-based investigations)
will provide further insight into microbiome development and functionality. Ultimately, a holistic understanding
of host-microbe interactions in kogiid whales may yield insights into the impacts of gut dysbiosis on health and
aid in health assessments and rehabilitation eorts for these species, which currently exhibit very low success rates
due to mortality from gastric and intestinal stasis35.
Methods
Ethics Statement. All research activities were carried out under a NOAA Stranding Agreement to UNCW
and research protocols were approved by UNCW’s Institutional Animal Care and Use Committee (protocols
A0809-019, A1112-013 and A1415-015). ere is considerable uncertainty surrounding the status of Kogia brev-
iceps and K. sima, with both species categorized as “Data Decient” on the IUCN Red List of reatened Species
(http://www.iucnredlist.org). is study relied solely upon postmortem sampling of stranded kogiid whales
from North Carolina, responded to under authorization of the US Marine Mammal Protection Act. Animals
were either found dead (n = 4), died during initial response (n = 4) or underwent humane euthanasia (n = 5)
for reasons unrelated to this study following consultation with the National Marine Fisheries Service and under
the supervision of a licensed veterinarian in accordance with the American Veterinary Medical Association
Guidelines for the Euthanasia of Animals (2013 Edition).

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Sample Collection, DNA Extraction and Illumina Sequencing. Colonic fecal samples were collected
during necropsy of stranded individuals of K. breviceps (n = 9) and K. sima (n = 4) from the mid-Atlantic United
States (North Carolina, Table3, Fig.4) between 2009 and 2014 and stored at −80 °C. All sampled individuals were
adults (length range = 220–328 cm, weight range = 184–515 kg) stranded in fresh to moderate carcass condition
(Carcass Classication Codes 1–3)51 and exhibited no signs of direct, human-induced mortality (e.g. boat colli-
sions, shery interactions). Sampled individuals included both sexes (Table3).
DNA extracts were prepared from 200 mg of fecal material using the Powersoil DNA Extraction kit
(MoBio), following Earth Microbiome Project standard protocols (http://press.igsb.anl.gov/earthmicrobi-
ome/protocols-and-standards/dna-extraction-protocol/). DNA extracts were sent to Molecular Research LP
(Shallowater, TX) for amplication, library construction and multiplexed sequencing of partial (V4) 16S rRNA
gene sequences on an Illumina MiSeq platform. DNA extracts were used as templates for PCR amplications
using the HotStarTaq Plus Master Mix kit (Qiagen) and the universal bacterial/archaeal forward primer 515 f
and reverse primer 806r52, with a multiplex identier barcode on the forward primer. ermocycler conditions
consisted of an initial denaturation step at 94 °C for 3 min; 28 cycles of 94 °C for 30 s, 53 °C for 40 s, and 72 °C for
1 min; and a nal elongation step at 72 °C for 5 min. Samples were pooled in equimolar concentrations, puried
using Agencourt Ampure XP beads (Beckman Coulter) and sequencing on an Illumina MiSeq following the man-
ufacturer’s guidelines. For comparative analyses, previously characterized cetacean gut microbiome datasets25
were downloaded for bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), hump-
back whales (Megaptera novaeangliae), right whales (Eubalaena glacialis) and sei whales (Balaenoptera borealis,
TableS5). ese datasets were constructed using the same extraction kit, primer pair and sequencing platform as
the data generated herein.
DNA Sequence Processing. Combined datasets from this study and Sanders et al.25 were processed
simultaneously in mothur53 using a modied version (full details and mothur code in TableS6) of a previ-
ously described pipeline54. Briey, sequence reads were quality-ltered, aligned to the SILVA database (release
119, non-redundant, mothur-formatted) and trimmed to the V4 region, screened for sequencing anoma-
lies (e.g. chimeras) and errors, then assigned to taxonomic groups using a naïve Bayesian classier55 and
Greengenes taxonomy (May 2013 release, mothur-formatted). Following the removal of non-target amplicons
(chloroplasts, mitochondria and eukaryotic reads), the remaining high quality sequences were clustered into
operational taxonomic units (OTUs) at 97% sequence identity (average neighbor algorithm) and taxonomy
assigned to each OTU by majority consensus56. Singleton OTUs (occurring once in the global dataset) were
removed and datasets were subsampled to lowest read count (n = 24, 389) to avoid artifacts of varied sam-
pling depth on diversity calculations. Raw sequence data were deposited as FASTQ les in the Sequence
Read Archive of the National Center for Biotechnology Information (SRA NCBI) under the accession no.
SRP097888.
Statistical Analyses. To compare community diversity among cetacean hosts, alpha-diversity indices were
calculated for OTU richness (observed richness, S), diversity (inverse Simpson, 1/D), evenness (Simpson, E1/D)
and dominance (Berger-Parker, d), as implemented in mothur. Analyses of variance (ANOVAs) were used to
compare diversity index means across cetacean host species, followed by Tukey’s honest signicant dierence
(HSD) tests for multiple pairwise post hoc comparisons.
To compare community structure among cetacean hosts, beta-diversity indices were calculated based
on an OTU-dependent metric (Bray-Curtis similarity) and an OTU-independent (i.e. phylogenetic) met-
ric (UniFrac distance)57. Two iterations of each beta-diversity metric were performed to differentiate
between the effects of taxon abundance (OTU relative abundance Bray-Curtis, weighted UniFrac) and
Lab
ID Field ID Species Sex Pregnant Length
(cm) Weight
(kg) Strand Date Latitude Longitude Condition*
K1 KLC-113 K. breviceps FYes 286 n.a. 16-Sep-2011 36.04357 N −075.67401 W 1, 2
K2 KLC-135 K. breviceps FNo 252.5 183.6 5-Oct-2012 35.67985 N −075.48023 W 1, 2
K3 NCARI-012 K. breviceps FYes 296 n.a. 14-Oct-2011 36.41728 N −075.83416 W 1, 2
K4 KLC-211 K. breviceps FNo 295 314.8 16-Sep-2014 35.87716 N −075.57759 W 1, 2
K5 KLC-106 K. breviceps MNo 261 257.6 4-May-2011 36.04387 N −075.67439 W 1, 2
K6 SWT-009 K. breviceps MNo 328.5 515 9-Dec-2012 33.87536 N −077.95721 W 1, 3
K7 WAM-644 K. breviceps MNo 307 392 16-Aug-2008 33.90623 N −078.34224 W 1, 2
K8 MDB-056 K. breviceps MNo 263.5 n.a. 15-Dec-2009 35.71547 N −075.49213 W 2, 3
K9 KLC-212 K. breviceps MNo 293.5 418 1-Oct-2014 36.06975 N −075.69198 W 1, 2
K10 CAHA-002 K. sima FNo 233.5 n.a. 24-Aug-2010 35.35057 N −075.49969 W 2, 3
K11 CAHA-065 K. sima MNo 226 187.3 6-Jul-2011 35.77310 N −075.52691 W 1, 2
K12 CAHA-003 K. sima MNo 236.5 n.a. 24-Aug-2010 35.44597 N −075.48259 W 2, 3
K13 CAHA-004 K. sima MNo 220 n.a. 25-Aug-2010 35.45683 N −075.48248 W 2, 3
Table 3. Stranded individuals of K. breviceps and K. sima examined in this study and associated metadata
for each sample (n.a. = data not available). *Carcass condition at stranding (le) and at examination (right):
1 = Alive, 2 = Fresh Dead, 3 = Moderate Decomposition.

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taxon membership (OTU presence-absence Bray-Curtis, unweighted UniFrac) on the structure of cetacean
microbiomes. Bray-Curtis similarity values were calculated from OTU tables in PRIMER (version 6.1.11)
and visualized in cluster dendrograms. UniFrac distances were calculated in mothur based on an approxi-
mate maximum-likelihood phylogeny constructed in FastTree 2.1.558 with unique sequence reads (n = 52,
867). Permutational multivariate ANOVAs (PERMANOVA+, version 1.0.1) were used to test for dierences
in microbiome structure among host species, with significance determined by Monte Carlo asymptotic
P-values corrected for multiple pairwise comparisons (Benjamini-Yekutieli false-discovery rate control59 and
an experiment-wise error rate of α = 0.05), and to estimate components of variation ascribed to the factor
‘host species’ and to residual variation. Permutation multivariate analyses of dispersion (PERMDISP) were
conducted to test for homogeneity of multivariate dispersions (i.e. deviations from centroids) among host
species, with signicance determined by permutational P-values similarly corrected for multiple pairwise
comparisons.
Additional analyses of kogiid microbiome data were conducted to identify individual symbiont taxa contrib-
uting to community-level dierences in microbiome structure between K. breviceps and K. sima. At the phylum
level, signicant dierences in the relative abundance of bacterial phyla between kogiid hosts were determined
using Student’s t tests. At the OTU level, a one-way similarity percentage species contributions (SIMPER) analysis
was conducted to investigate the contribution of each symbiont OTU to the observed community dissimilarity
between kogiid hosts. In addition, symbiont OTUs that were dierentially abundant between kogiid hosts were
identied using Metastats (non-parametric t tests)60 and LefSe (non-parametric Kruskal-Wallis sum-rank tests)61.
To test for microbiome dierentiation based on sex and carcass condition, separate two-way analyses of similarity
(ANOSIM) were conducted for the factors ‘sex’ and ‘carcass condition’, each crossed with the factor ‘host species’
to control for dierences between kogiid species. To exclude potentially transient members of kogiid symbi-
ont communities, core microbiomes were identied for each kogiid host and strictly dened as symbiont OTUs
detected in all samples within a host species.
Figure 4. Stranded individuals of Kogia breviceps (A) and K. sima (B). Photo credits: UNCW Marine Mammal
Stranding Program (A) and the Virginia Aquarium (B).

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Acknowledgements
For access to samples, we thank the North Carolina Wildlife Resources Commission (Karen Clark), Cape
Hatteras National Seashore (Michelle Bogardus, Paul Doshkov), Virginia Aquarium and Marine Science Center
Foundation (Susan Barco) and volunteers of the UNCW Marine Mammal Stranding Program. Work carried out
under NOAA Stranding Agreement to UNCW, UNCW IACUC Protocols A0809-019, A1112-013, and A1415-
015. Stranding response supported in part by NOAA Prescott Grants to UNCW.
Author Contributions
P.M.E., R.G.R., K.B.K., T.F.K., W.A.M., D.A.P. designed research: T.F.K., W.A.M., D.A.P. collected samples; P.M.E.
analyzed data; P.M.E., R.G.R., K.B.K., T.F.K., W.A.M., D.A.P. wrote the paper.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-07425-z
Competing Interests: e authors declare that they have no competing interests.
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