on March 22, 2017http://rsbl.royalsocietypublishing.org/Downloaded from rsbl.royalsocietypublishing.orgResearch
Cite this article: Ralls K, McInerney NR,
Gagne RB, Ernest HB, Tinker MT, Fujii J,
Maldonado J. 2017 Mitogenomes and
relatedness do not predict frequency of
tool-use by sea otters. Biol. Lett. 13:
20160880.
http://dx.doi.org/10.1098/rsbl.2016.0880Received: 7 November 2016
Accepted: 28 February 2017Subject Areas:
behaviour, ecology, evolution
Keywords:
tool, sea otter, mitogenome, diet specializationAuthor for correspondence:
Katherine Ralls
e-mail: rallsk@thegrid.netElectronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.fig-
share.c.3711958.& 2017 The Author(s) Published by the Royal Society. All rights reserved.Evolutionary biology
Mitogenomes and relatedness do not
predict frequency of tool-use by sea otters
Katherine Ralls1, Nancy Rotzel McInerney1, Roderick B. Gagne2,
Holly B. Ernest2, M. Tim Tinker3, Jessica Fujii4 and Jesus Maldonado1
1Center for Conservation Genomics, Smithsonian Conservation Biology Institute, Washington, DC 20008, USA
2Wildlife Genomics and Disease Ecology, Department of Veterinary Sciences, University of Wyoming,
Laramie, WY 82071, USA
3US Geological Survey, Western Ecological Research Center, Santa Cruz, CA 95060, USA
4Monterey Bay Aquarium, Monterey, CA 93940, USA
RBG, 0000-0002-4901-5081; HBE, 0000-0002-0205-8818
Many ecological aspects of tool-use in sea otters are similar to those in
Indo-Pacific bottlenose dolphins. Within an area, most tool-using dolphins
share a single mitochondrial haplotype and are more related to each other
than to the population as a whole. We asked whether sea otters in California
showed similar genetic patterns by sequencing mitogenomes of 43 otters
and genotyping 154 otters at 38 microsatellite loci. There were six variable
sites in the mitogenome that yielded three haplotypes, one found in only a
single individual. The other two haplotypes contained similar percentages
(33 and 36%) of frequent tool-users and a variety of diet types. Microsatellite
analyses showed that snail specialists, the diet specialist group that most
frequently used tools, were no more related to each other than to the popu-
lation as a whole. The lack of genetic association among tool-using sea otters
compared with dolphins may result from the length of time each species has
been using tools. Tool-use in dolphins appears to be a relatively recent inno-
vation (less than 200 years) but sea otters have probably been using tools for
many thousands or even millions of years.1. Introduction
Relatively little is known about tool-use in marine animals: the best-studied
species are sea otters (Enhydra lutris) and Indo-Pacific bottlenose dolphins
(Tursiops aduncus), both of which bring tools to the ocean’s surface where they
can be easily observed [1]. There are several intriguing parallels between the ecol-
ogy of tool-use in California sea otters and dolphins. First, not all individuals in a
population use tools [2,3]. Second, tool-use is related to consumption of prey that
are difficult to access. Sea otters use rocks or other hard objects to break open well
armoured prey such as marine snails [4]. Dolphins use conical sponges as tools to
protect their sensitive snouts while probing among rocks for small, burrowing
fish that live at the bottom of deep ocean trenches [3,5]. Third, tool-use appears
to be a response to resource limitation from high population density, which
leads to the development of dietary specialization [6,7]. In California sea otters,
food limitation results in individuals in the same area specializing on different
prey. Some eat mainly large prey such as abalones and crabs, others mainly urch-
ins andmussels, and others mainly small marine snails [7]. Individuals belonging
to every diet type sometimes use tools but tool-use is most frequent in those that
prey heavily on snails [4]. Tool-use in dolphins occurs in a subset of individuals
that specialize on small fish that cannot be accessed without the use of tools
and only individuals feeding on these fish use tools [8–10]. Finally, diet prefer-
ences appear to be transmitted by mothers to their female offspring in both sea
otters [11] and dolphins [12].
Table 1. Mitogenomes in southern sea otters. The three complete mitogenome haplotypes and the four D-loop haplotypes found by Larson et al. [15] are
shown for comparison. Individual genotypes are in table S1, electronic supplementary material.
snp
location: 1491 bp 11679 bp 13547 bp 14101 bp 15365 bp 15592 bp 15593 bp 15609 bp
gene: tRNA tRNA-Ser NDS ND6 tRNA-Thr D-loop D-loop D-loop
HAPLOTYPE T/C A/G A/G A/G C/T A/G C/T C/T N
1 C A A A C G C T 9
2 C G A A C G C T 1
3 T G G G T G T T 33
A G T C Larson et al. [15]
B A T T Larson et al. [15]
C G T T Larson et al. [15]
D G C C Larson et al. [15]
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revealed that almost all the tool-using dolphins in an area
belong to a single matriline [12,13] and nuclear markers indi-
cated that tool-users were more related to other tool-users
than expected by chance (although, this findingwas not signifi-
cant for the western gulf of Shark Bay). We asked whether sea
otters showed similar genetic patterns by analysing mito-
chondrial genomes and nuclear microsatellite genotypes of
individual sea otters together with behavioural data on their
diet and frequency of tool-use.2. Material and methods
Individual sea otters were captured and tagged from 2000 to
2014 along the California coast (electronic supplemental material,
table 1). We used focal animal sampling [14] to opportunistically
record foraging data on individual otters. For each feeding dive,
we recorded whether or not prey was captured, prey identifica-
tion and the presence or absence of tool-use (see Tinker et al. [7]
for detailed methods). Each otter was assigned to one of six
diet specialist groups—ABALONE, CRAB, MUSSEL, CLAM,
URCHIN or SNAIL—using fractional composition analysis as
detailed in Tinker et al. [7]. We excluded abalone captures from
analyses of tool-use because we could not consistently determine
the frequency of underwater tool-use to obtain abalone [2]. Indi-
viduals were considered frequent tool-users if they used tools for
at least 40% of observed prey captures, based on a gap in the dis-
tribution of tool-use frequency between the individuals that used
tools on 1–27% of prey captures and those that used them on
44–90% of prey captures (electronic supplementary material).
Dependent pups were not considered in any analyses.
We used massively parallel multiplexed sequencing to obtain
complete mitochondrial genomes from 43 otters (electronic sup-
plementary material) in the hopes of finding new haplotypes, as
only three are known from D-loop [15]. We performed a x2 test
and a Goodman–Kruskal t test to assess the relationship between
the mitochondrial haplotype and diet type. In addition, we geno-
typed 38 microsatellite loci from 154 otters. Mitochondrial
haplotypes were identified and compared with diet type and
whether individuals used tools frequently or infrequently. Micro-
satellite genotypes were used to determine if frequent tool-users
and otters belonging to the same diet specialist group were more
likely to be related to each other than to the population asa whole using a permutation test implemented in the ‘related’
package in R [16] (electronic supplementary material).3. Results
Mitochondrial sequencing revealed a 16 431 bp mitogenome
with a mean read depth ranging from 21 to 1100. The cytosine
homopolymer in the 16 s rRNA starting at position 2644 bp
was excluded from analyses because it could not be aligned
accurately. Themitogenome contained six variable sites reveal-
ing three mitogenome haplotypes (table 1). One haplotype
was found in only a single individual. The other two haplo-
types contained similar percentages of frequent tool-users
(33% in haplotype 1 and 36% in haplotype 3) and there
was no significant relationship with diet type (x2 ¼ 18.3, p ¼
0.405; Goodman–Kruskal t value ¼ 0.055) (table 2). Locations
of individual haplotypes are shown in electronic supplemen-
tary material, figure S1. We recovered one new D-loop
haplotype (GCT) in 23% of the samples (n ¼ 10). The rest of
the samples (77%) had D-loop haplotype GTT, congruent
with the Larson et al. [15] finding that ‘C’ haplotype for
D-loop was most common in California. We did not find
their haplotypes ‘A’ or ‘D’, which were less common in their
sample (A, 10% and D, 20%), probably by chance. Larson
et al. [15] found haplotype ‘B’ only in Alaska.
The microsatellite data did not have any geographical
structuring (R. B. Gagne 2017, unpublished data). A summary
of locus information (e.g. average number of alleles) is included
in the electronic supplementary material. Neither the Queller &
Goodnight (Q&G) [17] nor the Lynch and Ritland (L&R) [18]
estimator found that frequent tool-users (n ¼ 21) were more
likely to be related to each other than to the population
as a whole (n ¼ 133; L&R expected r ¼ 20.0066, observed
r ¼ 20.0067; Q&G r ¼ 20.031, 95% CI 0.021, p. 0.4) (see elec-
tronic supplementary material). The Q&G method found no
association with any diet type and relatedness, whereas the
L&R estimator found the otters specializing upon clams
(expected r ¼ 20.0065, observed r ¼ 20.0016, p, 0.05) and
crabs (expected r ¼ 20.0066, observed r ¼ 20.0040, p, 0.05)
were significantly more likely to be related to those with the
same diet type than to those with other diet types (electronic
supplementary material, figure S3). However, these values of
Table 2. Number of sea otters by diet type and mitogenome haplotype.
Number of frequent tool-users (more than or equal to 40% of dives) in
parentheses.
diet type
mitogenome haplotype
1 2 3 total
snail 3(3) 0 9(9) 12(12)
clam 2 1 4 7
mussel 2 0 3 5
abalone 1 0 1 2
crab 1 0 13(3) 14(3)
urchin 0 0 3 3
total 9(3) 1 33(12) 43(15)
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imal place) and can likely be interpreted as zero or no
relatedness. On a sample of 11 known mothers and pups, the
mean value of the Q&G estimator was closer to the expected
value of 0.5 (0.46+0.04) than that of the L&R estimator
(0.37+0.03).4. Discussion
Despite the many ecological similarities between tool-use in
dolphins and sea otters, we found that the genetic patterns
in these two species are different. In otters, diet types and
tool-use are dispersed across both common mitogenome
haplotypes and neither otters that specialize on snails (the
diet type that contains most of the frequent tool-users) nor fre-
quent tool-users are more related to each other than to the
population as a whole. By contrast, tool-use in the dolphins
and its associated diet type (small cryptic fish) are predomi-
nantly confined to a single mitochondrial matriline in each of
the two geographical areas in which it occurs and individuals
that use tools are more related to each other than expected
(however, relatedness was not statistically significant for the
western gulf of Shark Bay) [12,13,19].
The lack of genetic association with tool-use in sea otters,
compared with dolphins, may result from the length of time
each species has been using tools. Tool-use in dolphins is
thought to be a recent innovation [20]; however, it is likely a
much older behaviour in sea otters. This is supported by evi-
dence that, as in some tool-using birds [21] but unlike
dolphins, all young otters appear to be innately predisposed
to use tools; orphaned otter pups raised in captivity exhibit
rudimentary pounding behaviour without training or previous
experience [4,22] and wild pups develop tool-use behaviourbefore weaning, regardless of their mother’s diet type [22].
However, this behaviour only becomes a regular part of adult
foraging behaviour under certain ecological conditions [2,4].
Moreover, similar tool-use behaviours occur in all three sea
otter sub-species [23], so it is likely that tool-use developed
prior to sub-speciation [2]. Conversely, tool-use in dolphins
has only been regularly observed within Shark Bay in western
Australia. The Miocene ancestors of modern sea otters, Enhy-
driodon and Enhydritherium, had already developed large, flat
molars suitable for crushing shells and exoskeletons of macro
invertebrates [24]. Very hard-shelled prey, such as snails and
large clams, are not easily accessed without the use of tools:
recent research on sea otter bite-force indicates that the armour-
ing of marine snails gives them a hardness that is at the upper
limits of what a typical otter can crush with its jaws (C. Law
2017, personal communication). Thus, if the ancestors of
modern sea otters were eating hard-shelled molluscs, they
were likely using tools to open them. It may be possible not
only to confirm that the ancestors of modern sea otters used
tools but to estimate the approximate period when this behav-
iour emerged if some fossil sea otters exhibit morphological
signs of tool-use, such as the depressed sternum seen in some
modern individuals [24].Ethics. All fieldwork was conducted with authorization by the US Fish
and Wildlife Service under permits issued to M. T. Tinker, and with
oversight by the Institutional Animal Care and Use Committee at the
University of California Santa Cruz.
Data accessibility. Mitogenomes for individual otters are in the electronic
supplementary material file. Mitogenomes and microsatellite data
were submitted to the DRYAD repository with the accession number
#(doi:10.5061/dryad.6vf7j, URL:http://datadryad.org/review?doi=-
doi:10.5061/dryad.6vf7j) [25]. Field data have been archived in USGS
Sciencebase, DOI http://doi.org/10.5066/F78050S9
Authors’ contributions. K.R. conceived the study and wrote the first draft.
N.R.M., R.B.G., H.B.E. and J.M. supervised and conducted genetic
laboratory work and analyses. J.F. and M.T.T. supervised and con-
ducted fieldwork and assigned the otters to diet types. All authors
edited and approved the final draft of the manuscript and agree to
be accountable for all content.
Competing interests. We have no competing interests.
Funding. K.R. received Smithsonian Capstone funding. H.B.E. received
funding from Monterey Bay Aquarium and the California Depart-
ment of Fish and Wildlife. Funding and logistical support for
fieldwork were provided by the US Geological Survey, Monterey
Bay Aquarium, US Fish and Wildlife, California Coastal Conser-
vancy, University of California Natural Reserve System and the
California Department of Fish and Wildlife.
Acknowledgements. We thank the many dedicated individuals who col-
lected the field data and genetic samples. Dave Jessup and Melissa
Miller supported the microsatellite project in many ways over a
decade, and Lisa Dalbeck, Tracy Drazenovich and Tamala Gilliland
assisted with microsatellite genotypes. We thank Palle Villesen and
an anonymous reviewer for helpful comments. Any use of trade, pro-
duct or firm names in this publication is for descriptive purposes
only and does not imply endorsement by the US government.References1. Mann J, Patterson EM. 2013 Tool use by aquatic
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