MARINE ECOLOGY PROGRESS SERIES
Mar Ecol Prog Ser
Vol. 403: 255–267, 2010
doi: 10.3354/meps08417
Published March 22
Is multiple mating beneficial or unavoidable?
Low multiple paternity and genetic diversity in
the shortspine spurdog Squalus mitsukurii
Toby S. Daly-Engel1, 5,*, R. Dean Grubbs2, Kevin A. Feldheim3, Brian W. Bowen4,
Robert J. Toonen4
–
Department of Zoology, 2538 The Mall, Edmondson 152, Honolulu, Hawaii 96822, USA
University of Hawaii at M anoa,
2
Florida State University Coastal and Marine Laboratory, 3618 Hwy 98, St. Teresa, Florida 32358, USA
3
Field Museum, Pritzker Laboratory for Molecular Systematics and Evolution, 1400 S. Lake Shore Drive, Chicago,
Illinois 60605, USA
4
Hawaii Institute of Marine Biology, PO Box 1356, Kaneohe, Hawaii 96744, USA
1
5
Present address: University of Arizona, Forbes 410, 1140 E. South Campus Drive, Tucson, Arizona 85721, USA
ABSTRACT: Proposed benefits of multiple paternity include increased reproductive output, elevated
fitness of progeny, and maintenance of population genetic diversity. However, another consideration
is whether multiple paternity is simply an unavoidable byproduct of sexual conflict, with males seeking to maximize mating encounters while females seek to minimize the stress of copulation. Here we
examined the polyandrous mating system in sharks, with a focus on the reproductive genetics of the
shortspine spurdog Squalus mitsukurii. Members of the genus Squalus are long-lived, slow-growing,
and employ among the longest gestation periods of any vertebrate. To evaluate multiple paternity
and genetic diversity in S. mitsukurii, we genotyped 27 litters plus 96 individuals with 8 microsatellite loci. Further, 670 bp of the mtDNA control region were sequenced in 112 individuals to examine
population structure. S. mitsukurii in Hawaii showed low genetic diversity relative to other sharks
(π = 0.0010 ± 0.0008) and no significant population structure in the Hawaiian Archipelago. Direct
allele counts and Bayesian approximations returned concordant estimates of 11% multiple paternity,
the lowest observed in sharks to date. Considering the protracted reproductive interval of S. mitsukurii, sexual conflict that results from differential male and female reproductive strategies may favor
the development of female mating avoidance behavior to minimize trauma. In S. mitsukurii this
behavior includes segregation of sexes and an asynchronous reproductive cycle.
KEY WORDS: Elasmobranch · Polyandry · Control region · Microsatellite DNA · Population structure ·
Sexual conflict · Sexual segregation · Reproductive strategy
Resale or republication not permitted without written consent of the publisher
In sexually reproducing species, the existence of
conflicting fitness strategies between sexes can lead to
intense sexual selection and the establishment of sexual conflict, where coercive traits that arise in one sex
are countered by the evolution of resistance traits in
the other (Zeh & Zeh 2003). In the majority of verte-
brate mating systems, females bear the energetic burden of ova and parental care and are thus expected to
be the more ‘choosy’ sex in regards to mate selection.
Males, in contrast, are expected to be non-parental,
sexually competitive, and promiscuous (Smith 1984,
Birkhead 1998, Birkhead & Pizzari 2002). Contrary to
the historical assumption of monogamy in the choosy
sex, there is abundant evidence of multiple mating by
*Email: tengel@email.arizona.edu
© Inter-Research 2010 · www.int-res.com
INTRODUCTION
256
Mar Ecol Prog Ser 403: 255–267, 2010
females with conventional sex roles (reviewed by Zeh
& Zeh 2003). Polyandry (females mating with more
than one male) and multiple paternity (a single brood
of offspring sired by multiple males) are now recognized as common strategies in widely divergent taxa
including amphibians, mammals, reptiles, insects,
crustaceans, and fishes (Evans & Magurran 2000, Toonen 2004, Adams et al. 2005, Bretman & Tregenza
2005, Daly-Engel et al. 2006, Dean et al. 2006, Jensen
et al. 2006). It is still unclear, however, what roles sexual conflict and intersexual selection might play in
polyandrous mating systems.
For males, the advantages to having multiple breeding partners are clear: the more females a male inseminates, the more offspring he fathers and the greater his
reproductive fitness. The benefits of polyandry to females are less obvious. Potential direct benefits to the
female include nuptial gifts or parental care on the part
of the male. No direct benefits have been shown in
shark mating systems, though there is potential for indirect or genetic benefits through polyandrous mating. If
there is little or no opportunity to evaluate males prior
to copulation, a female may hedge her bets by mating
promiscuously and therefore increase her chances that
one of these matings may lead to higher survivorship
for offspring (genetic bet-hedging; Watson 1991, Madsen et al. 2005). Alternatively, polyandry may result in
inbreeding avoidance or increase the likelihood that a
female’s offspring will be sired by a male whose genes
are compatible with hers (genetic compatibility hypothesis; Zeh & Zeh 1997, 2001, Neff & Pitcher 2005).
However, multiple mating can also be disadvantageous
to females due to exposure to disease or risk of injury
during mating events; female sharks may sustain serious injury or even die as a result of harm incurred during copulation (Pratt & Carrier 2001).
Apart from benefits to offspring, there is ongoing
debate over whether multiple paternity might confer
benefits to a population by maintaining genetic diversity, depending on whether it increases or decreases
variance in reproductive success (Sugg & Chesser
1994, Zeh & Zeh 2003, Karl 2008). One school of
thought maintains that multiple paternity may buffer
against the loss of allelic diversity by increasing the
effective population size (Sugg & Chesser 1994, Newcomer et al. 1999, Martinez et al. 2000, Hoekert et al.
2002). This is countered by theoretical results indicating that by increasing the variance in male reproductive success (because each mating may result in fewer
offspring per male than with genetic monogamy), multiple paternity will reduce effective population size
and, consequently, limit population genetic diversity
(Nunney 1993, Ramakrishnan et al. 2004, Karl 2008).
Reproductive strategy can have considerable effect on
genetic diversity, which in turn affects the ability of
populations to respond to selection pressures like
changes in environmental conditions (Rowe & Hutchings 2003, Frankham 2005). For this reason, loss of
genetic diversity has been associated with increased
vulnerability to population depletion and extinction
(Dulvy et al. 2003, Rowe & Hutchings 2003, Frankham
2005). Though a speciose group, sharks in particular
exhibit slower rates of genetic evolution than other
vertebrates (Martin et al. 1992), as well as lower rates
of growth and reproduction, which may limit their ability to recover from population depletion.
The frequency in a population with which a gravid
female carries a brood sired by more than one male
(multiple paternity) can be estimated by inferring the
minimum number of fathers per brood from genotypes
of mothers and their offspring. Previous work on multiple paternity in elasmobranchs has shown a large
degree of inter- and intraspecific frequency variation
(Ohta et al. 2000, Saville et al. 2002, Chapman et al.
2004, Feldheim et al. 2004, Daly-Engel et al. 2007,
Lage et al. 2008). Given that Lage et al. (2008) found
low (30%) multiple paternity in the congener Squalus
acanthias, a low level of multiple paternity in S. mitsukurii could indicate genus-level concordance in
squalid sharks. However, recent studies have further
shown that rates of multiple paternity can vary even
between populations of a single species (Daly-Engel et
al. 2007, Portnoy et al. 2007), indicating high levels of
behavioral trait plasticity. Though the number of studies on polyandrous mating in elasmobranchs continues to increase, multiple paternity has not yet been
shown to confer either direct or indirect benefits to
sharks (DiBattista et al. 2008a), leading some investigators to hypothesize that multiple paternity in elasmobranchs may be influenced by sexual conflict (DalyEngel et al. 2007, Portnoy et al. 2007, DiBattista et al.
2008b).
We assessed the frequency of multiple paternity in
27 litters of the shortspine spurdog Squalus mitsukurii
from throughout Hawaii using a suite of 8 polymorphic
microsatellite DNA markers, including 6 novel speciesspecific markers developed for the present study and
2 previously published loci developed for Squalus
acanthias (McCauley et al. 2004). In addition, we examined the link between genetic diversity and reproductive strategy by estimating genetic diversity using
a 670 bp segment of the mitochondrial control region
for comparison to other studies. We also calculated
allelic richness for the microsatellite loci in all published surveys of shark multiple paternity to determine
whether genetic diversity correlates with multiple
paternity in sharks. This is the first estimation of
genetic polyandry in S. mitsukurii coupled with one of
the few direct measures of genetic diversity (allelic
richness) in any squalid, a globally distributed family
Daly-Engel et al.: Multiple paternity in Squalus mitsukurii
of small sharks known collectively as dogfish. These
data, generated from an unfished population, will
serve as a foundation for future studies examining natural reproductive strategies and genetic diversity in
both exploited and unexploited populations of elasmobranch fishes.
MATERIALS AND METHODS
Study species. The shortspine spurdog Squalus mitsukurii aggregates on or near the bottom at a depth of
100 to 950 m in temperate, subtropical, and tropical
seas, particularly along coastlines, continental shelves,
and on seamounts (Wilson & Seki 1994, IUCN 2003).
The species is ovoviviparous with low fecundity.
Females give birth to an average of 6 pups ~25 cm in
length at birth every 2 to 3 yr (IUCN 2003, Compagno
et al. 2005). S. mitsukurii is widely distributed in the
Pacific Ocean (Last & Stevens 1994) and is likely a species complex. Age at maturity is between 4 and 7 yr for
males and between 14 and 16 yr for females (Wilson &
Seki 1994, Taniuchi & Tachikawa 1999) and generation time is more than 25 yr (Compagno et al. 2005).
S. mitsukurii has no known quiescent period between
gestations, with ova maturing concurrently with
embryos, such that when pups are at term the new ova
are ready for fertilization (T. S. Daly-Engel & R. D.
Grubbs pers. obs.).
Squalus mitsukurii is currently listed as endangered
on the IUCN Red List (IUCN 2003), based primarily on
data taken from the Australian population. There,
257
S. mitsukurii populations declined as much as 97%
between 1976 and 1997 due to fishing mortality as
bycatch from commercial trawling (Graham et al. 2001,
IUCN 2003). The status of other populations of S. mitsukurii is unknown due to the high likelihood of misidentification and the lack of data from most of the
world. In Hawaii, S. mitsukurii is rare as bycatch in the
bottomfish fishery, and little is known about its range,
population structure, or stock status. A study of large
aggregations of S. mitsukurii from the Hancock seamount in the Hawaiian–Emperor seamount chain is
the only published report of this species from the central Pacific (Wilson & Seki 1994). Anecdotal data and
catch rates from the present study indicate a robust
Hawaiian stock which is largely unaffected by fishing
mortality, making Hawaii an ideal location to acquire a
baseline understanding of the genetic mating system
and allelic diversity of this species. A note on species
identification: though the species of dogfish most common in Hawaii is widely accepted to be S. mitsukurii,
recently published morphological keys (White et al.
2007) appear to exclude Hawaiian dogfish from S. mitsukurii (R. D. Grubbs unpubl. data). Until additional
studies are done, however, we will continue to use
accepted nomenclature.
Sampling. We collected sharks near Oahu and 5
other locations throughout the Hawaiian Archipelago
between August 2005 and November 2008 (Fig. 1). Of
the 27 litters collected, 4 were sampled from the newly
–
–
akea
Marine National
established Papahanaumoku
Monument in the Northwest Hawaiian Islands
(NWHI). The NWHI includes 10 small atolls, pinnacles,
Fig. 1. Hawaiian Archipelago. Stars indicate the 6 sampling sites. N: corresponding sample sizes were analyzed for mitochondrial
diversity. Base map reproduced from www.oar.noaa.gov/spotlite/archive/images/bottomfishing_NWHI.jpg
258
Mar Ecol Prog Ser 403: 255–267, 2010
and islands and encompasses 360 000 km2 of ocean
water northwest of Kauai. The remote location of the
NWHI combined with high level of protection made it
difficult to acquire specimens, which were opportunistic bycatch from bottom fishing vessels. The remaining
23 litters were obtained from the Main Hawaiian
Islands (MHI) off the islands of Oahu and Maui (Penguin Banks, Table 1) using monofilament research
lines (shortened longlines, ~0.8 to 1.2 km) anchored at
each end and marked with buoys. Approximately 17%
of the sharks caught by longlining were pregnant
females. We used branch lines or gangions 4 m in
length composed of stainless steel tuna clips attached
to 2.5 m of 250 kg monofilament line. The line was
attached to 1.5 m of stainless steel aircraft cable with
8/0 stainless steel swivel and 11/0 circle hooks baited
with Japanese mackerel Scomber japonicus or squid
(Loligo spp.). Each line consisted of 50 to 80 gangions
spaced approximately 15 m apart. Sharks were measured and weighed, litter size was recorded, and small
samples of fin or muscle tissue were taken using scissors from mothers and pups. Tissue was stored in 20%
dimethylsulfoxide (DMSO) saturated salt buffer
(Seutin et al. 1991) or > 75% ethanol (EtOH). DNA was
extracted from tissue using a salting-out protocol
adapted from Sunnucks & Hales (1996). Samples
stored in EtOH were dried in a speed vacuum for
30 min at 55°C before extraction.
Microsatellite fragment analysis. We developed
microsatellite markers using an enrichment protocol
(Glenn & Schable 2005). This protocol, which employs
streptavidin-coated magnetic beads and biotin-labeled
repetitive probes — here, (AGAT)8, (AAAG)8, and
(AAAC)6 — was followed as described previously
(Feldheim et al. 2007). Six species-specific primers
were developed using the default settings in 0Primer3
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.
cgi). These 6 plus 2 primer pairs developed for Squalus
acanthias (T289 and U285; McCauley et al. 2004) were
found to be highly variable and therefore informative
for parentage analysis (Table 2). Following optimization, unlabeled reverse primers were obtained from
Integrated DNA Technologies. Forward primers were
labeled with 6-FAM, VIC, NED, and PET proprietary
dyes (Applied Biosystems). PCR reactions consisted of 0.1 U Biolase Taq
Table 1. Squalus mitsukurii. Date of capture and location is shown for each of
DNA polymerase (Bioline), 1× Taq
27 litters, as well as the size of the mother (TL: total length, cm), number of pups
buffer, 0.25 to 0.0625 µm of each primer
per litter, average TL of pups, maximum number of paternal alleles detected
across 8 microsatellite loci, and minimum number of sires indicated by the pres(see Table 2), 200 µm each dNTP, and
ence of these alleles in each litter. PB: Penguin Banks; nd: not determined
2.0 mm MgCl2. PCR amplification on a
MyCycler (Bio-Rad) consisted of an iniLitter Capture Capture Maternal
No.
Mean Max. no. Min.
tial denaturation at 95°C for 4 min folID
date
location
TL
pups in
TL of paternal
no.
lowed by 35 cycles of 1 min at 95°C,
litter
pups
alleles
sires
30 s at optimal annealing temperature
(Ta) (Table 2), and 30 s at 72°C, folB
Aug 2005
Oahu
60.0
6
21.5
2
1
C
Aug 2005
Oahu
66.5
7
14.6
2
1
lowed by a final extension at 72°C for
D
Sep 2005
Oahu
68.5
5
23.6
2
1
20 min. PCR products were resolved
E
Feb 2006
Oahu
78.5
8
16.3
2
1
with an ABI 3100 automated sequencer
F
Feb 2006
Oahu
78.0
5
12.7
2
1
and visualized using ABI PRISM GenG
Feb 2006
Oahu
84.0
5
10.3
2
1
H
Feb 2006
Oahu
74.0
5
24
2
1
eMapper Software 3.0 (Applied BiosysI
Feb 2006
Oahu
77.0
5
22.3
2
1
tems). Negative and positive controls
J
May 2007
Oahu
nd
3
7.9
2
1
consisted of extraction and amplificaK
Feb 2008
Oahu
87.5
10
nd
2
1
tion of known samples and DNA
L
Mar 2008
PB
72.5
4
17.4
2
1
sequencing of randomly selected indiN
Mar 2008
PB
79.0
7
3.5
2
1
O
Mar 2008
PB
81.0
6
4.2
2
1
viduals.
P
Mar 2008
PB
70.0
5
2.3
2
1
We estimated heterozygosity and
Q
Apr 2008
Oahu
nd
7
nd
2
1
tested
for deviation from Hardy-WeinR
Nov 2008
Oahu
88.0
9
12.8
2
1
berg Equilibrium (HWE) in 96 unreS
Nov 2008
Oahu
87.5
10
2.5
2
1
T
Nov 2008
Oahu
84.5
10
9.3
2
1
lated individuals, including mothers
U
Nov 2008
Oahu
79.5
5
8.4
2
1
and all individuals from the NWHI,
V
Nov 2008
Oahu
88.0
9
14.7
4
2
using Genepop 3.4 (Raymond &
W
Nov 2008
Oahu
92.0
9
1.9
2
1
Rousset 1995), and tested for linkage
X
Nov 2008
Oahu
86.0
10
18.4
2
1
disequilibrium using Arlequin 3.11
Y
Nov 2008
Oahu
83.0
6
4.2
2
1
NA
Jun 2006 Lisianski
90.0
6
20.2
4
2
(Excoffier et al. 2005). We used MicroNB
Jun 2006 Lisianski
101.0
7
10.9
2
1
Checker 1 (van Oosterhout et al. 2004)
NC
Oct 2006 Gardner
84.5
5
7.6
2
1
to infer genotyping errors due to null
ND
Dec 2007
Nihoa
78.5
4
11.9
4
2
alleles, short PCR dominance (large
259
Daly-Engel et al.: Multiple paternity in Squalus mitsukurii
allele dropout), the scoring of stutter peaks, and typographic errors. We inferred the minimum number of
sires from the number of non-maternal alleles detected
among all pups following the methods of Neff et al.
(2002). For each litter, we removed the maternal alleles
and counted the number of unique non-maternal alleles (Toonen 2004). Since the genotypes of the sires are
unknown in these field-collected animals, we used the
conservative assumption that every female mated with
only heterozygous males. Given this assumption, the
minimum number of sires per litter is one-half the
number of non-maternal alleles. If an odd number of
non-maternal alleles were detected among the pups,
the minimum number of males was rounded up. For
example, if 3 non-maternal alleles were detected, the
minimum estimated number of sires was rounded up
from 1.5 to 2 males. Mendelian inheritance of maternal
alleles was tested in each litter using a chi-squared
goodness-of-fit test against an expected 1:1 inheritance ratio.
We used the program PrDM 1 (Neff & Pitcher 2002)
to calculate the probability of detecting multiple mating (PrDM) in a sample of offspring based on (1) the
number of loci, (2) the number of alleles per locus, (3)
allele frequencies in the natural population (obtained
from the 96 unrelated individuals), (4) the conservative
estimate of number of sires contributing to each brood,
and (5) reproductive skew of each sire (Vieites et al.
2004). The model assumes single-sex multiple mating
(polygyny or polyandry), where all offspring in a brood
were either full-siblings or half-siblings. We used an
initial model of only 2 sires, each with the probability
of mating equal to 0.5, because this is the most conservative estimate. Adding sires to the model would
increase the statistical power to detect multiple paternity, but could lead to false overestimation of multiple
matings (Neff et al. 2002). We performed 8 replicates of
the analysis for the range observed in our samples (3 to
10 pups).
The Bayesian program FMM 1 (Neff et al. 2002) was
used to estimate expected frequency of multiple paternity in this population. Because not all of the males in
the population are heterozygous for alleles other than
those carried by the mother, an estimate based solely
on the observed number of non-maternal alleles may
underestimate the true frequency of multiple paternity
(Neff et al. 2002, Toonen 2004). The Bayesian method
used in FMM takes the allele frequency distribution of
the population into consideration when calculating the
most likely frequency of multiple paternity, and
assigns a 95% confidence interval to that estimate.
Statistical correlations between the total length (TL) of
the mother, number of pups per litter, and number of
paternal alleles detected were tested with Minitab 14.
Genetic structure and diversity. Because we sampled
across a broad geographic range (2000 km), we needed
Table 2. Squalus mitsukurii. Details on microsatellite loci used in the present study. Locus name, primer sequence (F: forward; R:
reverse), repeat motif, and size (bp) of the allele from which primers were developed, plus annealing temperature (Ta, °C) and
primer concentration (µmol reaction–1). Also shown are allelic diversity (k), allelic richness (A), observed and expected heterozygosities (Hobs and Hexp), probabilities (p-values) from Hardy-Weinberg Equilibrium tests for homozygote excess based on multilocus genotypes from 96 unrelated S. mitsukurii, and values of Jost’s estimated D (Dest). Dye labels were applied to forward primers
Locus
Primer sequence
Smi033 F: GAAAGCAGAAATGCCCACAT
R: GGGATATATGAACCCTTTTAAGTCA
Smi063 F: GGACAATTCAAACAATCTAAACAATG
R: AGTGCTGGACCATCATAGCC
Smi242 F: CATGTTTCAAGGAAGGATGG
R: TAGTTGGGCACATGCAAGAA
Motif
Size Ta Primer k
conc.
(AC)22
223 62
0.5
Imperfect
191 62
Imp. AAAG/ 286 62
AAGG repeat
A
p
Hobs
Hexp
Dest
13 11.20 0.311
0.352
0.229 –0.003
0.125 21 19.12 0.756
0.806
0.352 –0.034
0.25
2
1.93 0.607
0.514
0.000a –0.007
Smi292 F: TATATGGGGAATGASATTAAG
R: AAAAGGAGATGGAATAACTATGGTG
Imperfect
249 56
0.15
8
8.00 0.385
0.373
0.097 –0.005
Smi294 F: AACATAGCCACCCAATCACC
R: TTCAATGCACGTCAACAAGG
Imperfect
158 62
0.15
2
2.00 0.674
0.502
0.000a –0.007
Smi327 F: CCGCTTCAGATCAGCTTTTT
R: CCAAGGATTTGTACGGCATC
(TAGA)17
202 62
0.125 13 11.64 0.846
0.866
0.574 –0.044
T289a
F: GGGCGTCTGTGAACGCAGAC
R: ATAGTCCAGTAACATAACCTG
(TCC)7
191 56
0.25
6
5.39 0.489
0.519
0.556 –0.011
U285a
F: CTGTCCATGGTCACTTTT
R: GATACTTTTGTTCAGAGC
(CT)11
240 56
0.125 8
7.56 0.551
0.602
0.138 –0.016
a
McCauley et al. (2004)
260
Mar Ecol Prog Ser 403: 255–267, 2010
to first confirm that we were assaying a single breeding
population. To this end, mitochondrial haplotype diversity was calculated in all 112 unrelated individuals collected across the sampling locations represented in the
present study (Fig. 1). A fragment of the control region
(670 bp) was amplified from each sample using the
ProL2 (5’-CTG CCC TTG GTC CCC AAA GC-3’) and
PheCaCaH2 (5’-CTT AGC ATC TTC AGT GCC AT-3’)
primers (Pardini et al. 2001). Target DNA was amplified
using the protocol outlined above, with a Ta of 60°C.
PCR products were cycle sequenced using Big Dye
chemistry on an ABI 3100 automated sequencer (Applied Biosystems) at the Hawaii Institute of Marine Biology EPSCoR Sequencing Facility, aligned by eye, and
edited using Sequencher 4.6 (Gene Codes Corporation). Arlequin was used to generate nucleotide and
haplotype diversities. PAUP* 4.0b10 (Swofford 2000)
was used to calculate genetic distance and Structure
2.2 (Pritchard et al. 2000) was used to calculate the
likely number of distinct populations (K ) using microsatellite data. In Structure we used the admixture
model with a 10 000 burn-in length and 10 000 simulations to test K = 1 – 5 with 10 repetitions each. The relationships between haplotypes are described with a parsimony network based on TCS 1.21 (Clement et al.
2000) (see Fig. 2). We also used SMOGD 1.2.0 (Crawford 2009) to calculate Jost’s D for unrelated individuals
at 8 microsatellite loci. Jost’s D is a measure of genetic
differentiation that is independent of within-subpopulation heterozygosity (Jost 2008).
For the analysis comparing allelism at microsatelliteloci to frequency of multiple paternity, results from
7 studies were compared: Chapman et al. (2004),
Feldheim et al. (2004), Daly-Engel et al. (2007), Portnoy et al. (2007), DiBattista et al. (2008b), Lage et al.
(2008), and the present study. Allelic richness was calculated using FSTAT 2.9.3.2 (Goudet 1995). FSTAT
applies a rarefaction method to standardize alleles per
locus to a uniform sample size, in this case, 60 to
70 individuals. In studies where the number of unrelated individuals genotyped was already 60 to 70 individuals, rarefaction was not performed. Percent multiple paternity was arcsine square root-transformed for
linearity, and Pearson correlation on these data was
done using Minitab 14.
Dest = 0.016, Table 2). The TCS parsimony network of
haplotypes (Fig. 2) showed 11 variable sites and 6
haplotypes (GenBank accession no. GU192450–
GU192455). Two of these were exhibited among the
vast majority of individuals (107 out of 112), with the
other 4 haplotypes distributed among 5 remaining
individuals. No more than 2 mutational steps separated any haplotype from another except for the divergent type found in a single specimen from Gardner
Pinnacles, which was separated from the ancestral
type by 8 mutations (a genetic distance of d = 1.2%).
The parsimony network (Fig. 2) shows that the 2 most
common haplotypes were observed at every sampling
site where more than 1 sample was obtained, indicating high maternal gene flow throughout the sampling
range.
MicroChecker detected no microsatellite scoring
errors resulting from DNA degradation, low DNA concentrations, or primer-site mutations. There was evidence of deviation from HWE at Smi242 and Smi294,
which showed significant heterozygote excess in the
sample of 96 unrelated individuals (Table 2). Maternal
RESULTS
Using the program Structure, we found no evidence
for more than one population within Hawaii (K = 1)
with estimates of posterior probability approaching 1,
which is consistent with a lack of genetic structure.
Similarly, within-population tests of genetic differentiation showed little differentiation across loci (average
Fig. 2. Squalus mitsukurii. Parsimony network of control region haplotypes from 112 unrelated individuals. Size of circles
or wedges represents the number of samples within each
haplotype, and uninterrupted branches represent single
mutational steps
261
Daly-Engel et al.: Multiple paternity in Squalus mitsukurii
alleles at these loci were inherited in expected 50:50
ratios in all offspring of the 27 litters, so heterozygote
excess at these loci did not affect our estimate of multiple paternity. Smi033 was out of HWE due to heterozygote deficiency until we excluded the specimens from
Lisianski and Raita Banks (the 2 atolls at the distal
northwest end of our sampling range), possibly indicating a null allele at these locations. Though heterozygote deficiency may indicate a Wahlund effect,
we eliminated this possibility because the discrepancy
in HWE was limited to a single locus. Exclusion of the
5 individuals or this locus from any of our analyses did
not significantly change our results, so we retained
them in our analysis. HWE at Smi033 was based on 91
rather than 96 unrelated individuals. There was no evidence of linkage disequilibrium among pairs of loci
after Bonferroni correction.
We found evidence of multiple paternity (3 or 4 paternal alleles at each of 2 to 3 loci) in 11% of the litters
sampled (3 of 27 litters; Table 1). Each of the 178 pups
had at least one maternal allele, and chi-squared tests
confirmed that inheritance of these alleles did not vary
from predicted 1:1 Mendelian inheritance ratios within
each litter (df = 1, p > 0.05). The program FMM estimated the expected Bayesian frequency of multiple
mating to be 9% in this population (excluding Smi242
and 294; Neff et al. 2002), which closely approximated
our estimate of 11% based on direct count of nonmaternal alleles. The 95% confidence interval (CI) was
1 to 24% mixed paternity. When we removed Smi033
from this analysis, the results were essentially
unchanged (expected frequency of multiple mating =
12%, 95% CI = 2 to 27%).
Litters of Squalus mitsukurii ranged in size from 3 to
10 pups, and mean litter size was 6.6 (Table 2). The
program PrDM (Neff & Pitcher 2002) assigned a 90%
probability of detecting multiple paternity in litters of
this size (Neff & Pitcher 2002), hence we had good
power to detect multiple paternity in S. mitsukurii. If
we adjusted our calculation of multiple paternity to
conservatively assume that it occurred in the 10% of
cases where we lacked statistical power to detect it,
then the frequency of multiple paternity in this population was approximately 12%, well within the 95% CI
calculated by FMM. Among these 27 litters we found a
significant correlation (Spearman’s test, df = 1, α =
0.05) between the TL of the mother and the number of
pups per litter (R2 = 0.34, ρ = 0.59, p = 0.002). There was
no significant correlation between the TL of the mother
and the number of paternal alleles found among the
pups (R2 = 0.06, ρ = 0.26, p = 0.216), or between litter
size and the number of paternal alleles detected (R2 =
0.01, ρ = –0.06, p = 0.76).
Arlequin yielded a haplotype diversity value of h =
0.5412 ± 0.0221 and nucleotide diversity value of π =
0.0010 ± 0.0008. Table 3 shows the results of all known
studies documenting nucleotide and haplotype diversities in the mitochondrial control region for elasmobranch species. Haplotype diversity in Squalus mitsukurii is the third lowest among sharks to date, and
nucleotide diversity was the second lowest measured
in an elasmobranch.
To examine the relationship between mating strategy
and genetic diversity we performed correlation analysis
on all 7 data points from shark paternity studies published to date. Fig. 3A reports the results from the 5
studies that used only species-specific microsatellite
loci (Feldheim et al. 2004, Portnoy et al. 2007, DiBattista
et al. 2008b, Lage et al. 2008, present study), and Fig.
3B reflects the same analysis of these 5 studies plus 2
that did not use species-specific loci (Chapman et al.
2004, Daly-Engel et al. 2007). Microsatellite loci that
Table 3. Genetic diversity in the mitochondrial control region among 13 elasmobranch species. Nucleotide diversity (π), haplotype diversity (h), sequence length (bp), and sample sizes (N) are shown. nd: not determined. *Studies encompassing more than
one geographic region. SA: South Africa; WA: Western Australia
Species
Squalus mitsukurii
Galeorhinus galeus*
Negaprion brevirostris
Negaprion acutidens
Rhincodon typus
Cetorhinus maximus*
Carcharias taurus (SA)
Carcharias taurus (WA)
Sphyrna lewini*
Carcharhinus limbatus
Raja clavata
Raja miraletus
Raja asterius
Carcharadon carcharias
π ± SD
h ± SE
Sequence length
N
Source
0.0010 ± 0.0008
0.0025
0.0059
0.0006
0.0110 ± 0.006
0.0013 ± 0.0009
0.0030 ± 0.0001
0.0031 ± 0.0001
0.0130 ± 0.0068
0.0021 ± 0.0013
0.0072
0.0031
0.0092
0.0203
0.541 ± 0.022
0.805
0.780
0.280
0.974 ± 0.008
0.720 ± 0.028
0.717 ± 0.010
0.458 ± 0.024
0.800 ± 0.020
0.805 ± 0.018
0.610
0.170
0.290
nd
670
~990
1090
1090
1236
1085
700
700
548
1070
335
330
329
nd
112
116
80
58
70
62
26
16
271
323
26
12
18
88
Present study
Chabot & Allen (2009)
Schultz et al. (2008)
Schultz et al. (2008)
Castro et al. (2007)
Hoelzel et al. (2006)
Stow et al. (2006)
Stow et al. (2006)
Duncan et al. (2006)
Keeney et al. (2005)
Valsecchi et al. (2005)
Valsecchi et al. (2005)
Valsecchi et al. (2005)
Pardini et al. (2001)
262
Mar Ecol Prog Ser 403: 255–267, 2010
DISCUSSION
Population structure
Fig. 3. Correlation of allelic richness with percent multiple
paternity (% MP; square root-arcsine transformed) in all elasmobranch multiple paternity studies to date for which allele
frequency data was available. (A) Includes data points from 5
studies with species-specific microsatellite markers (Feldheim et al. 2004, Portnoy et al. 2007, DiBattista et al. 2008b,
Lage et al. 2008, present study); (B) shows the same correlation including 2 studies that used non-species-specific loci
(Chapman et al. 2004, Daly-Engel et al. 2007)
are cross-amplified across species may be less polymorphic than they are in target species, though the number
of loci that successfully cross-amplify in sharks is often
higher than in other taxa, presumably due to their
slower rate of nucleotide mutation (Martin et al. 1992).
Although the removal of 2 studies leaves us with only 5
data points in Fig. 3A, we chose to present both sets of
data because we thought that the effect of this variable
cannot be sufficiently resolved within the scope of the
present study (though the present study used 2 loci developed for a congener, these loci were not considered
when calculating allelic richness). The correlation between multiple paternity and genetic diversity in the 5
species-specific studies returned an R2 value of 0.40
(p = 0.184; Fig. 3A). When we included the 2 studies
that did not use species-specific markers (Chapman et
al. 2004, Daly-Engel et al. 2007), the R2 dropped only
slightly, to 0.32 (p = 0.249; Fig. 3B). While preliminary
and not statistically significant, these data indicate that
a relationship may exist between allelic richness and
multiple paternity in sharks, though more data points
are needed to provide thorough analysis.
We analyzed 670 bp of the mitochondrial control
region to characterize population structure and
nucleotide diversity in the shortspine spurdog Squalus
mitsukurii in Hawaii. Overall, our observation of several common haplotypes distributed among nearly all
sampling sites indicates that S. mitsukurii throughout
the Hawaiian Archipelago is composed of a single
breeding population (K = 1). Given the low mtDNA
diversity and low sample sizes at most locations, however, the conclusion of no population structure in
S. mitsukurii from Hawaii must be regarded as provisional. Although a robust test of this conclusion would
require larger sample sizes, the finding of no genetic
structure is consistent with reef fish studies that show
high connectivity across the Hawaiian Archipelago
(Craig et al. 2007, Eble et al. 2009). Interestingly, the
single specimen obtained from Gardner Pinnacles had
the most divergent haplotype, 1.2% from the nearest
related haplotype. This divergence is notable because
Gardner Pinnacles in the central Hawaiian Archipelago is near Johnston Atoll, a suspected entry point for
colonization into Hawaii (Gosline 1955). These data
indicate that dispersal in S. mitsukurii is greater than
their known habitats would indicate (see Schultz et al.
2008), because maternal gene flow appears to occur
across depths greater than the maximum depth (954 m)
reported for this species (Compagno et al. 2005).
Multiple paternity in sharks
Our observation of 11% multiple paternity (3/27) in
Hawaiian Squalus mitsukurii is the lowest level estimated in an elasmobranch species to date, with a maximum of 4 paternal alleles found at any single locus.
Number of paternal alleles detected per litter was not
correlated with TL of the mother or number of pups per
litter, though significant correlation was found between TL of the mother and number of offspring, a
finding consistent with other shark species (Cortes
2000). Estimates of the frequency of multiple paternity
in natural shark populations have included a predominance of genetic monogamy in the present study, as
well as in the bonnethead shark Sphyrna tiburo (18%;
Chapman et al. 2004). Two studies have returned intermediate values of multiple paternity, for the spiny dogfish shark Squalus acanthias (30%; Lage et al. 2008)
and the Hawaiian population of sandbar sharks Carcharhinus plumbeus (40%; Daly-Engel et al. 2007).
Very high prevalence of multiple paternity was reported in lemon sharks Negaprion brevirostris from the
Daly-Engel et al.: Multiple paternity in Squalus mitsukurii
Bahamas (87%; Feldheim et al. 2004) and Florida
(85%; DiBattista et al. 2008a), and in the Northwest
Atlantic population of sandbar sharks C. plumbeus
(86%; Portnoy et al. 2007). No shark species examined
to date has shown a complete absence of multiple
paternity. The ubiquity of multiple paternity in sharks
indicates that this strategy is beneficial or unavoidable,
or possibly both.
Portnoy et al. (2007) proposed that females with
longer reproductive cycles may employ polyandrous
mating behavior, effectively increasing the cumulative
genetic variation in progeny. Lemon sharks and Northwest Atlantic sandbar sharks, which show a high rate
of multiple paternity, mate once every 2 yr, while the
predominantly monogamous bonnethead sharks have
an annual reproductive cycle (Chapman et al. 2004,
Compagno et al. 2005). Our current results and those
from a previous paper (Daly-Engel et al. 2007) do not
support the long reproductive cycle–high multiple
paternity hypothesis, since sandbar sharks in Hawaii
have the same reproductive cycle as those in the
Atlantic, but a much lower rate of multiple paternity.
Encounter rate theory and sexual conflict
The simplest explanation for the multiple paternity
observed in natural populations is the encounter rate
theory (Lopez-Leon et al. 1993, Daly-Engel et al. 2007),
which holds that rate of multiple mating should
depend on the number of male conspecifics a female
encounters over the course of a breeding season. In
high density populations, therefore, a female should
have more opportunities to encounter males, and the
rate of multiple paternity should increase (Kokko &
Rankin 2006, Daly-Engel et al. 2007). For example, in
nesting populations of olive ridley sea turtles Lepidochelys olivaceus, Jensen et al. (2006) found that the
frequency of multiple paternity was highly correlated
with the density of reproductive adults.
In sexually reproducing species, differing fitness
strategies can lead to conflict between males and
females. Though we did not directly measure sexual
conflict, the frequency of multiple paternity may be
determined not only by the ecological conditions that
affect encounter rate, but the sex ratios under which
those encounters occur. Among roving predators such
as sharks, the social interplay between the sexes can
strongly influence encounter rate. Though mating
behavior in sharks is difficult to observe, female sharks
do exert mate choice in the wild, largely through mating avoidance (Pratt & Carrier 2001, Whitney et al.
2004). In contrast, male sharks are expected to exhibit
a fitness strategy that favors promiscuity. Because
many sharks exhibit sexual segregation as well as sex-
263
ually differential migration, the conflict between the
sexes is played out largely during mating encounters.
Shark mating is usually characterized by the male biting the female, especially around the base of the fins
and flank, until he succeeds in grasping one of her pectoral fins, wrapping his body around her, and inserting
1 of 2 intromittent organs (claspers) into her cloaca for
insemination (Pratt & Carrier 2001, Hamlett 2005).
Though mortality is rare, it is common for females to
incur serious injury during mating (Carrier et al. 2004)
and to be more vulnerable to predation during and
immediately after mating attempts. In sharks, the
female is the larger of the 2 sexes, and could theoretically avoid mating with a conspecific male in a one-onone encounter. In encounters where males outnumber
females, which may occur within a mating aggregation, males can overcome the size disadvantage with
cooperative behavior (mobbing or herding) to induce
otherwise unwilling females to mate (Pratt & Carrier
2001, Whitney et al. 2004). In the case of coercive mating, a female may capitulate to avoid incurring more
harm, resulting in convenience polyandry (Thornhill &
Alcock 1983, Lee & Hayes 2004, DiBattista et al.
2008a). For example, populations of sandbar sharks
Carcharhinus plumbeus in the Northwest Atlantic are
sexually segregated throughout much of the year, but
aggregate in the warmer water of the Gulf of Mexico in
the winter (Musick 1999). These aggregations may
create opportunities for cooperative behavior on the
part of the males to induce mating. In the Hawaiian
sandbar shark population, males and females mix
throughout the year (Daly-Engel et al. 2006, 2007) and
no large aggregations for the purposes of mating have
been observed. The encounter rate theory predicts that
because sexual segregation is less stringent in Hawaii
than in the Atlantic, there should be a higher rate of
multiple paternity in Hawaii. Instead, the rate of multiple paternity in Hawaii is about half that observed the
Northwest Atlantic (Daly-Engel et al. 2007, Portnoy et
al. 2007), indicating that aggregative behavior which
facilitates male coercion may have a disproportionately large effect on rate of multiple paternity.
Genetic polyandry and mating avoidance
The discrepancy between predictions based on the
encounter rate theory and observations from Pacific
and Atlantic sandbar sharks indicates that the sex ratio
during mating encounters (male-biased aggregations
versus one-on-one encounters) may play a role in determining the prevalence of multiple paternity. Even
when a population does not include mating aggregations, predictability in the mating behavior of one sex
(e.g. female dependence on coastal nursery grounds,
264
Mar Ecol Prog Ser 403: 255–267, 2010
or philopatry; Feldheim et al. 2004, Grubbs et al. 2007)
may create the opportunity for seasonally elevated
density. Such predictable behavior may account for the
high (81 to 87%) multiple paternity observed among
populations of philopatric lemon sharks Negaprion
brevirostris. Feldheim et al. (2004) and DiBattista et al.
(2008a) suggest that high multiple paternity in lemon
sharks is more likely a result of convenience polyandry
than of indirect genetic benefits such as inbreeding
avoidance.
Squalus mitsukurii in Hawaii have a number of
physiological and life history traits which, taken together, may reduce genetic polyandry. Compared with
oviparous sharks, the squalid oviducal gland (the
organ of elasmobranch sperm storage) is relatively
reduced (Hamlett 2005), suggesting that long-term
sperm storage may not play a large role in the squalid
mating system. Ecologically, S. mitsukurii inhabit a
slope habitat (100 to 950 m depth), aggregating around
pinnacles, canyons, and seamounts. Within these aggregations, males segregate from females, and adults
from both subadults and juveniles (Wilson & Seki
1994). This sexual segregation is common to almost
every shark species examined to date, and is thought
to be a mechanism for both mating and cannibalism
avoidance (Cortes 2000). Mating aggregations which
facilitate convenience polyandry are unlikely in species like S. mitsukurii, whose asynchronous, ovoviparous reproductive strategy makes it difficult for
males to predict when females might be receptive to
mating. Lack of opportunity for male coercion could
lead to potentially low rates of multiple paternity in
species that demonstrate asynchronous reproduction.
For example, Lage et al. (2008) recently estimated the
rate of multiple mating to be 30% in 10 litters of the
congener species S. acanthias, which has the same
asynchronous, ovoviviparous reproductive strategy as
S. mitsukurii.
The protracted reproductive cycle may provide further incentive for female Squalus mitsukurii to avoid
incurring harm from multiple copulations (Siva-Jothy
2006). S. mitsukurii gestate their young for 24 mo
(Compagno et al. 2005) and give birth to average of
6 pups per litter (Table 1). Every mature female S. mitsukurii caught for the present study had either fertilized ova or embryos; like the congener S. acanthias,
S. mitsukurii appears to have little or no quiescent
period between pregnancies (Fischer et al. 2006,
T. S. Daly-Engel & R. D. Grubbs unpubl. data), such
that successful copulation most likely occurs very soon
following parturition. In squalid sharks, all fertilized
ova in each uterus are encased in a single membranous
casing or ‘candle’ that fills the uterus. The distal ends
of this candle plug the oviduct cranially and the uterine
sphincter caudally, such that any copulation following
fertilization would be unsuccessful, likely causing a
rupture in the candle leading to the death of the existing embryos. This physiology likely results in decreased opportunity for multiple mating in squalid compared to carcharhinid sharks, which can mate while
gravid over a period of several months without harming the embryos, and added incentive for male avoidance in female S. mitsukurii.
Genetic diversity and multiple paternity
A frequently proposed benefit of multiple paternity
is its potential for increasing effective population size
by increasing the number of males that mate successfully, thereby maintaining population genetic diversity
(Nunney 1996, Ramakrishnan et al. 2004, Frankham
2005). Elasmobranchs have lower genetic diversity
than most other taxa (Hoelzel et al. 2006), perhaps
because of their slow rate of molecular evolution (Martin et al. 1992). Multiple paternity at some frequency
has been observed in every elasmobranch species
examined to date, indicating that multiple paternity
may serve as a stable evolutionary strategy to maintain
genetic diversity in elasmobranch populations. Metabolism might also play a role in lowering genetic diversity in Squalus mitsukurii, which inhabits deeper,
cooler waters than the other species examined, and
whose correspondingly slower metabolic rate may
confer a lower than normal rate of genetic evolution
(Brown et al. 1979).
Multiple paternity may result in increased genetic
diversity in a single litter, but at the population level,
this effect is likely to be mitigated by a corresponding
increased variance in male reproductive success (Karl
2008). Our comparison of published estimates of
multiple paternity in sharks (Fig. 3) yielded a nonsignificant correlation of R2 = 0.40 between genetic
diversity and multiple paternity. Though this test has
arguably low power because of the sample size of only
5 studies, as more of these studies are done, the relatively high R2 value indicates that there may well be a
relationship between these 2 variables and that further
investigation is warranted. It is possible that allelic
richness itself might account for some of this pattern,
since increased allelic diversity enhances the probability of detecting multiple paternity across loci (Neff &
Pitcher 2002). However, the ability to detect multiple
paternity in most of these studies is quite good (> 90%).
It is possible that even in studies reporting a high
PrDM, multiple paternity may be underestimated due
to lack of allelic diversity across loci or sampling sites,
but most studies incorporate an interpretation of
allelism and PrDM in their discussions when reporting
on rate of multiple paternity.
Daly-Engel et al.: Multiple paternity in Squalus mitsukurii
CONCLUSIONS
Here we report the lowest level of multiple paternity
(11%) observed to date in an elasmobranch, the shortspine spurdog Squalus mitsukurii. This is the first survey of genetic polyandry in a deep-water vertebrate.
While frequency of genetic polyandry in shark populations is likely influenced by sexual conflict, the findings for S. mitsukurii also indicate a potential role for
physiology and encounter rate in determining the frequency of multiple paternity. Under this hypothesis,
the predominance of genetic monogamy in this species
results from life history characters such as asynchronous reproduction, lack of mating aggregations,
and an ovoviviparous reproductive mode where all
embryos initially develop in a common casing. S. mitsukurii also exhibited low nucleotide and haplotype
diversity relative to other elasmobranchs (π = 0.0010 ±
0.0008, h = 0.5412 ± 0.0221). Given that the S. mitsukurii in Hawaii represent a healthy, unfished population yet show low levels of genetic diversity, it is possible that populations elsewhere may experience low
levels of diversity made even lower by exploitation.
Though the case for a causative relationship between
polyandry and genetic diversity has yet to be made, it
is known that both reproductive strategy and genetic
diversity can influence a species’ ability to rebound
from population depletion, and these factors should be
considered in efforts to conserve and manage these
taxa.
Acknowledgements. The authors extend special thanks to
K. Holland, whose support made this project possible. Thanks
to J. Musick, J. Romine, C. Cotton, Y. Papastamatiou, J. Dale,
D. Itano, B. Alexander, C. Kelley, S. Lee, K. Kawamoto,
L. Litherland, M. Gaither, T. Timoney, G. Dill, B. Kikkawa,
and L. Yamada for help with sample collection. C. Lage, J. DiBattista, and D. Chapman generously contributed data for the
diversity meta-analysis. Z. Szabo, C. Bird, S. Daley, M. Mizobe, A. Eggers, T. Trejo, G. Concepcion, J. Puritz, J. Eble,
J. Franks, K. Andrews, and J. Coffey helped with genetic and
statistical analysis, and J. Eble gave valuable input that
greatly improved this manuscript. Thanks to all the members
of the Holland, Toonen, and Bowen Labs for their support.
Genetic analyses were made possible by the EPSCoR Evolutionary Genetics Facility at the Hawaii Institute of Marine
Biology, and funding was provided by the Ecology, Evolution
and Conservation Biology (EECB) Program at the University
of Hawaii, the National Science Foundation (NSF Graduate
K-12 program grant to EECB No. 0232016, OCE-0453167 to
B.W.B., OCE-0623678 to R.J.T., and EPS-0554657 to University of Hawaii), the PADI Foundation, the American Association of University Women, and Sigma Xi. Research in the
Northwest Hawaiian Islands is supported by NOAA National
Marine Sanctuaries Program MOA grant No. 2005-008/66882
(B.W.B. & R.J.T.). Microsatellite enrichment was partially
funded by the Grainger Foundation and was carried out in the
Pritzker Laboratory for Molecular Systematics and Evolution
operated with support from the Pritzker Foundation, and writing was supported by grant No. 2 K12 GM000708 to the PERT
265
Program at the University of Arizona from the National Institute of General Medical Sciences division of NIH. We also
thank reviewers and editor J. H. Choat for helpful comments
and improvements to the manuscript. This is contribution
No. 1357 from the Hawaii Institute of Marine Biology and contribution No. 7786 from the School of Ocean and Earth Science and Technology at the University of Hawaii.
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Submitted: August 18, 2009; Accepted: November 11, 2009
Proofs received from author(s): March 14, 2010