Molecular Assessment of Mating Strategies in a Population of Atlantic Spotted Dolphins
Molecular Assessment of Mating Strategies in a Population of Atlantic Spotted Dolphins
Michelle L. Green 0 1 2 3 4
Denise L. Herzing 0 1 2 3 4
John D. Baldwin 0 1 2 3 4
0 Accepted: January 9 , 2015
1 Received: August 1 , 2014
2 Academic Editor: Hanping Wang, The Ohio State University , UNITED STATES
3 . Current address: Department of Animal Sciences / Illinois Natural History Survey, University of Illinois , 1503 S. Maryland Drive, Urbana, Illinois, 61801 , United States of America
4 1 Department of Biology, Florida Atlantic University , 3200 College Avenue, Davie, Florida, 33314 , United States of America, 2 Wild Dolphin Project , P.O. Box 8436, Jupiter, Florida, 33468 , United States of America, 3 Department of Biology, Department of Psychology, Florida Atlantic University , 777 Glades Road, Boca Raton, Florida, 33431 , United States of America
Similar to other small cetacean species, Atlantic spotted dolphins (Stenella frontalis) have been the object of concentrated behavioral study. Although mating and courtship behaviors occur often and the social structure of the population is well-studied, the genetic mating system of the species is unknown. To assess the genetic mating system, we genotyped females and their progeny at ten microsatellite loci. Genotype analysis provided estimates of the minimum number of male sires necessary to account for the allelic diversity observed among the progeny. Using the estimates of male sires, we determined whether females mated with the same or different males during independent estrus events. Using GERUD2.0, a minimum of two males was necessary to account for the genetic variation seen among progeny arrays of all tested females. ML-RELATE assigned the most likely relationship between offspring pairs; half or full sibling. Relationship analysis supported the conservative male estimates of GERUD2.0 but in some cases, half or full sibling relationships between offspring could not be fully resolved. Integrating the results from GERUD2.0, ML-RELATE with previous observational and paternity data, we constructed two-, three-, and four-male pedigree models for each genotyped female. Because increased genetic diversity of offspring may explain multi-male mating, we assessed the internal genetic relatedness of each offspring's genotype to determine whether parent pairs of offspring were closely related. We found varying levels of internal relatedness ranging from unrelated to closely related (range -0.136-0.321). Because there are several hypothesized explanations for multi-male mating, we assessed our data to determine the most plausible explanation for multi-male mating in our study system. Our study indicated females may benefit from mating with multiple males by passing genes for long-term viability to their young.
Competing Interests: The authors have declared
that no competing interests exist.
Differences in parental investment , and ecological factors affecting spatial and temporal
distribution of mates and resources play an important role in shaping the mating system of a
population . Mating systems develop depending on interactions between costs and benefits
of behaviors within and between sexes. From the female perspective, mating bonds in
mammalian mating systems include monogamy where females maintain exclusive mating bonds with a
single male throughout most of their life, polyandry where females mate with a specific group
of the same males in successive breeding attempts, and promiscuity where males and females
mate with multiple individuals in successive attempts when no social bond between mating
individuals exists . Most mammals have an open polygynous system where one or both sexes
mate with multiple individuals .
Long-term observations of toothed whales and dolphins (Suborder Odontoceti) have led to
a better understanding of life history and behavior in these complex and highly social groups
. In most behavioral studies, male dolphins seek out receptive females, but spend little
time with females and new calves [5, 8]. In some locales, males have been observed guarding
and coercing receptive females  but this observation has not been universal. Because
observations typically indicated that males spent little time with females except to mate, mating
systems were labeled as promiscuous in species including dusky dolphins (Lagenorhynchus
obscurus ), estuarine dolphins (Sotalia guianensis ), common dolphins (Delphinus
delphis ), Hectors dolphins (Cephalorhynchus hectori ) and bottlenose dolphins (Tursiops
sp. [9, 14]). Given variable social behaviors and the advancement of molecular techniques, it is
now common to distinguish between social and genetic mating systems . The genetic
system describes the relatedness of individuals resulting from copulation and breeding, which can
differ from the breeding expectations derived from social mating system behaviors such as
mate guarding and pair bonding [2, 16]. Discerning between social and genetic mating systems
of marine mammals is especially challenging because of the difficulties associated with
longterm observation, delayed sexual maturity, long inter-birth intervals and collection of genetic
material. Thus only a handful of genetic mating system studies have been completed for
In this paper we present data on whether observable genetic patterns support a promiscuous
mating system, specifically from the perspective of the females. Prior to describing the
objectives and methods of this study, it is useful to review the social and demographic factors of the
study population that relate to reproductive behavior and provide a basis for a genetic mating
Study population demographics
A well-studied population of Atlantic spotted dolphins (Stenella frontalis) has been the focus of
intense observational study since 1985 and many demographic and social aspects of the
population are well characterized [4, 2326]. The study site (approximately 480 km2) is located on
the northwest side of Grand Bahama Island on Little Bahama Bank. The habitat is a shallow
sand bank (616 m depth) that borders a steep drop off of > 500 m depth into the Gulf Stream.
Over the past 25 years, more than 200 individuals have been identified in the study area and
the resident population consists of roughly 90100 individuals each year.
Atlantic spotted dolphins display four developmental color phases [4, 27]. Two tone
calves (03 yr) are born without spots. At roughly three years, dark ventral spots develop and
individuals advance to the speckled age class (39 yr). The mottled age class (916 yr)
begins when white spots form on the dorsal surface and ventral spotting has increased. The oldest
age class (fused) occurs at approximately 16 years of age and is identified when spots are no
longer discrete but fuse together into an overall coloration pattern .
Many individuals in the study population have been observed for more than 20 years and
reproductive histories of females and offspring are well documented. Females become sexually
mature near 10 years of age and on average, give birth to a calf every 2.96 years (range 15
years ). Length of gestation is estimated between 11 and 12 months and two calving peaks
(early spring and late fall) have been identified . However, females are not completely
synchronized because very young neonates have been observed in August and January.
Both sexes demonstrate natal philopatry and immigration has been consistently low (i.e.,
only a few individuals per year) for the community , indicating a relatively closed
population. The sex ratio (M:F) is close to parity with an average ratio across years of 0.97:1 [4, 24].
Age class structure generally consists of 32% fused, 27% mottled and 21% each of speckled and
two tone animals . Site fidelity has been relatively high, some individuals in the study
population have been sighted regularly for more than two decades . Atlantic spotted dolphins
are long-lived and some individuals that were in the fused age class (at least 16 yr) when first
sighted in 1985 have been resighted as recently as 2014, indicating an estimated age of at least
Study population social system
Atlantic spotted dolphins live in fission/fusion  societies exhibiting dynamic group
membership [25, 28]. The study population is considered a single community that is behaviorally
self-contained  and includes female networks, male alliances, but lacks long-term
associations between sexes . The community is subdivided into three social clusters (North,
Central and South)  consisting of sets of individuals with social associations that are stronger
within clusters than between, even though geographic ranges overlap.
Group size in the study population is variable, ranging from 160 individuals with a mean
group size of nine or fewer animals . Groups may consist of all combinations of sex and
age classes, and often consist of associated individuals . Most strong associations occur in
same sex groups . Female-female associations are influenced by reproductive status, calf
care, and social familiarity . Females associate closely with calves during the first years of
the calfs life .
Males form strong associations within and between male pairs/trios that remain relatively
constant over time . Males in the study population exhibit two levels of alliance formation;
long-term first order alliances and short-term second order alliances which combine two or
more first order alliances . Alliance formation is typically attributed to increased female
access  and male Atlantic spotted dolphin alliances have been observed displaying
cooperative female tending behaviors , although few observations exist. During a cooperative
monopolization event in the study community, 34 males followed a female, surrounded her,
escorted her during feeding and fended off other male groups [26, 33]. Such cooperative events
are not necessarily wide-spread among all male alliances nor do these interactions last for
extended periods of time. Compared to bottlenose dolphins in Shark Bay, Australia, males may
cooperatively tend females for months , whereas cooperative tending events in the study
population are typically observed over a time scale of minutes to hours and rarely over
Although male coalition formation (the joining of forces by two or more parties during a
conflict of interests with other parties ) is typically attributed to female access benefits
, male alliance and coalition formation are also important in interspecies conflicts in
the study population. Sympatric bottlenose dolphins exist in the study area and male Atlantic
spotted dolphin alliances form coalitions during agonistic encounters with male bottlenose
dolphins . The dynamic interaction between the species is not fully understood but it may
be that intraspecies coalitional behavior among Atlantic spotted dolphin males intercepts
mating activity between the species and maximizes the reproductive success of potential sires
What is the genetic expectation?
Most mammalian systems are polygynous and multi-male mating is common . It is
expected that females will mate with multiple males potentially due to the benefits of paternity
confusion , or genetic benefits to offspring . Based on limited genetic investigation,
there is evidence that female cetaceans mate with different males throughout their reproductive
life. Genetic analysis of humpback whale females and offspring revealed promiscuous mating
. In Sarasota, Florida, molecular investigation of bottlenose dolphins (T. truncatus)
indicated that individual males may have sired more than one calf with a given female but a single
male had not sired all of the calves from a female . In Shark Bay, Australia, paternity was
assigned to different bottlenose dolphin (Tursiops sp.) males for two offspring from the same
mother . In the study population, mating behaviors have been documented in all age
classes and between both sexes . Mating behavior occurs often and it can be difficult to observe
all courtship behavior in large groups of animals. Given promiscuous social behavior and
evidence from the small amount of literature available, we generally expect females to mate with
different males during each estrus event. However, characteristics of the study population raise
questions about the validity of that expectation.
In the study population, approximately 100 individuals are sighted each year , and the
sex ratio is near parity, indicating roughly 50 males in the population each year. Females
preferentially copulate with older males  and there is genetic evidence that males must reach a
minimum age (fused age class) prior to obtaining successful mating opportunities . If only
fused males obtain mating opportunities and one third of the males in the community are in
the fused age class category , then approximately 1617 males are available for mating.
Couple this with the fact that females may avoid mating with some males either because individuals
in this small community are genetically related  or male quality differs , the number of
suitable males may be reduced to a small number (< 16). Furthermore, the cohort of suitable
males may not change much from one estrus event to the next because the animals are
longlived and immigration in the population is low. Could these factors cause females to mate with
the same male during each estrus? Could a low number of males force a promiscuous species
into a system where only a few males obtain the majority of mating opportunities? Although
the expectation based on socially promiscuous behavior is that females will mate with different
males, these concepts raised a reasonable question that has implications for fragmented
populations as well as small populations of threatened species.
We tested whether females mate repeatedly with the same male for multiple conception
events, or whether females mate with different males. The duration of observation and
knowledge of maternal relationships provided a unique opportunity to collect genetic material from
sexually mature females and multiple calves, determine whether calves born to a single female
were sired by one or more males, and consider the hypotheses that best explain the observed
genetic mating patterns. As such our goals were to (1) genetically determine the minimum
number of males necessary to explain the allelic diversity of a progeny array, (2) construct
pedigrees based on the genetic data, and (3) assess genetic diversity among offspring.
Materials and Methods
We collected fecal samples from all possible individuals (20002007) to address multiple,
concurrent genetic investigations of the population. Field seasons consist of approximately 80100
field days every year from May through September utilizing a 21 m power catamaran as the
research platform. Observations and collections occurred from 07002000 during days in the
study area, with shorter observation times on days of travel to and from the study area or
during severe weather. During collection, researchers entered the water for direct observation of
dolphins and noted individual information including sex, age class, reproductive status, and
behavioral interactions whenever possible. We used underwater observation of spot and
coloration patterns as well as nicks, cuts and scars to reliably identify and age individual spotted
dolphins. Researchers familiar with the resident population confirmed identifications through
digital photographs or video taken of the animals. We compared photos of sampled individuals
to a master catalog of all known animals in the population. We assigned unique four-letter
identifiers to individual animals and used the identifiers to refer to specific individuals in this
study. We based mother-calf assignments on observational data of close association and
nursing behavior . We used sighting records to determine the year of birth for each calf and
estimated year of conception as one year prior to year of birth. We based the mothers estimated
age at first parturition and age at the birth of each calf on previously reported age estimates .
The mothers age of conception for each calf was based on an estimated 12 month
Fecal sample collection, storage and DNA extraction was carried out as previously described
by Green et al. . We amplified ten polymorphic microsatellite loci to generate genotypes
for females and offspring: EV37, EV01 , D08 , Ttr04, Ttr11, Ttr19, Ttr34, Ttr48 ,
Ttru AAT44  and KWM12 . All amplification reactions followed previously reported
protocols [EV37, D08 and Ttr48 reported by Green et al. ; EV01, Ttr04, Ttr11, Ttr19,
Ttr34, Ttru AAT44 and KWM12 reported by Green et al. ]. We initially visualized all
microsatellite fragments on a 6% polyacrylamide gel stained with ethidium bromide and sized
each fragment on an ABI Prism 310 genetic analyzer using GENESCAN ANALYSIS v. 3.1 and
GENOTYPER v. 2.1 (Applied Biosystems, Foster City, California, USA).
Because the current study was part of a larger study, the population-level data set yielded
sample sizes appropriate for checks of heterozygote deficiency, deviations from
Hardy-Weinberg and genotyping error as a result of null alleles, large allele drop-out and stutter bands. We
tested for deviation from Hardy-Weinberg equilibrium and linkage disequilibrium using
Fishers exact tests and the Markov chain method (10,000 dememorization steps, 1,000 batches,
and 10,000 iterations per batch) using GENEPOP v. 3.4  and Bonferroni corrected for
multiple comparisons. We constructed input files for GENEPOP using CONVERT v. 1.31 . We
calculated allele frequencies, number of alleles per locus, estimates of null allele frequency and
polymorphic information content using CERVUS v. 3.0.3 . We used MICRO-CHECKER
v. 2.2.3  to assess genotyping error as a result of null alleles, large allele drop-out and stutter
bands. In addition, because noninvasive tissues may lead to genotyping errors [55, 56], we took
specific precautions throughout the project to reduce genotyping error. We completed multiple
amplifications based on the multiple tubes approach [56, 57] for those samples containing low
quantity or excessively degraded DNA. We generated a consensus genotype from all
amplifications and used the consensus in subsequent analyses. We used GENOTYPER v. 2.1 to assign allele
sizes and all chromatograms were checked by eye to verify the allele sizes. We estimated the
genotyping error rate in the data set by a double-blind study. An individual without prior
knowledge of the sample genotypes randomly selected seven samples (approximately 54% of
the female-offspring subset) for blind analysis. The blind study samples followed the same
protocol used to generate the original genotypes in the data set, in that questionable
amplifications were re-amplified until a consensus was reached. Questionable samples were re-tested if
allele amplitude was close to the baseline or if differences in peak sizes were unclear.
To evaluate the power of our microsatellite loci to detect multiple paternity, we ran
simulations in GERUDsim2.0 . GERUDsim simulates sets of offspring genotypes based on specified
progeny sizes, draws a sample of offspring from population level observed allele frequencies
and estimates the number of sires present in each array. The program used a single multilocus
maternal genotype and 14 randomly generated paternal genotypes (based on the potential
number of male sires in a progeny array of 24 calves) to evaluate the probability of correctly
determining the number of fathers within an array. Our simulations consisted of individual
runs of 24 offspring per array generated from 14 fathers (100 iterations per run). Exclusion
probabilities were generated by GERUD2.0  for array comparisons based on all ten loci when
the mothers genotype was known.
Within the female-offspring data set used for this study, we tested for evidence of multiple
paternity using GERUD2.0 which uses a computer algorithm to reconstruct parental genotypes
based on multi-locus data from a progeny array when the genotype of one parent is known.
Because microsatellite inheritance is biparental, the maternal allele was first subtracted, leaving
the paternally inherited allele at each locus for each calf. In some cases, the mother and calf
carried identical alleles, meaning the paternal allele could be either allele size. The possible
paternal alleles at each locus were combined to generate all possible paternal genotypes for each calf.
The reconstructed genotypes were then compared to the progeny array to determine the
minimum number of males necessary to explain the alleles in the offspring array. Multiple paternity
was assigned as a result of more than two different paternal alleles found among the offspring
at a given locus.
To determine whether half or full sibling relationships were more likely between offspring
pairs, we analyzed calf arrays using ML-RELATE . We estimated population allele
frequencies using CERVUS v. 3.0.3  and all available animals genotyped for the larger, concurrent
study. We limited relationship matrix analysis to only the progeny arrays in the dataset. The
analysis reported the most likely relationship category (unrelated, parent-offspring, half
siblings or full siblings) and specific hypothesis tests were also run to determine whether a half or
full sibling relationship was significantly more likely (based on 10,000 simulations).
Based on male sire estimates and previous paternity assignments reported by Green et al.
, we constructed hypothetical pedigrees. Pedigrees for each female and offspring array were
constructed for the minimum and maximum number of males. If paternity was previously
assigned for any offspring in an array, the father was included. We estimated male age at time of
conception using previous analysis . If the exact year of birth was unknown, a minimum
age was calculated based on age class and the number of years sighted. For example, if an
individual first sighted in 1985 was in the fused age class, their age range in 2009 was estimated as
40+ years because the individual was at least 16 years old in 1985, plus an additional 24 years to
2009. We used field sighting data to consider whether successful male sires (as indicated by
paternity analysis) were available for mating during conception years of sibling offspring in
We calculated three microsatellite measures of genetic diversity to reflect the levels of
individual inbreeding as a result of closely related parents. We tested standardized multilocus
heterozygosity (SH), internal relatedness (IR) and homozygosity by loci (HL; [41, 60, 61]) for all
calves using the IR macroN3 developed by W. Amos (http://www.zoo.cam.ac.uk/directory/
william-amos). Aparicio et al.  suggested that HL performed the best of all three methods,
however, we present all three for comparison to other studies. Internal relatedness is similar to
Queller and Goodnights  measure of genetic relatedness (R) between two individuals but
looks within a single individuals genotype to compare two alleles at a single locus rather than
compare two pairs of alleles. The value is calculated over all loci; negative values indicate
outbred parents, zero values indicate unrelated parents and positive values indicate related
parents. SH reflects heterozygosity scores at each locus that are weighted by the overall
heterozygosity at the locus and HL goes even further by weighing the contribution of each
locus to the homozygosity index depending on allelic variability, giving more weight to more
informative loci. HL values vary from zero (all loci are heterozygous) to one (all loci are
homozygous) and intermediate values depend on the expected heterozygosity levels.
The field work conducted for this study was approved and completed under a research permit
granted by the Bahamian Department of Fisheries. The Institutional Animal Care and Use
Committee at Florida Atlantic University reviewed all protocols and approved the field work
for this study.
Fecal samples were collected from 85 (35 male, 50 female) individual Atlantic spotted dolphins.
Tests of multiple paternities among calves from a single mother required genetic samples from
females and at least three of her offspring. Three females with at least three offspring each were
successfully sampled for this study. Females FLYI and LGSH were sampled, each with three
offspring, and female PAIN was sampled along with four calves. Estimated age of first parturition
ranged from 1012 years and birth ages ranged from 1027 years (Table 1).
All samples were successfully extracted and subsequently amplified across all ten loci.
Among all samples included in the blind study, the replicates resulted in allele typing that
matched with 100% accuracy to the previously assigned genotypes, providing an estimated
genotyping error rate of 0%. There was no evidence of heterozygote deficiency, deviations from
Hardy-Weinberg equilibrium, no evidence of null alleles, allelic dropout or genotyping error as
a result of stutter bands. The number of alleles per locus for the ten loci ranged from 26
(mean = 4.3) and the mean polymorphic information content was 0.49. The overall exclusion
probability based on all ten loci combined was 0.98 when the mother was known.
GERUDsim reconstruction accuracy varied with the number of fathers and number of
offspring allotted to each generated paternal genotype. In 100% of simulations testing 24
offspring from a single father, the reconstructed number of fathers matched the real number.
Accuracy was reduced to 65% when testing four calves from two fathers and further reduced to
42% when testing three offspring from two fathers. In simulations testing three offspring from
three fathers and four offspring from three or four fathers, the program always underestimated
the number of fathers necessary to explain the progeny array. In no circumstances was the
number of real fathers overestimated by the heuristic search algorithm utilized in GERUDsim.
Using ten polymorphic loci in GERUD2.0 analysis, it was determined a minimum of two
males was necessary to explain the progeny arrays of all three females (Table 1). Analysis with
ML-RELATE categorized all offspring of FLYI as half siblings, indicating a different male sired
each calf. ML-RELATE identified one full sibling pair (LHAL LHAS, full sibling p = 0.0344) and
two half sibling pairs (LHAL LAGU, LHAS LAGU) among the three offspring of LGSH, indicating
two potential fathers. Among the four offspring of PAIN, ML-RELATE was unable to resolve a
series of plausible relationships and, as a result, was unable to determine if two or three males
best explained the offspring array. One calf, PICA, was more likely to be a half sibling to all
other offspring (mean p = 0.032). BRUS PIGM (p = 0.041) and PIGM PORT were more likely to
Year of birth (YoB), estimate of mothers age at time of birth of young (MB).
* mothers estimated age of first parturition (mothers ID in bold, followed by the ID of her calves)
Minimum number of males required to account for progeny array, internal relatedness (IR), standardized heterozygosity (SH) and homozygosity by loci
(HL), average of offspring array (ave).
be full siblings (p = 0.041). However, the relationship between PORT and BRUS could not be
resolved (half sibling p = 0.145, full sibling p = 0.400).
Because ML-RELATE indicated half-sibling relationships among all three offspring, we
constructed a three-male model pedigree for FLYI (Fig. 1A). Based on previous paternity
assignments , FLYI mated with SICK in 2001 to produce FREE. SICK was estimated to be 26 or more
years old in 2001. Siblings KP and FLAM were conceived in 1991 and 1998, respectively. SICK
was first observed in the study population in 1991 and re-sighted every year through 2002,
We constructed two-male and three-male model pedigrees for LGSH. LGSH mated with BIGG
in 2000 to produce LHAS. BIGG was estimated to be at least 30 years old in 2000. ML-RELATE
indicated a full sibling relationship between LHAL and LHAS (Fig. 1B), however, previous paternity
assignments did not support BIGG as the most likely father of LHAL. The three-male model
reflects a half sibling relationship between LHAL and LHAS, rather than a full sibling relationship
(Fig. 1C). Estimated conception years of siblings LHAS and LAGU were 1992 and 1996,
respectively. BIGG was first sighted in the study population in 1986 and re-sighted every year until
2012, except 2007.
We constructed two-, three- and four-male model pedigrees for PAIN. PAIN mated with BIGG
in 1998 to produce PICA, siblings BRUS, PIGM and PORT were conceived in 1989, 1993 and 2006,
respectively. BIGG was estimated to be at least 28 years old at the time PICA was conceived. As
mentioned previously, BIGG was first sighted in the study population in 1986 and re-sighted
every year until 2012, except 2007. ML-RELATE could not resolve the relationship between PIGM
and two siblings, BRUS and PORT. A two-male model reflects full sibling relationships between
Fig 1. Hypothetical pedigree construction of Atlantic spotted dolphin (Stenella frontalis) families
based on the integration of previous paternity analysis, observational data and results of GERUD2.0,
which estimates the number of males necessary to explain the allelic diversity of the progeny array,
and ML-RELATE, which determines the most likely relationship (i.e., half or full sibling) between
offspring pairs; (a) three-male model to explain the progeny array of female FLYI; (b & c) two- and
three-male models to explain the progeny array of LGSH; (d g) two-, three- and four-male models to
explain the progeny array of female PAIN.
BRUS, PIGM and PORT (Fig. 1D). A three-male pedigree represents both scenarios where either
BRUS and PIGM (Fig. 1E) or BRUS and PORT are true full siblings (Fig. 1F). If neither of the full
sibling relationships is true, a four-male pedigree best explains the array (Fig. 1G).
The average heterozygosity measure within each offspring array varied (Table 1). The
genotype of FLYI was similar to those of the males that sired two of her offspring (KP and FLAM,
HL = 0.771 and 0.755, respectively), whereas the sire of offspring FREE was less genetically
similar (HL = 0.384). The offspring of LGSH were more heterozygous (mean HL = 0.335) than the
offspring of FLYI (mean HL = 0.637), indicating that the male sire genotypes were less similar to
LGSH than the males that successfully mated with FLYI. Among the four offspring of PAIN, two
calves had relatively low heterozygosity (PIGM HL = 0.789, PORT HL = 0.883) while the other
two calves indicated more heterozygosity (BRUS HL = 0.506, PICA HL = 0.474).
We confirmed that multiple paternity was common in Atlantic spotted dolphins. Given
observational data and the fact that our result was consistent across all three females, this pattern
likely represents the majority of the population. Interestingly, ML-RELATE indicated the
possibility of full sibling relationships rather than half sibling relationships as might be expected in a
promiscuous system. It is important to resolve full versus half sibling relationships because
each scenario has important and unique implications for the mating system. Inclusive fitness
theory proposes that individuals will aid genetic relatives as a mechanism to increase their own
fitness . Inclusive fitness benefits may influence alliance formation because male alliances
are thought to function in mate access. Relatedness as a factor in male alliance formation is
variable among populations. Male alliances in bottlenose dolphins studied in Sarasota, Florida, do
not appear to be based on relatedness  whereas male alliance members in both Shark Bay,
Australia  and the Bahamas  were closely related. It is not known whether Atlantic
spotted dolphins form male alliances based on relatedness. Although full siblings Atlantic
spotted dolphins are unlikely to be alliance members because of age differences, preferential
coalition formation between alliances containing full siblings may provide inclusive fitness benefits
if coalitions function in increasing mating access to females.
Conservative male sire estimates provided by GERUD2.0 were supported by full sibling
relationships in ML-RELATE. However, GERUDsim2.0 often underestimated the true number of
males raising concern that ML-RELATE also underestimated the number of half sibling
relationships among offspring. Careful consideration is necessary prior to accepting a full sibling pair
over a half sibling pair. Available methods for estimating relatedness have a tendency to
mislabel relationships . Csillry et al.  found that dyads of previously known pedigree
relationships were often misclassified when based exclusively on genetic information. In fact,
Csillry et al.  found consistently high misclassification rates; relationships were
overestimated and dyads were classified as closer kin than they actually were. In this study population,
Green et al.  reported overestimates of related dyads among known maternal half siblings
based on relatedness analysis that assigned an average of nearly six additional maternal half
siblings per individual. It is possible that the full sibling relationships in the current offspring
arrays were also overestimated and full sibling relationships may not be true.
Overestimates of close relationships likely stems from low allelic diversity. The population
of Atlantic spotted dolphins consists of approximately 90100 individuals per year. The
animals are considered resident and stable with discovery rates indicative of a relatively closed
population . Given these factors, it is not surprising that allelic and genetic diversity among
individuals was relatively low. A study of Atlantic spotted dolphins encompassing a much
larger geographic scale averaged 11 alleles per locus (range 715; ) compared to 4.3 in the
current study population. Given low allelic diversity, similar genotypes may arise in non-related
individuals. Consider a scenario where two males with similar genotypes each sire a calf with a
single female. Because the males share alleles common in the population, those alleles may
assort into similar patterns in two offspring, resulting in R values that incorrectly indicate full
siblings rather than half siblings. Although it is not impossible for full siblings to arise in small
populations, these close relationships are likely overestimated in the current study.
Whether we accept full or half sibling relationships, more than one male was necessary to
explain to the progeny arrays. Given the fact that the number of suitable males may be low, the
reason(s) why females mate with different males are likely important. One reasonable
consideration to explain why females mate with different males is that the same male was not available
for subsequent mating. Female Atlantic spotted dolphins reach sexual maturity between 811
years of age . Gestation lasts approximately one year and females care for calves for 23
years, resulting in a typical calving interval of three years . Therefore, the same male must be
available for several years to mate with the same female when she is receptive. While this study
did not assign paternity to specific males, paternity was previously assigned for one calf in each
progeny array . In nearly all cases, the same male was sighted during the year each female
was in estrus. An exception occurred in 1998 between SICK and FLYI. SICK was not sighted in
1998 when FLYI conceived FLAM. However, it is important to note that a gap in sighting does
not definitively indicate that the individual was not active in the study population throughout a
given year. It is possible that SICK was active but simply not observed, making him a potential
mate for FLYI. However, it is also possible that SICK was not available as a mate option to FLYI.
Overall, sightings suggest the same males were available for mating throughout several estrus
cycles but the females did not repeatedly mate them.
If the same male was available for repeated mating after a female successfully raised a calf,
why wouldnt the female mate with the male again? It is important to note that females will not
benefit unreservedly from promiscuous behavior. For example, if paternity is confused, females
will not receive help from males in raising offspring [4, 33]. Second, if females actively seek out
multiple males with which to mate as has been reported for bottlenose dolphins in the Bahamas
, they may incur energetic costs of travel and costs associated with increased predation risk
. Females also increase their chance of contracting a venereal disease  such as genital
lesions, papillomas , or squamous cell carcinomas . What benefits outweigh costs
incurred from mating with multiple males [38, 72]? We offer discussion on the most plausible
hypotheses for the species.
Hrdy  first postulated that females mate with multiple males to confuse paternity and
avoid infanticide. The hypothesis fits well in species that give birth to altricial young and where
killing offspring results in a mating opportunity for the male . If females are using
multimale mating as a tactic to confuse paternity and therefore avoid infanticide, we expect males to
exhibit guarding behaviors to ensure access to the female when she is sexually receptive
following the death of her calf [3, 75]. Although cetaceans give birth to relatively precocial calves
, violent interactions resulting in cetacean infanticide, although rare, do exist. Patterson
et al.  suggested infanticide among bottlenose dolphins in Moray Firth based on a large
proportion of stranded juveniles exhibiting injuries consistent with bottlenose attack. Similarly,
Dunn et al.  reported stranded calves died from blunt-force trauma consistent with
bottlenose attack. The only reported observation of a violent encounter that appeared to lead to the
death of a calf occurred among tucuxi dolphins (Sotalia guianensis) . In our study
population, no violent actions towards calves have been observed over 26 field seasons. Overall,
several independent research groups observe a variety of dolphin behavior in unique populations,
and if infanticide was common, we expect more reports.
Furthermore, it is unclear whether male Atlantic spotted dolphins will gain a mating
opportunity following the loss of a calf. The average interbirth interval of female Atlantic spotted
dolphins with successful calves is 3.56 years, but females that lose a calf significantly decrease their
interbirth interval and become pregnant the same or following year [4, 80]. Previous research
identified a spring and fall calving peak in the study population , indicating that
synchronized estrus in the wild may limit female receptivity following the loss of a calf. However,
isolated observations of young neonates during off-peak times have occurred and may indicate
female receptivity, although it is unknown whether these calves were born to females that
previously lost offspring. In Shark Bay, Australia, female bottlenose dolphins that have lost calves
became receptive within a few days (based on attractiveness to males; ) but it is not known
whether the same is true for Atlantic spotted dolphins. When females become receptive, male
behaviors such as herding and tending are expected in order to control the mating opportunity.
Among Atlantic spotted dolphins, sporadic events of female herding have been reported [26,
In addition to the published reports that may indicate males attempt to control mating
opportunities, males have also been observed spending time with very late gestation females and
females with new calves. While it does not completely rule out the possibility that males are
attempting to control mating opportunities, the lack of aggression towards calves in these
interactions does not support infanticide. Rather this may indicate supportive behavior by the
males as young members of the social group are extremely vulnerable to predation. Conversely,
the males may hedge their bets and stay close to a female with a very young calf in the event
that a predator takes the calf and the female becomes receptive. Precocial young and a lack of
violent observations does not support paternity confusion as an explanation for multiple
mating in the case at hand but it does indicate the possibility of alternative male tending tactics.
The genetic benefits hypothesis proposes several reasons females might mate with multiple
males . It is known that multiple mating reduces the proportion of young dead at birth [81,
82] and higher levels of internal relatedness can lead to decreased survival [83, 84] and
decreased reproductive success in inbred offspring . Furthermore, animals with higher than
normal parental relatedness were more susceptible to illness and post-illness recovery time
increased . Among the offspring in our study, we found diverse levels of individual
heterozygosity ranging from outbred parents to related parents in the same progeny array. Calf
mortality in the study population is approximately 34%  and those calves do not survive
long enough for genetic sampling. Therefore, we do not have an estimate of individual
heterozygosity among failed calves. However, given the low population diversity, females may benefit
from even slight increases in genetic variation among her progeny array.
Females that mate with attractive males may pass those genetic characteristics on to male
offspring, ensuring sons will be attractive to females and ultimately, successful in reproduction.
If all females are attracted to the same characteristics, strong sexual selection on males results.
Although sexual size dimorphism occurs in several cetacean species , little sexual
dimorphism occurs among Atlantic spotted dolphins (e.g., white-tipped rostrum in older males ),
lending insignificant support the genetic benefits hypothesis. However, females may find
nonmorphological characteristics such as age or social status as attractive genetic quality indicators.
As stated previously, young female Atlantic spotted dolphins preferentially copulate with older
males and older males are more often seen attending female-calf pairs . Previous paternity
assignments indicated a minimum male age requirement for successful mating . The
average male conception age was 25+ years (range 1830+ years) and the youngest male assigned
paternity was 18 years old at the time of conception. SICK and FLYI mated to produce FREE in
2001 when SICK was 26+ years old. SICK was 16+ years when KP was conceived, perhaps too
young to successfully secure the mating opportunity. Similarly, BIGG was 19+ and 23+ when
BRUS and PIGM were conceived, potentially supporting the age bias hypothesis. However, our
model pedigrees also indicate conception opportunities when the male was older than the
average age of successful males (e.g., BIGG was 36+ when PORT was conceived), but was not
identified as the father of the calf. Therefore, age and long-term viability may be important to
receptive females but several additional factors may be significant as well.
In conclusion, our study confirms that females mated with different males over their
reproductive life even though the number of suitable males may have been small. The simple
explanation of varying male availability during subsequent estrus events cannot explain the
observed pattern of multiple mating. Paternity confusion also seems unlikely to explain
promiscuous mating among female Atlantic spotted dolphins but alternative tending tactics used
by males may occur among the study population. Our data suggests females potentially gain
genetic benefits from mating with different males. Because evidence suggests males must reach
a minimum age before successfully siring offspring, it is possible that females pass genes for
long-term viability to their young. Offspring arrays of females may also benefit from slight
increases in genetic diversity but further research is needed. The genetic quality of offspring is
not considered a universal factor affecting the development of multi-male mating across all
taxa, however, our study presents a species that may garner genetic benefits from
The authors would like to thank CR Hughes, DM Binninger and EO Keith for their helpful
comments on this project. Special thanks to the staff, crew and supporters of the Wild Dolphin
Project for their help and support. Finally, the authors would like to acknowledge the lab staff
and students who assisted with this project.
Conceived and designed the experiments: MLG DLH JDB. Performed the experiments: MLG
JDB. Analyzed the data: MLG JDB. Contributed reagents/materials/analysis tools: DLH JDB.
Wrote the paper: MLG DLH JDB. Collected samples for analysis: MLG DLH.
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