SC/F14/J07

Paternity analysis on Antarctic minke whales using
JARPA and JARPAII samples
Naohisa Kanda, Mutsuo Goto, and Luis A. Pastene.
The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Contact email: kanda@cetacean.jp
ABSTRACT
Applications of paternity analysis are very wide from documentation of pedigrees based on genetic relationships
of individuals, exploration of migration patterns based on position data, to estimation of stock sizes bymark-recapture approach. Paternity analysis in a wild stock is however challenging because stock size andgeographic distribution of the wild animals tend to be too large and wide to obtain an appropriate sample. This
study is the first attempt to conduct the paternity analysis in a large stock of Antarctic minke whales using
JARPA and JARPAII samples. Genotypic data of a maximum of 12 microsatellite DNA loci was prepared from
137 fetuses that were collected from pregnant females captured during 2003/04 JARPA, and their potential
fathers were sought among 1,779 males collected from 2001/02 JARPA to 2010/11 JARPAII. One matchingcase of a fetus-mother-father trio was found. Although its statistical significance was varied depending on thelevel of uncertainty considered, other information such as the area they collected (Area IIIE), and father’s bodylength (8.66m) and estimated age (12 years) did not necessarily deny the match.
Detection of at least one match of a fetus-mother-father trio in the partial, yet relatively large, samples of
JARPA and JARPAII increases the potential for paternity analysis in our future research as an additional
approach to estimate the stock size as well as infer the stock distribution. For large stock like Antarctic minke
whales, the effectiveness of this approach depends on size and randomness of samples, and our samples from thecomprehensive large-scale JARPA and JARPAII surveys should meet the requirements.
KEYWORDS: ANTARCTIC, FEEDING GROUNDS, GENETICS, ANTARCTIC MINKE WHALE

INTRODUCTION
Applications of paternity analysis are very wide, for instance, from documentation of pedigrees based on genetic
relationships of individuals, exploration of migration patterns based on position data, to estimation of stock sizes
by mark-recapture approach. In addition, once fetus-mother-father trios are identified, information of theirsampling locations allows us to determine stock boundaries, and the stock origins of the parents tell us the levelon genetic exchange within or between stocks.
Recently, the genetic mark-recapture approach has been used in whale studies to estimate stock size andexamine the migration pattern (Palsbøll, 1999; Pearse et al., 2001). Use of biopsy samples may be popular forthis purpose, but it is not practical especially for the large stocks because the amount of effort involved to obtainthe large sample size is excessive. Paternity testing in catches appears to be an alternative way to compensate forsuch difficulty. For the paternity analysis, DNA profiles, usually analyzed by a set of microsatellite loci, of themother-fetus pairs are used to look for potential fathers of the fetuses in the sample. If their fathers are found, thenumber of the matches can be applied to the traditional mark-recapture analysis such as the Petersen method for
estimation of stock size (e.g., Skaug and Øien, 2004).
Paternity analysis in a wild stock is however challenging because the stock size and the geographic
distribution of the wild animals tends to be large and wide. For such large stocks, a small sample from a narrow
scale sampling may not contain the potential fathers by chance. Antarctic minke whale samples obtained fromour comprehensive large-scale JARPA and JARPAII provide us a good opportunity to conduct the paternity
analysis in the large stock. This study is the first attempt to conduct the paternity analysis in the large stock of
Antarctic minke whales using JARPA and JARPAII samples.

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MATERIALS AND METHODS
Samples
Mother-fetus pairs of 2003/04 JARPA survey were used in this study, and potential fathers of the fetus weresought in the male samples of JARPA and JARPAII from 2002/03 to 2010/11 (Table 1). The males from the
2002/03 JARPA sample were used because considering the pregnancy period of females (Kato, 1990), some ofthem could have been fathers of fetuses collected in 2003/04. The mothers of the mother-fetus pairs werecollected from Areas IIIE and IV, and all other whales in the samples were collected from Areas IIIE to VI.
Microsatellite DNA data
The JARPA samples (2002/03 to 2004/05) were analyzed with six microsatellite loci: EV1, EV104 (Valsecchi
and Amos, 1996), GT023, GT195, GT211 (Bérubé et al., 2000) and DlrFCB14 (Buchanan et al., 1996), whereas
the JARPAII samples were analyzed with 12 loci: old set + AC045, AC082, AC087, AC137, CA234, andGT129 (Bérubé et al., 2005). Details of laboratory work can be found in Kanda et al. (2014). The power todetect the potential fathers depends on the number of loci used for the paternity analysis, so we recognized theimportance of analyzing the JARPA samples with the additional six loci. Because of a time constraint, however,it was difficult for us to genotype all of the 2002/03-2004/05 JARPA samples with the additional six loci. We,
therefore, conducted an initial screening with the first six microsatellite loci to select whales from the JARPA
samples that would require an increased data set. Only these screened individuals from the JARPA samples wereanalyzed with the additional six loci. It is important to note that because the condition of extracted DNA of someindividuals was not very good these individuals had the genotypic data obtained from less than six loci in the
JARPA samples and 12 in the JARPAII samples. Those individuals that had the data from at least three loci wereused. MICRO-CHECKER (van Oosterhout et al., 2004) was used to check for null alleles and reading/typing
errors.
Paternity analysis
The Paternity analysis was conducted using CERVUS (Marshall et al., 1998), a computer program forassignment of parents to their offspring using genetic markers. Because this was a preliminary analysis, we
looked for the mother-fetus-father trio that showed complete matches over the loci in Mendelian fashion. Thismeans we ignored the effects of null alleles, typing error, and point mutation that could show allelicdisagreement even between mother and fetus. This treatment could have underestimated the number of matches.
RESULTS AND DISCUSSION
For the initial screening with the data from the six microsatellite loci, the 137 mother-fetus pairs and the 1,779
males were analyzed using CERVUS (Table 1). The initial run selected 82 mother-fetus pairs and 189 males.
Among them, some pairs matched to one male and other pairs to more than two males most likely due to the low
resolution power of using the six loci (maximum). After the initial screening, these 82 mother-fetus pairs and
189 males were then genotyped with new six loci (maximum), and after the paternity analysis, one match of amother-fetus-father trio was found (Table 2). Although some of the individuals in the samples had theincomplete dataset, these three individuals had the genotypic data from all of the 12 loci (Fig. 1). According to
Kato (1987), body length and age at sexually maturity for males is 7.9 m and around 7 years old. The biologicalinformation of the matched potential male did not exclude the possibility of this male mating with 0304427
female some time in 2003.
It is important to note, however, that the level of statistical significance of this match varies depending on thelevel of uncertainty considered. For instance, proportions of the loci typed and mistyped per individual are
critical especially when the proportion of missing data is high. Considering the effects caused by these factors,
the complete matches from only a few loci (others had no data) as well as matches containing one mismatchedlocus were excluded from the final outcome. This may have caused an underestimate for the number of matches.
The case with one mismatched locus occurred even between the mother and fetus in our study probably due to anull allele. In addition to that, because it is impossible to collect all of the males in the stock, the significance isinfluenced by the average number of potential fathers per offspring present at the time of breeding and the
proportion of these potential fathers that are sampled and analyzed. These factors should be estimated from fieldobservations that were not available for Antarctic minke whales at the time. Nevertheless, the detection of atleast one match of a fetus-mother-father trio in the partial, yet relatively large, samples of JARPA and JARPAII
increased the potential for paternity analysis in our future research as an additional approach to estimate stock
size as well as infer stock distribution. For the large stock like Antarctic minke whales, the effectiveness of thisapproach depends on size and randomness of samples, and our samples from the comprehensive large-scale
JARPA and JARPAII surveys should meet the requirements.

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This single match was used to tentatively estimate the mature male number (Nm) in the I stock using thePetersen mark-recapture method modified by Chapman (referred from Van Den Avyle, 1993):( M + 1)(C + 1)
( M + 1)(C + 1)( M − R)(C − R )Nˆ m =− 1 with variance V ( Nˆ m) =,( R + 1)
( R + 1) 2 ( R + 2)

where M is the number of the fetus-mother pairs, C is the total number of the mature males, and R is the numberof the males in C that matched to the fetus in M. Among all of the male samples, we used only mature males
collected from IIIE and IV because the mother-fetus pairs were collected from those areas. This method we used
estimates mature male abundance in the stock. We plugged M=137, C=677, and R=1 into the formula andobtained Nm=46,782 (CV=0.572).
This method with the paternity analysis yielded the estimate of the mature male number, so that total stock
size has to be converted from 46,782. Assume mature/immature ratio and sex ratio is 1:1, respectively, then total
stock size is estimated as 187,128. The average abundance estimate of Areas IIIE and IV over all of the JARPA
and JARPAII surveys is 51,474 (Hakamada and Matsuoka, 2014) and the average abundance estimate of Areas
III and IV from two IDCR surveys was 151,174 (IWC, 2013). Our estimated stock size is larger than what we
expected from these two reported estimates. This could be because this paper estimated total stock size, while theother two estimated the number of individuals distributed in the research area based on the sighting survey. The
large difference between the two could be real because it has been reported that many whales are distributed
outside the research area such as polynea (e.g., Murase et al., 2014). Another reason for the difference could bethat this paper overestimated the total stock size. Several important assumptions must be met for a valid
estimation using the Petersen method. Any reasons for underrepresentation of marked individuals in the second
sample will lead to overestimation. Among these factors, because our samples were collected from multiple-year
samplings in the open ocean, mortality of the marked individuals, recruitment of new individuals, and migrants
from different stocks might not be negligible. At this point, we were not able to determine which factor(s) was
responsible for the difference.
It is important to note that we found only one match. As indicated by the large CV, the precision of theestimation was low. Because we used only the mother-fetus pairs from the 2003/04 season among those fromseveral different surveys, the estimate was tentative. We should wait till all of the samples will be analyzed for abetter estimate with higher precision.
ACKNOWLEDGEMENTS
We would like to thank all the researchers and crew members of JARPA/JARPA II surveys involved in thesampling of the Antarctic minke whale samples used in this study. We also would like to thank H. Hatanaka for
his useful comment on an earlier version of this manuscript.
REFERENCES
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Bérubé, M., Rew, M.B., Skaug, H., Jørgensen, H., Robbins, J., Best, P., Sears, R., and Palsbøll, P.J. 2005.
Polymorphic microsatellite loci isolated from humpback whale, Megaptera novaeanglliae and fin whale,balaenoptera physalus. Cons. Genet. 6: 631-636.
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Table 1. The number of fetus-mothers and males used in the study.
SampleSeason

1st set (I stock)

2nd setFetus/Mother

03/04

137

82

02/03-10/11

1779 (677)189

Candidate Father

Table 2. Sampling and biological information of matched individuals.
ID0304 427F

Sampling

Lat.

Long.

LengthSex

Age0304 427

3/1/2004

67.39S77.00E

8.71

F

x0708AM120 12/29/2007

64.40S

60.03E

8.66

M

12

ID
0304 427F
0304 427
0708AM120

Microsatellites
EV104
GT211
GT195
GT23
AC045
AC082
AC087
AC137
EV1
DlrFB14CA234
GT129
129 133 141 147 100 112 269 271 153 157 107 107 183 187 127 143 171 173 125 135 188 206 110 112
129 129 141 157 100 112 269 269 153 153 105 107 185 187 133 143 173 181 123 125 188 196 104 112
133 137 139 147 100 110 269 271 157 159 107 109 183 183 127 137 169 171 105 135 194 206 110 114

Fig. 1. Genotypic data at 12 microsatellite loci of the mother-fetus-father trio found in this study.

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