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Polar Biology
https://doi.org/10.1007/s00300-018-2354-x
ORIGINAL PAPER
Fish consumption ofharbour seals (Phoca vitulina) innorth western
Iceland assessed byDNA metabarcoding andmorphological analysis
SandraM.Granquist1,2,3· RodrigoEsparza‑Salas4· ErlingurHauksson1,2· OlleKarlsson4· AndersAngerbjörn3
Received: 21 November 2017 / Revised: 24 May 2018 / Accepted: 6 June 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Understanding ecological relationships between humans and marine predators is crucial for the implementation of sustain-
able management practices. Comprehensive estimation of pinniped diet is essential for assessing interaction with fisheries
and often has an important conservational value. Due to uncertainty regarding the accuracy of methods traditionally used
to estimate harbour seal (Phoca vitulina) diet it is necessary to improve analysis methods. We investigated the diet of har-
bour seals hauling out in an estuary area in north-western Iceland between May and August of 2010 and 2011 by genetic
(molecular) analysis of prey in faeces using DNA metabarcoding. The results were compared to previously published results
from morphological analysis. Our results showed that species consumed were mainly sandeels (Ammodytes sp.), flatfishes
(Pleuronectidae), gadoids (Gadidae), herring (Clupea harengus) and capelin (Mallotus villosus). The results from molecu-
lar and morphological analyses were similar in regards to important prey species, but species diversity was lower in the
morphological analysis and 38% of the samples included prey items that were unidentifiable in the morphological analysis.
Notably, despite Atlantic salmon (Salmo salar), brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) availability
in the study area, neither of the methods found evidence of salmonids in the harbour seal diet. Recently, a severe decline
has been observed in the Icelandic harbour seal population. Since the main reason for culling harbour seals in Iceland is to
reduce predation on salmonids, findings presented in this paper have essential conservation implications and suggest that
culling needs to be reassessed.
Keywords Prey DNA· Metabarcoding· Salmon· Harbour seal· Phoca vitulina
Introduction
The relationship between marine mammal conservation and
fisheries management is complex and friction between dif-
ferent stakeholders arises frequently. Many pinniped species
are considered to be generalists preying on fish species that
are easily available in their home range. In areas where com-
mercial fisheries occur, the fish species eaten by seals may
overlap with species targeted by the fishing industry, which
often creates management conflicts regarding how pinnipeds
affect harvest by “eating all the fish” (Yodzis 2001; Cook
etal. 2015; Houle etal. 2016).
Due to the complicated nature of predator and prey rela-
tionships in an open marine ecosystem, it is hard to pinpoint
the exact effects of seal predation on commercial fish stocks.
In the North Atlantic, some studies suggest that seal pre-
dation can affect fish mortality and stock recovery (Bundy
2001; Trzcinski etal. 2006; Cook etal. 2015; Swain and
Benoît 2015). Other studies suggest that seal predation is
Accession numbers Raw sequence data in fastq-format has been
submitted to the Sequence Read Archive under the accession
number PRJNA471262 (https ://www.ncbi.nlm.nih.gov/Trace s/
study /?acc=SRP14 6085).
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0030 0-018-2354-x) contains
supplementary material, which is available to authorized users.
* Sandra M. Granquist
sandra.magdalena.granquist@hafogvatn.is
1 Marine andFreshwater Research Institute, Skúlagata 4,
101Reykjavík, Iceland
2 The Icelandic Seal Center, Brekkugata 2, 530Hvammstangi,
Iceland
3 Department ofZoology, Stockholm University,
10691Stockholm, Sweden
4 Swedish Museum ofNatural History, Box50007,
10405Stockholm, Sweden
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often of a smaller magnitude than harvest by commercial
fisheries, implying that seal predation affects human harvest
to a small extent (Carter etal. 2001; Hansen and Harding
2006; Houle etal. 2016).
In addition to direct predation on commercial stocks,
indirect effects of seal activity on the fishing industry have
often been described, such as damage to fishing gear caus-
ing additional work and costs for fishing companies (e.g.
Kauppinen etal. 2005; Suuronen etal. 2006). The fishing
industry can also affect seal populations as seals drown in
fishing gear or get entangled in discarded nets (McIntosh
etal. 2015) and further, depredation (pinnipeds eating fish
directly from fishing nets), also causes harvest losses for the
fisheries (Königson etal. 2009; Cosgrove etal. 2015). West-
erberg etal. (2008) reported that losses due to seal induced
damage were about 15–20% of annual catch value for the
total coastal fisheries in Sweden. Kauppinen etal. (2005)
presented a similar scenario concerning trap-net fisheries
in the northern Baltic Sea and showed that seals damaged
37% of the salmon (Salmo salar) catch in the Botnian Bay,
although the damage varied between locations in the study.
Despite uncertainty about the effects of seal removals
in the ecosystem, seals are often culled in the vicinity of
commercial fish stocks; either with the purpose of allowing
fish stock recovery or to reduce the effect that seal preda-
tion is believed to have on harvest. This is, for example,
the case regarding the interaction between salmonid (Sal-
monidae) angling activities and seal populations in north-
eastern Scotland (Thompson etal. 2007; Graham 2015) and
Iceland (Granquist and Hauksson 2016a), where culling or
harassment of seals occurs in estuaries in spite of sufficient
knowledge on effect on salmonid populations and fisher-
ies. Stakeholder conflicts between the angling industry and
harbour seal (Phoca vitulina) management and conservation
often become heated. Harbour seal colonies are frequently
found near important salmon river estuaries, and due to their
opportunistic feeding habits, it is often suspected that the
reason for their local abundance is the availability of sal-
monids (Middlemas etal. 2006; Graham 2015). Previous
studies indicate that salmonids are sometimes common prey
species in harbour seal diet, especially around estuaries (e.g.
Carter etal. 2001; Butler etal. 2006; Wright etal. 2007).
However, other studies have found that salmonids are not
generally a preferred prey item (Matejusová etal. 2008), and
sometimes only few individual harbour seals are responsible
for the salmonid predation (Wright etal. 2007).
In Iceland, angling is a popular activity among locals and
tourists. In 2015, 17% of the 1.3 million tourists visiting
Iceland stated that they went fishing (Icelandic tourist board
2016) and river angling of salmonids generates a financially
valuable industry. Atlantic salmon (S. salar) fishing occurs
in rivers, mainly by angling, while salmon fisheries in the
ocean are not permitted in Iceland. Brown trout (Salmo
trutta) and Arctic char (Salvelinus alpinus) are caught by
river angling or in nets in lagoons (Gudbergsson 2015). For
many local landowners, the yield from the salmon angling
business is crucial in terms of the annual income (Toivonen
etal. 2004), which creates a concern about the potential
indirect effect of seal predation on the salmon harvest.
During recent decades, traditional sealing for meat and
fur has decreased in Iceland, although culling is still con-
ducted in estuaries to reduce potential predation on salmo-
nids. As an example, in 2015 82% of the culled harbour
seals in Iceland were killed around river mouths (Granquist
and Hauksson 2016a). The Icelandic harbour seal population
has recently suffered a decline of over 30%, from 12,000
to 7700 individuals between 2011 and 2016 (Granquist
etal. 2011, 2015; Thorbjörnsson etal. 2016). It has there-
fore become urgent to investigate the diet of seals in estua-
rine ecosystems. In particular it is pressing to analyse the
importance of salmonids in harbour seal diet in the area, to
determine whether the culling of seals in estuaries is feasi-
ble as a means of reducing predation on salmon, but also
from an ethical perspective in the light of the declining seal
population.
To solve stakeholder conflicts, an important first step is
to elucidate ecosystem functions, such as illustrating preda-
tor–prey relationships with empirical evidence. Several
methods have been developed to estimate pinniped diet,
such as morphological analysis of otoliths and bones from
the digestive tract or from faeces (hard-part analysis) (Pierce
and Boyle 1991), stable isotope analysis (Hobson etal. 1996;
Young etal. 2010) and fatty acid analysis (e.g. Iverson etal.
2004). More recently, genetic (molecular) analysis of faeces
and/or digestive tract contents to investigate pinniped diet
has been successful (Purcell etal. 2004; Parsons etal. 2005;
Matejusová etal. 2008; Deagle etal. 2009; Thomas 2015).
Each method has advantages and biases, providing informa-
tion over different time intervals: While stable isotope and
fatty acid analysis can reveal long term dietary patterns, it is
often challenging to obtain taxonomic information on prey
species consumed. In contrast, morphological and molecular
analyses provide taxonomic prey information, but typically
represent only the most recent meal (Bowen and Iverson
2013). This temporal penalty may, however, be mitigated by
a rigorous and repetitive sampling programme. Faecal analy-
sis also has the advantages of facilitating sample collection
more than once from each individual, as well as being non-
invasive and hence ethically feasible. The classical mor-
phological hard-part analysis of prey has been criticised in
previous literature for its potential shortcomings (Pierce and
Boyle 1991). For instance, otoliths of some species, includ-
ing salmonids, are fragile and can easily break and degrade
in the digestive system, potentially obscuring the presence of
such species in diet. Furthermore, seals might not eat whole
large fish such as adult salmonids or gadoids (Gadidae), but
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rather consume only the more nutritious parts, such as the
liver and muscle (Orr etal. 2004), so called “belly biting”.
In such cases, otoliths and bones might not be ingested, mak-
ing the fish non-detectable using morphological analysis.
By contrast, consumed prey can be detected by DNA analy-
ses even when the head and bones of a fish have not been
ingested. Further, molecular analysismay detect a higher
diversity of prey species in faeces, when compared with
morphological analysis of the same samples (Deagle etal.
2009; Méheust etal. 2015), given that the taxonomic reso-
lution can be higher (Purcell etal. 2004; Matejusová etal.
2008) than for morphological analysis. Analysing prey using
DNA barcoding to investigate pinniped diet has previously
been successfully used in several studies (Deagle and Tollit
2007; Méheust etal. 2015). As an example, Méheust etal.
(2015) used DNA barcoding for the identification of soft
remains of prey in the stomach contents of grey seals (Hali-
choerus grypus) and found that the identification of prey
species was improved compared to morphological analysis
of stomach content.
The aim of this paper was to estimate the diet of harbour
seals in an estuary area in north-western Iceland, with spe-
cial emphasis on harbour seal predation on salmonids in the
area. To obtain species-level prey identification and relative
prey contribution, molecular analysis (prey-DNA metabar-
coding) was used. Subsequently, the molecular analysis was
compared with diet composition estimates based on more
traditional morphological analysis previously conducted on
the same samples (published in Granquist and Hauksson
2016b).
Materials andmethods
The study area
The study was conducted between May and August in 2010
and 2011 in the harbour seal haul-out site in the estuaries
of Osar and Bjargaós, north-western Iceland (Fig.1). The
habitat type is sandy beaches and the distance between these
estuaries is approximately 1.5km. Bjargaós is the estuary
of three large rivers, Vididalsa, Fitja and Gljufura, where
angling of Atlantic salmon, Arctic char and brown trout
is conducted from May to October. Osar is the estuary of
the Sigridarstadavatn lagoon, where Arctic char and brown
trout are abundant throughout the year. Previous studies have
shown that Osar is an important harbour seal breeding and
haul-out site with a bimodal annual haul-out distribution,
with one peak during the pupping season in June and a sec-
ond peak during the moulting season in August (Granquist
and Hauksson 2016c). The abundance in Bjargaós has its
peak between the two peaks in Osar (late June) and Gran-
quist and Hauksson (2016c) suggested that the harbour seals
may use Bjargaós as a foraging site between the high energy
expenditure periods of pupping and moulting, respectively.
The monthly average number of seals hauling out in Osar
ranged between 108.6 (SD = 86.6) and 251.3 (SD = 79.6)
during the time of this study, while in Bjargaós, the monthly
average number of hauling-out seals was between 0 and 34.0
(SD = 36.7) (Granquist and Hauksson 2016c). In 2010, no
hunting occurred due to the ongoing research, and in 2011
only a few animals were culled in the area (Fig.1).
Sampling andanalysing faeces
Throughout the study period, the estuaries were visited
during low tide once every spring tide (approx. twice per
month). During high tide the haul-out site is covered by
water and hence, only samples that were fresh of the day
at low tide were accessible. All samples were collected
(n = 116) and stored at −20°C. Before the DNA extraction,
samples were thawed and dissolved in water and a subsam-
ple was taken from the solution.
DNA was isolated from all samples using the QIAmp
DNA stol kit (Qiagen), following the “Isolation of DNA
from stool for human DNA analyses” protocol to maxim-
ise extraction of prey-DNA while minimising bacterial
Arctic circle
ICELAND
VATNSNES
PENINSULA
Osar
Bjargaós
Fig. 1 Location of the sampling site Osar and Bjargaós eastuary area
on Vatnsnes peninsula, north-western Iceland. The Arctic circle is at
latitude 66°33′47.1″ North
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endosymbionts. An approximate volume of 200µl of faeces
solution was used as starting material. DNA was eluted in
a 200µl volume.
The primers 16sPreyF (5′-CGT GCR AAG GTA GCG-3′)
and 16sPreyR (5′-CCT YGG GCG CCC CAAC-3′) were used
to amplify an approximately 270 base pair section of the
mitochondrial 16s rDNA gene by polymerase chain reac-
tion (PCR). As a strategy to avoid over-amplification of the
predator DNA, the nucleotide at the 3′ end of the forward
primer was chosen to mismatch the 16s sequence of phocid
seals, while being conserved in jawed fish and other verte-
brates, including birds.
Each of the primers was synthesised in eight variations,
each including a unique six nucleotide “barcode” sequence
at the 5′ end, as suggested by Binladen etal. (2007) for mul-
tiplexing of individuals in DNA libraries, and posterior de-
multiplexing of the output data. PCR reactions were carried
out in 25µl volumes, including 12.5µl HotStart Taq master
mix (Qiagen), 10pmol of each primer and 2µl of DNA
extract solution. Cycling conditions included initial dena-
turation at 95°C for 5min; 40 cycles of 94°C for 30s, 54°C
for 30s and 68°C for 45s; and a final extension step of
72°C for 10min. PCR products from different samples were
normalised to approximately equal concentrations using the
SequalPrep normalisation plate kit (Life Technologies), and
subsequently pooled in sets of 64, each corresponding to
unique combinations of forward and reverse “barcoded”
primers. Sets of up to 64 PCR products were used to prepare
different DNA libraries.
Pooled PCR products were concentrated to a 20µl vol-
ume using the MinElute kit (Qiagen). Amplicon libraries
were prepared following the “Rapid Library Preparation
Method Manual” for the GS-Junior titanium series instru-
ment protocols (Roche 454 sequencing) with modifica-
tions as follows: The nebulization step was omitted, for
the “fragment end repair”, the volumes of RL dNTP, RL
T4 polymerase and RL Taq polymerase were substituted
by EB buffer (Qiagen), and size selection was carried out
by agarose gel excision. A different multiplex identifier
(MID) adaptor was ligated to each set of pooled PCR prod-
ucts. Libraries were diluted to a working concentration of
1 × 106 molecules per µl.
Emulsion PCR was carried out following the “emPCR
Amplification Method Manual—Lib-L” for the GS-Junior
Titanium Series instrument (Roche 454 sequencing). Parallel
pyrosequencing was carried out following the “Sequencing
method manual” for the GS-Junior Titanium Series instru-
ment (Roche 454 sequencing).
The sequence output in FastA format and its respective
quality scores were combined into a FastQ file using Gal-
axy (Blankenberg etal. 2010). Sequence reads with either
a < 60bp length, a quality score of < 15 or a non-defined
base call (N-bases) of > 2% were filtered out from the dataset
using PRINSEQ (Schmieder and Edwards 2011). The sort-
ing of the sequencing output file into individual libraries
and individuals within libraries, respectively, was carried
out using the programme 454 tag sorting by Johan Nylander
(https ://githu b.com/nylan der/454_tag_sorti ng). Species
identification from the output sequences was carried out
using the BLAST+programme (Camacho etal. 2009), with
the individually tagged DNA sequences (in FastA format)
as a query database, and the nucleotide collection (nr) as a
reference subject database. Only the highest matching result
was kept in the BLAST output. Representatives of match-
ing sequences were then compared individually in order to
identify eventual ambiguous matches. Individual samples
that produced less than 100 valid matched prey sequence
were discarded from further analyses. Out of 116 faeces
samples analysed for prey-DNA, 80 samples (Fig.2) could
be used for further analysis (> 100 valid sequences). Hence,
the PCR amplification success was 68.97%. Samples were
subsequently analysed with morphological analysis (see
Granquist and Hauksson 2016b), which enabled method
comparison on the same individuals (n = 72).
Fig. 2 The distribution of sam-
pling dates for samples produc-
ing > 100 valid sequences in the
sampling years 2010 (n = 41)
and 2011 (n = 39)
0
2
4
6
8
10
12
14
16
18
14 Ma
y
2 June
20 June
28 June
30 June
5 July
13 July
15 July
27 July
2 August
17 August
23 August
27 August
2010
2011
Numbers of samples collected each sample day
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Statistical analysis
The importance of different fish species was investigated in
two ways; (1) the frequency of occurrence (the proportion
of all analysed samples that included the species) and (2)
the relative contribution of each species in the harbour seal
diet (the proportion of sequences of each species in the sam-
ples). Differences in frequency of occurrence between the
two sampling years were investigated by Fishers exact test.
The results from the molecular analysis were compared
to results from morphological analysis with respect to the
frequency of occurrence of species (Granquist and Hauksson
2016b) by Fishers exact test. When investigating diet with
morphological analysis it was not possible to define gadoids
into species, and therefore gadoid species were pooled when
results from the molecular analysis were compared with
results from the morphological analysis.
Results
Frequency ofoccurrence ofsh species inthediet
The molecular dietary analysis revealed that sandeel (Ammo-
dytes sp.) occurred in the highest amount of samples for
the years combined (46%), while cod (Gadus morhua) and
flatfishes (Pleuronectidae) were the second (33%) and third
most prominent species (31%) respectively. Capelin (Mal-
lotus villosus), herring (Clupea harengus), whiting (Mer-
langius merlangus) and haddock (Melanogrammus aeglefi-
nus) were also important species in the diet, each occurring
in > 10% of the samples. In 10% of the samples, bird DNA
(e.g. Eider Duck, Somateria mollissima, n = 4 and White
Wagtail, Motacilla alba, n = 1) was detected (Fig.3, Online
resource 1).
Annual differences in frequency of occurrence for capelin
and cod were found. Capelin occurred in 34% of the sam-
ples in 2010, while only one sample (2.6%) from 2011 con-
tained capelin (Fishers exact test, p < 0.001). Cod was found
in more samples in 2011 (49% of the samples) compared to
2010 (17%, Fishers exact test, p = 0.004). (Figure3, Online
resource 1). We found no evidence of salmon, char or trout
DNA in the diet.
The results from both molecular and morphological
analyses revealed that the main prey of harbour seals in this
study were sandeel, flatfish, gadoids and capelin, with sand-
eel occurring in the highest frequency of samples (50 vs.
53% respectively). However, the results from the molecu-
lar analysis did not completely match the results from the
morphological analysis of the same samples (Granquist and
Hauksson 2016b). The number of samples including gadoids
(Fishers exact test, p = 0.007) was higher according to the
molecular analysis (36%) compared to the morphological
analysis (15%). Other species did not vary significantly
between the methods, although fish species diversity was
higher in the molecular analysis (average 2.06 ± 1.25 SD,
n = 72. Maximum number of species detected in a sam-
ple was 7) compared to the morphological analysis (aver-
age 1.15 ± 0.87 SD, n = 72. Maximum number of species
detected in a sample was 3). Some species (e.g. mackerel;
Scomber scombrus and bullrout; Myoxocephalus scorpius)
were not detected at all in the morphological analysis. Nev-
ertheless, in the morphological analysis, 38% of the sam-
ples included some unidentifiable otoliths or bones (Fig.4,
Online resource 2).
Relative contribution ofsh species inthediet
The greatest contributor to relative diet using molecu-
lar analysis were sandeel, with an average of 26% of all
Fig. 3 Frequency of occurrence
of species found in the faecal
samples in 2010 (n = 41), 2011
(n = 39) and in total for the
years combined (n = 80) using
molecular analysis
0
5
10
15
20
25
30
35
40
45
50
Ammodytes
Pleuronecdae
Gadus morhua
Merlangius merlangu
s
Melanogrammus
aeglefinus
Mallotus villosus
Clupea harengus
Anarhichas sp.
Cyclopterus lumpus
Amblyraja radiata
Pholis gunnellus
Other
Unidenfied fish specie
s
Bird
2010
2011
Total
Proporon of samples including
Gadidae
species (%)
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sequences identified across samples. Further, flatfishes
comprised in total to 18% of the relative diet, and cape-
lins were 15%. The gadoid species cod, whiting and had-
dock comprised between 4.2 and 15% each when the years
were combined (Fig.5). The relative diet of harbour seals
shifted somewhat between the two sampling years. In
2010, capelin (28%) was equally important in the relative
diet as sandeel (28%), while capelins were rather seldom
ingested in 2011. However, in 2011 cod (26%) dominated
the diet together with sandeel (24%). Herring was only
contributing to 0.3% of the diet in 2010, while in 2011 it
amounted to 6.8% of the diet (Fig.5).
Discussion
Diet ofharbour seals hauling outintheestuaries
The molecular analysis showed that the main prey of harbour
seals hauling out in the estuaries of Bjargaós and Osar was
sandeel occurring in almost half of all samples (46%) and
contributing 26% of the relative diet in total for both years.
Flatfish, capelin and cod were also important prey species,
occurring in 19–33% of the samples, and each correspond-
ing to 15–18% of the relative diet. Interestingly, we found
no evidence that harbour seals prey on salmonids despite
our regular sampling scheme during the time of year when
salmon migrate through the estuary area (Figs.3, 5).
Fig. 4 Frequency of occurrence
of species found in the faecal
samples in total for all samples
(2010 and 2011) using molecu-
lar analysis in comparison with
data from morphological analy-
sis of the same samples (n = 72).
Data combined for 2010 and
2011 for both methods
0
10
20
30
40
50
60
Ammodyte
s
Pleuronecda
e
Gadidae
Mallotus villosu
s
Clupea harengus
Anarhichas sp
.
Cyclopterus lumpus
Amblyraja radiata
Pholis gunnellu
s
Other
Unidenfied
Molecular analysis
Morphological analysis
Proporon of samples including
species (%)
Fig. 5 Relative diet contribution
of different fish species in har-
bour seal diet in 2010 (n = 41),
2011 (n = 39) and in total for all
samples (2010 and 2011) using
molecular analysis (n = 80)
0
5
10
15
20
25
30
35
40
45
50
Ammodytes sp.
Pleuronecdae
Gadus morhua
Merlangius merlangu
s
Melanogrammus
aeglefinus
Mallotus villosus
Clupea harengus
Other fish
Birds
2010
2011
Total
Relave diet proporon (%)
Gadidae
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Catches of salmonids from the rivers flowing into our
study estuaries suggest that the availability of salmon,
brown trout and char was normal during the period over
whichfaeces were collected (Gudbergsson 2012). We used
the exact protocol for the prey-DNA barcoding analysis as
used in grey seal dietary analysis in the Baltic Sea (ECO-
SEAL 2016; unpublished data), where salmonids were found
in 6 out of 161 samples, (approximately 3% of the relative
grey seal diet on average). Further, several other studies have
successfully detected salmonid DNA using DNA barcoding
analysis (Deagle and Tollit 2007; Deagle etal. 2010). Hence,
if the harbour seal aggregation in the estuaries was related to
salmonid abundance, we would have found salmonid DNA
in our analysis. Although we cannot exclude that individual
harbour seals occasionally consume salmonids in the study
area, our results suggest that in general salmon, trout and
char were not the main prey for harbour seals in these estu-
aries during the time of study. Since the main reason for
culling harbour seals in Iceland is to prevent seal predation
on salmonids, these findings are highly important in terms
of conservation and management implications.
The present study, together with the study conducted
by Granquist and Hauksson (2016b), is the first attempt
to investigate predation on wild salmonids in estuaries by
the Icelandic harbour seal population. In addition, Gran-
quist (2014) found that seal induced wounds on caught
salmon and trout in the adjacent rivers of Bjargaós and Osar
wasrare, implying harbour seal predation on salmonids was
low. However, Thórisson and Sturlaugsson (1995) suggested
that individual harbour seals specialised in salmonid preda-
tion in a former salmon aquaculture site in W-Iceland. On
the other hand, this was not supported by Osmond (2013),
who studied potential seal effects on salmon aquaculture
pens and found no effects of seal predation.
Notably, a severe decrease in the sandeel population in
other parts of Iceland during the time of study has been
reported, for instance causing poor breeding of Atlantic puf-
fin (Fratercula arctica) colonies in southern Iceland in 2010
and 2011 (Lilliendahl etal. 2013). Despite the collapse in
other areas of Iceland, sandeel was found to be the most
important prey species for harbour seals in the Osar and
Bjargaós estuaries. Annual shrimp research surveys con-
ducted in the area (Ingibjörg Jónsdóttir, personal commu-
nication) confirm that codfish (haddock, cod and whiting),
flatfish, capelin and herring were common fish species in
the area for the years of study. Although it should be noted
that the shrimp research survey is carried out in September
and October and hence slightly later than the present study,
it indicates that harbour seal diet is in accordance with avail-
able fish species in the area. However, this was not the case
for salmonids, which were not found in the diet of harbour
seals, despite documented salmonid availability in the area
during the time of sampling (Gudbergsson 2012).
Similarly to the current study, previous studies of seal
diets from the NW-coast of Iceland have revealed that flat-
fishes, Ammodytes, gadoids, herring, catfish and capelin
are the main prey species of harbour seals, while salmo-
nids were not important in the diet (Hauksson and Bogason
1997; Nebel 2011). Harbour seal fish predation and salmo-
nid importance in their diet has been frequently studied in
other geographical areas outside of Iceland and findings
often indicate opportunistic feeding behaviour with feeding
to some extent being correlated to fish availability in the area
(Härkönen 1987; Ramasco etal. 2017). Studies from other
areas of the North Atlantic, such as Scotland, have found that
salmonids may be consumed by harbour seals hauling out
in estuaries (Carter etal. 2001; Butler etal. 2006), although
salmonid species often are a minor contributor to the total
diet (Middlemas etal. 2006; Matejusová etal. 2008). Fur-
ther, Middlemas etal. (2006) found a correlation between
harbour seal aggregations in an estuary and salmon returning
to the river. In a review of harbour seal diet in the Pacific
by Steigrass (2017), salmonids were found to be more com-
mon, occurring in 13–24% of the samples. Wright etal.
found indication of harbour seals consuming 21% of the
estimated prespawning population of coho salmon (Onco-
rhynchus kisutch) in the Pacific North-west. Hauser etal.
(2008) found that during the peak of sockeye salmon (Onco-
rhynchus nerka) spawning seasonin Iliamna Lake, Alaska,
the frequency of occurrence of sockeye salmon was 98%in
resident harbour seal diet.
The results presented in the paper suggest that the main
feeding area of the seals may not be in the estuaries, but
further out at sea. Data on the geographical range of feeding
trips of harbour seals in the study area are lacking, how-
ever, Hauksson (2005) suggested that seals hauling out in
the estuaries of Hamarsörður and Alftaörður in eastern
Iceland were foraging in a more marine environment. Har-
bour seals are known to make foraging trips to divergent
areas from where they haul-out (Thompson and Miller 1990;
Carter etal. 2001). As an example, Thompson and Miller
(1990) reported that radio-tagged harbour seals in Scotland
travelled up to 45km from their haul-out sites on feeding
trips for up to 6days. Harbour seal abundance in Bjargaós
and Osar estuaries is likely connected to other factors than
salmonid availability. The sandy beaches of the estuaries
are easily accessible for the seals during low tide and are
excellent pupping and moulting sites, and the pupping and
moulting of harbour seals coincide with salmonid migration
through the area.
Methodological considerations
Both molecular and morphological analyses used in this
study showed that the most important prey species for har-
bour seals in this study were sandeel, flatfishes, gadoids
Polar Biology
1 3
and capelin. In addition, neither method found evidence
to support harbour seals preying on salmon, char or trout.
Nevertheless, some differences were found when comparing
results from the different methods. The molecular analysis
detected a higher number of species and indicated a higher
occurrence of flatfishes, gadoids and herring, as compared
to the morphological analysis. However, in the morphologi-
cal analysis 38% of the samples included otoliths that were
unidentifiable, mainly due to digestion.
All currently available methods for reconstructing pin-
niped diet (morphological and genetic analysis, as well
as fatty acid and stable isotope analysis) have advantages,
but all also incorporate several biases. Genetic analysis
approaches, such as meta-barcoding analysis as used in this
study, are taxonomically sensitive and hence suitable for
exploring diet and species occurrence. The higher species
detection obtained by our molecular analysis indicates that
the morphological analysis provided a lower taxonomical
resolution than the molecular analysis. Digestion of otoliths
could partly explain the difference in frequency of occur-
rence (lower for several species in the morphological analy-
sis) and the lower species detection for the morphological
analysis, when compared to the molecular analysis. On the
other hand, a clear benefit associated with using morpho-
logical analysis is that it offers the possibility to reconstruct
prey sizes, through otolith-length and fish-length (fish-
weight) relationships, including corrections for degradation
of otoliths. Subsequently, individual prey energy content
can be calculated. This is not possible with genetic analysis.
Toassess temporal changes based on analysis made on fae-
ces or stomach samples, a rigorous sampling scheme is nec-
essary. The optimal approach hence depends on the purpose
of the study. A higher resolution knowledge on pinniped
diet could be achieved by combining taxonomically sensi-
tive analysis from the present study with available methods
assessing greater temporal fluctuations, such as stable iso-
tope analysis (Chiaradia etal. 2014).
Another reason for the difference found between the
morphological and molecular analyses could be that if the
harbour seals pursued large fishes, “belly biting” could have
occurred. Belly biting, where only the most nutritious parts
of the fish (liver, etc.) is consumed while the head and other
less nutritious parts are left out, has been highlighted as a
potential problem when using the morphological method,
since otoliths and bones are not found in the faeces (e.g. Orr
etal. 2004, but see also Stenson etal. 2013). Large fishes
may therefore be under represented in the morphological
analysis. Results from the molecular analysis of the present
study suggest a higher consumption of cod compared to the
morphological analysis. Granquist and Hauksson (2016b)
found that the estimated size of cod built on size of discov-
ered otoliths was small (15.7cm on average), which supports
the theory that in the case of smaller gadoids, the whole
fish is consumed by the seals, while seals might consume
larger gadoids by belly biting without consuming the head/
otoliths. Such behaviour may partly explain the difference
in frequency of occurrence between molecular and morpho-
logical analysis in our study.
It should be noted that the barcoding marker used in this
study is part of the mitochondrial genome. It does not nec-
essarily represent the whole amount of tissue present in the
sample, but rather the amount of mitochondria. If the seals
indeed preferably consume energy-dense tissue such as liver
from larger fishes as we hypothesise, the mitochondrial den-
sity of the consumed tissue might be higher than that of
other tissue types (e.g. muscle). Hence, the results might
imply that the seal has eaten more of that particular spe-
cies, than if other tissues were eaten (Else and Hubert 1985;
Veltri etal. 1990). This means that, although, the consumed
weight of two species would have been the same, the relative
amounts of detected prey would be biassed toward the larger
speciesin such cases. Moreover, different fish species may
have different mitochondrial densities altogether, depending
on metabolic rate (Johnston etal. 1998).
We are confident that the method presented in this study
amplifies efficiently all bony fish species. We tested the PCR
amplification of the primers used in this study on reference
DNA of identified voucher specimens of several species,
including bony fish (Osteichthyes; 41 species including sal-
monids), cartilaginous fish (Chondrichthyes; three species)
jawless fish (Agnatha, three species) and birds (Aves, eight
species). All tested species with the exception of jawless fish
produced strong bands. As previously mentioned, salmonids
have been detected with this method in a different study
(ECOSEAL 2016; unpublished).
Several authors have already used molecular analysis
to investigate pinniped diet (e.g. Purcell etal. 2004; Par-
sons etal. 2005; Matejusová etal. 2008; Deagle etal. 2009;
Thomas 2015) and show the promising possibilities and
capability of this method. As an example, Deagle and Tollit
(2007) used real time PCR to quantify mtDNA and found
that the proportion of fish fed to the pinnipeds correspond
well to the relative amount of DNA in the analysis. However,
potential biases associated with the technique have not been
thoroughly investigated (Deagle etal. 2013). For example,
DNA density may differ between species (Pompanon etal.
2012; Deagle etal. 2013). Since there is an individual com-
ponent within the prey species as well, species specific cor-
rection factors could incorporate biases (Deagle etal. 2013).
In our study, we used the proportion of detected sequences to
estimate the relative diet of harbour seals. Although molecu-
lar diet analysis methods have improved during recent years,
it should be considered that there are many potential bio-
logical and technical biases regarding preservation, ampli-
fication and sequencing, when constructing relative diet
(Deagle and Tollit 2007; Deagle etal. 2010; Thomas etal.
Polar Biology
1 3
2014, 2016). Although further investigation to understand
the potential biases regarding relative diet contribution is
needed, molecular analysis provides relatively precise taxo-
nomic information regarding frequency of occurrence of dif-
ferent prey species, some of which are often missed using
other analysis methods. As discussed previously, in the pre-
sent study, we have shown that the species detection rate is
higher using molecular analysis compared to morphological
analysis, and in cases where seals do not ingest or excrete
prey otoliths and bones, prey species will still be detected in
the molecular analysis.
The lack of salmonids detected using morphological
analysis is not likely to be due to the fact that the otoliths
had been completely digested. A number of correction fac-
tors to account for the complete digestion of species when
estimating pinniped diet using morphological analysis were
presented in Bowen (2000). Although salmonid otoliths are
often considered fragile, the correction factor for salmonid
species (1.6) was similar to gadoids and lower than the cor-
rection factor for herring (3.0 on average). Both herring and
gadoids were frequently found in the present study. The rea-
son that herring was more important in the harbour seal diet
according to the molecular analysis than the morphological
analysis can partly be due to complete digestion since the
correction factor for herring was 3.0 on average according
to Bowen (2000).
Several species that had not been detected by analysing
otoliths and bones could be detected by analysing prey-
DNA. The identity match of DNA sequences against the
available databases was very high for all fish species (100%
match for the most common sequence variant in a given
species), with the exception of one fish species for which
the closest match was pike perch (Sander lucioperca) where
the match was 94–99% and for bird species (identity match
64–97%). Pike perch is not present in the Icelandic fauna
and therefore the most likely explanation is that the species
that these sequences correspond to does not exist in the fish
databases for the 16s rDNA gene, and instead it matched a
closely related species. The availability of reference data-
bases for non-stardard DNA barcoding markers including,
such as 16s rDNA in animals, is less comprehensive (Deagle
etal. 2014). Although given the usability of 16s and other
ribosomal markers in meta-barcoding studies, it is expected
that the respective reference databases will expand, which
in consequence might improve the taxonomic resolution in
future DNA meta-barcoding studies.
Since our study was designed to only detect fish and bird
species in the faeces, the results did not provide informa-
tion on potential invertebrate prey. Further, morphologi-
cal studies based on faeces samples have previously been
found to underestimate the importance of invertebrates in
the diet of pinnipeds (Hammill etal. 2005). However, the
morphological analysis that was carried out simultaneously
on the faeces samples of this study found evidence of inver-
tebrate occurrence in the harbour seal diet (Granquist and
Hauksson 2016b). This needs further investigation and in
future molecular analysis protocol invertebrates should be
included. There is anecdotal evidence of pinnipeds preying
on seabirds, such as Eider Ducks and in the literature there
are several examples of sea lions (Rey etal. 2012; Morrison
etal. 2017) and fur seals preying on penguins and other bird
species (Page etal. 2005). In our analysis, we found DNA
from a few bird species (e.g. Eider Duck and White Wag-
tail). In the estuaries, Eider Ducks are widely abundant and
White Wagtail can also be abundant in the study area and
hence could have either been ingested by the seals, or their
droppings could have been sampled with the seal faeces.
The presence of other fish DNA in the stomach of a prey
item (i.e. secondary predation) can be another source of
bias (Harwood etal. 2001; Sheppard etal. 2005). Currenlty,
prey-DNA analysis does not illuminate differences of pri-
mary and secondary prey. Consumption of primary prey of
smaller sizes would probably only cause trace amounts of
secondary prey in the seals diet. We discuss the possibility
that the harbour seals might have eaten larger fish of some
species (e.g. gadoids). Although it is unlikely that the seals
would have targeted the prey stomach in cases of belly bit-
ing, some secondary prey might have been consumed. It is
however important to note that even if secondary prey were
to be found in substantial amounts it would also be digested
by the seal and would therefore be a valid source of energy
that can be considered seal nutrition.
Concluding remarks
Despite the recent severe decline in the Icelandic harbour
seal population (Granquist etal. 2011, 2015; Thorbjörns-
son etal. 2016) culling of harbour seals occurs and the
main reason for the culling is currently to protect salmonid
angling. In this paper, we show by using a taxonomically
sensitive method and samples collected regularly across the
salmonid migration timeframe over 2years that salmonids
are not important prey species of the harbour seal colony in
the Bjargós and Osar estuaries at the time of study. These
findings are highly important in terms of evidence based
management and have conservation implications. The lack
of evidence of the importance of salmonids in seal diet in the
area suggests that removal of seals may not be an effective
measure to increase salmonid fisheries and hence, culling
needs to be reassessed due to ethical and monetary reasons.
The differences between the two methods of analysis
that we have identified in this paper call for further inves-
tigations, as well as continued development of methods for
estimating pinniped diet. Since the molecular analysis had
a higher species detection it is preferable in cases when
Polar Biology
1 3
occurrence of species is investigated, while morphologi-
cal analysis can add to important information on prey size
distribution.
Acknowledgements Hrafnhildur Laufey Hafsteinsdóttir, Ester Sánchez
Cacho, Laila Arranda Romero, Elsa Freschet, Halldór Jón Pálsson,
Gudmundur Jóhannesson and Haraldur Fridrik Arason assisted in the
field and/or in the lab. Johan Nylander provided assistance with the
454 data analyses. Ingibjörg G. Jónsdóttir, Alastair Baylis and Eric dos
Santos commented on an earlier version of this paper.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable national and institutional guidelines
for the use of animals were followed. All procedures performed in the
study were in accordance with the ethical standards of the institutions
at which the studies were conducted.
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