SC/67a/SDxx

Finding parent-offspring pairs among western North Pacificcommon minke whales
Ralph Tiedemann1, Magnús R. Tiedemann2, Mutsuo Goto3, Mioko Taguchi3 and Luis A. Pastene3
1

Unit of Evolutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of
Potsdam, D-14476 Potsdam, Germany

2

Faculty of Computer Science, University of Magdeburg, D-39016 Magdeburg, Germany

3Institute of Cetacean Research, Toyomi-cho 4-5, Chuo-ku, Tokyo 104-0055, Japan

ABSTRACT
A dataset of complete genotypes at 16 microsatellite loci, accompanied with mtDNA and biologicalinformation, in 4,554 North Pacific common minke whales was used to infer Parent-Offspring (P-O)
relationships, using a Maximum-Likelihood approach. The relationship between False Discovery Rate
(FDR) and Power (P) was evaluated by simulation. Of 145 inferred P-O pairs at an estimated FDR of 0.1,
141 were further evaluated by typing 10 additional microsatellite loci. 75 were confirmed (among them
26 Mother-Fetus pairs), 66 pairs were ranked “False Positives”, yielding an overall observed FDR of
0.468. FDRO was substantially reduced when J and O stock were analysed separately. While observed andestimated values for Power were in the same range of magnitude, observed FDR was always substantiallyhigher than estimated FDR. This was attributed to the fact that FDRE was estimated via simulation,
implicitly assuming a single panmictic population, an assumption clearly not met in the present data set.
This interpretation is corroborated by the reduced FDRO when stocks were analysed separately. The
dataset with 26 microsatellites clearly outperformed the 16 microsatellite data sets. At FDRE=0.001,
Power was at or close to 100% (PE=0.989 and PO=1.000) and the observed False Discovery Rate was
FDRO=0.128. Among the validated P-O pairs, O stock pairs were significantly overrepresented, whilepairs between J and O stock individuals were absent. Specimens neither assigned to J nor O stock(“unassigned”) exhibited a stronger affinity to the O stock. The J stock seems to appear on both sides ofJapan closer to the coast, while the O stock occurs mostly east of Japan, both close to the coast and far
offshore. This analysis provides no evidence for further stock structure in the area covered by this data set.
This study demonstrates that a modest increase in the number of loci investigated (here, from 16 to 26
microsatellite loci) may already substantially improve kinship inference under Maximum Likelihood. Itfurther addresses recommendations made at both the JARPNII final review and the 2016 IWC ScientificCommittee meeting regarding kinship analysis in North Pacific common minke whales.

INTRODUCTION
Results of the updated analyses on stock structure of western North Pacific common minke whales were
presented to the JARPNII final review workshop (IWC, 2017a), and a summary of the new information
was made by Pastene (2017). Most of the new analyses were conducted in response to recommendations
made during the 2009 JARPNII mid-term review (IWC, 2010).
One of the recommendations made by the mid-term review was the examination of ‘the spatial
distribution of related individual, which can provide information directly relevant to stock structure
consideration’ (IWC, 2010). Subsequently the analysis on close kin in western North Pacific common
minke whale started in 2015 applying the likelihood-based approach described in Tiedemann et al. (2014),
and preliminary results were presented during the open session of the JARPNII final review workshop.
In reviewing the preliminary results in 2016, the International Whaling Commission Scientific Committee(IWC SC) recommended that a paper to examine the spatial distribution of close kin in North Pacificminke whales be submitted for review at the 2017 annual meeting. The IWC SC made the following
specific suggestions:a)Emphasises the importance of evaluating the potential for false positive and false negative
detections of parent-offspring pairs (see Tiedemann et al., 2014);b) Encourages the authors to explore different approaches (e.g. software) to conduct kinship-based
analyses; andc) Recommends that the samples be genotyped at additional loci (microsatellites or SNPs tovalidate the putative Parent-Offspring pairs that were identified (IWC, 2017b).
The objective of the present study was to conduct a comprehensive analyses on kinship (Parent-Offspring
pairs) based on i) the total available genetic samples till 2015 from JARPN, JARPNII and Japanese bycatches; ii) microsatellite profiles; iii) the analytical approach described in Tiedemann et al., 2014; and iv)
the recommendations from the IWC SC in 2016.
Furthermore, this paper evaluates the effect of implicit assumptions inherent to LOD-score-based kinshipanalysis. Specifically, population simulations to estimate False Discovery Rate (FDRE) and Detection
Power (PE) based on observed genotype frequencies may implicitly assume random mating and lack of
population/stock structure, while real data sets frequently do not meet this assumption. Therefore, we also
evaluate the impact of data partitioning on kinship inference.

MATERIALS AND METHOD
Genetic and Biological data
Offshore samples of common minke whales from the western North Pacific were from JARPN/JARPNII
surveys from 1994 to 2013 at SA7, SA8, SA9, and SA11. Common minke whale samples obtained fromthe coastal JARPNII survey between 2002 and 2015 were also used in this study, Kushiro in SA7CN andSanriku in SA7CS. Samples from common minke whales that were bycaught on set net fishery along theJapanese coast from 2001 to 2015 were also used. The by-catches used were from subareas SA2, SA6,
SA7, SA10, and SA11 year-round.
This analysis was based on a dataset of a total of 4,554 North Pacific minke whales including fetuses forwhich information on 16 microsatellite loci (Pastene et al., 2016), mtDNA haplotype (M. Goto, unpubl.
data ), and biological information (e.g. sampling date and position, sex, sexual maturity and body length)
was available at ICR Japan. The entire data set was analysed for occurrence of Parent-Offspring (P-O)
pairs according to Tiedemann et al. (2014) (see below). For all P-O pairs inferred at an estimated False
Discovery Rate (FDRE) of 0.1, the respective specimens (n=275) were typed at an additional set of 10
microsatellites(GATA97408,
GATA91083,
GATA43950,
GATA38314,
GATA6059012,
GATA5946992, GATA6063862, GATA6064765, GATA5943219 and GATA5890064: P. J. Palsbøll,
pers. comm.). For four individuals, this typing was not complete, such that this analysis yielded fullgenotypes at in total 26 loci for 271 individuals.
Inference of Parent-Offspring Pairs
From the available genotype data for the 16 standard microsatellite loci, we formed five different data sets,i.e., the full data set, the “J stock only” dataset, the “O stock only” dataset, the “J stock+unassigned”
dataset, and the “O stock+unassigned” dataset, based on stock assignment in Pastene et al. (2016).
The five data sets were analyzed for occurrence of Parent-Offspring (P-O) pairs according to Tiedemannet al. (2014) with the following parameters:• Typing error rate per allele 0.0025 (Goto et al. 2016)• Number of random individuals simulated 5000 (corresponding to 12,497,500 random pairs for
estimation of the False Discovery Rate (FDRE)• Number of related pairs simulated 100,000 for estimation of Power PE
Only specimens with complete genotypes were included in the analyses and fetuses were excluded fromthe population-wise allele-frequency calculation (see Tiedemann et al. 2014 for details).

We ran the analysis with the following FDRE-values: 0.3; 0.2; 0.1; 0.05; 0.01; 0.001 to evaluate theimpact of FDRE on Power. P-O pairs inferred at an FDRE of 0.1 were retained for further validation.
Validation of Parent-Offspring Pairs
Validation of Parent-Offspring pairs was performed in two steps as follows:First, the results at the 10 additional microsatellite loci were considered:• For the dataset of all specimens involved in an inferred P-O pair based on 16 loci (FDR 0.1), theanalysis was repeated with 26 microsatellites, using again FDR-values of 0.3; 0.2; 0.1; 0.05;
0.01; and 0.001. Note that this data set allowed only for 36,585 pairwise comparisons. Further, it
is not a random data set, as the likelihood to be part of a P-O relationship is elevated: as any
specimen was part of a inferred P-O relationship, it could be as high as 0.000027 (if all inferred
P-O pairs were true positives). If correcting this rate for the observed False Discovery Rate
(FDRO=0.468 for the full data set at FDRE 0.1; see table 4), the rate of P-O pairs in this dataset isstill 0.000015, about 2 times higher than in the full data set.
• All inferred P-O pairs were checked for full compatibility with a P-O relationship at the 10
additional microsatellite loci, i.e., sharing at least one allele at any locus. Note that this
implicitely assumes no genotyping error and no mutation. It may hence produce False Negatives,
i.e., rejection of valid P-O pairs.
• Mother-Fetus pairs were considered as biologically validated true positives. Out of 26 MotherFetus pairs among the inferred P-O pairs, 24 were fully compatible with a P-O relationship at the
10 additional microsatellite loci, but two (8%) were not (cf. “no” for full fit in table 2), due to asingle mismatch each. The lowest LOD score for a Mother-Fetus pair in the 26 locimicrosatellite analysis was 4.11 – this pair was one of the two with a “no” for full fit (Table 2).
• All other inferred P-O pairs with a “no” and a LOD score below 4.11 were considered False
Positives (FP).
• 85 P-O pairs yielded a LOD-Score of at least 4.11, i.e., all 26 Mother-Fetus pairs and 59 otherinferred P-O pairs. Among these 59 pairs, 49 had a “yes” for full fit at the additional 10
microsatellite loci and were retained. 10 (17%) had a “no” here, which is about twice thepercentage for such cases when compared to the Mother-Fetus pairs (8% with a “no” for full fit).
We ranked those 10 pairs by LOD score and assigned the five pairs with the lowest LOD scoresas “false positives”. The other 5 pairs (all with a LOD score over 5.1) comprised one Father-Son
pair (retained) and 4 Mother-Offspring pairs. These 4 pairs were evaluated for their mtDNA
haplotype: 3 pairs exhibited a shared type (retained), while one pair had different mtDNA types(ranked False Positive; see Table 2 for details).
All inferred P-O pairs were independently checked for compatibility of the inference (true vs. false
positive) against the biological data, i.e., mtDNA in Mother-Offspring pairs and compatibility of year ofcatch and sexual maturity, i.e., at least one sexually mature adult in the pair.
••Out of the 63 pairs ranked False Positives by the microsatellite genetic inference (26 loci), 34
had been independently ranked “False Positives” by biological information incompatible with a
P-O relationship, i.e., both specimens immature or inferred Mother-offspring pair with different
mtDNA haplotype.
Out of the 78 pairs retained after microsatellite genetic inference, 3 were Mother-Offspring pairs
with incompatible mtDNA haplotype. They were additionally considered “False Positives”,
rendering a set of 75 pairs considered “True positives” (i.e., real P-O pairs). Only three of them(all bycatch) had some ambiguity with regard to whether their length measurement is fullycompatible with adulthood. As length measurements during bycatch-operations may be lessprecise and as two of these three pairs were Mother-Offspring pairs supported by shared mtDNAtype, these three pairs were retained as “True Positives” (see Table 2 for details).

Calculation of observed False Discovery Rate and Power
Observed False Discovery Rate (FDRO) was calculated for estimated FDRE rates of 0.1, 0.05, 0.01, and
0.001 for all datasets as the rate of inferred P-O pairs ranked “False Positives” after validation (see above).

Observed Power (PO) was calculated for estimated FDRE rates of 0.1, 0.05, 0.01, and 0.001 for all datasets
as the percentage of Mother-Fetus pairs in the data set which were detected by the analysis.

RESULTS
In total, we inferred – based on an FDR of 0.1 and 16 microsatellite loci - 145 Parent-Offspring pairs,
thereof 27 mother-fetus pairs. Genotyping additional 10 microsatellite loci of the involved 275
individuals yielded full genotypes for 271 individuals. For 4 pairs (thereof 1 mother-fetus pair), genotypes
were incomplete, such that 141 P-O pairs (thereof 26 mother-fetus pairs) were retained for validation(Tables 1, 2).
Of the 26 Mother-Fetus pairs, 24 were genetically fully compatible with a P-O relationship (i.e., one
shared allele at each of the additional 10 microsatellite loci), while in 2 pairs, there was a single mismatchat one of the loci. If we assume no mutation, this translates into a typing error rate of 0.0038 per allele,close to the error rate of 0.0025 previously estimated for the standard 16 microsatellite loci in the entire
NP minke whale data set (Goto et al., 2016).
Out of the 141 P-O pairs with full genotype at 26 microsatellite loci, 75 P-O pairs were validated as “True
Positives”: 26 were Mother-Fetus pairs (i.e., considered biologically validated True Positives), theremaining 49 were validated by the additional 10 microsatellite loci (Table 2; see methods for details). In
all cases, biological information was compatible with a Parent-Offspring relationship (i.e., at least one
specimen was an adult; time between catches was compatible with P-O relationship). 10 of thesevalidated P-O pairs were Mother-Offspring pairs, all confirmed by identical mtDNA haplotype (Table 2).
66 P-O pairs inferred based on 16 microsatellites at an FDRE of 0.1 were ranked “False Positives” after
validation. For 31 of them, this judgement was confirmed by biological information incompatible with a
P-O relationship, i.e., both specimens immature or inferred Mother-Offspring pair with different mtDNA
haplotype.
The observed False Discovery Rate (FDRO) of 0.468 was strongly biased towards J-stock individuals, i.e.,
only 9 out of 46 inferred P-O pairs originating from J stock were validated (FDRO=0.804), while for the
O-stock, 57 out of 83 were validated (FDRO=0.313). If stocks were analysed separately, FDRO dropped
dramatically in the J stock (i.e., to 0.000) and also substantially in the O stock (to 0.258; Table 3).
If FDRE was decreased in the initial analysis on the full data set (16 microsatellites) to 0.01, observed
FDRO was also considerably lower, i.e., 0.179 for the full data set, 0.000 for the J-stock, and 0.103 for the
O-stock (Table 3).
Observed Power (PO) was in the same order of magnitude as estimated power (PE). For the full data set,
PO was slightly higher than PE, in the data subsets involving the O-stock, this relation was reversed (Table3). In the data partitions “J stock only” and “J+unassigned”, there were only very few Mother-Fetus pairs(2 and 4, respectively; see Table 1). As none of these Mother-Fetus pairs was detected in the Maximum
Likelihood analysis, this nominally translates into PO of 0.000, but this estimate is likely imprecise andshould be treated with caution.
With an overall PO=0.5 for the full data set at FDRE=0.1 (the value used for the inference presented inTable 1), we can expect to have identified about half of the true P-O pairs present in the data set.
For the dataset with 26 microsatellites, both estimated and observed Power were considerably higher: At
FDRE=0.001, PE=0.989 and PO=1.000. Under these conditions, observed False Discovery Rate was
FDRO=0.128.
If we evaluate how validated P-O pairs were distributed across stocks, there were 3 significant deviations
from random expectations (Table 4):•

There were significantly more observed P-O pairs in the O stock than expected.

••There were significantly fewer (i.e., no) observed P-O pairs among the J stock and the O stock
than expected under a random association.
There were significantly fewer (i.e., no) observed P-O pairs among the J stock and unassigned
specimens than expected under a random association.

Geographically, many validated P-O pairs were identified across subareas, with J stock pairs centeredaround the coast at both sides of Japan, while the O stock pairs appeared mostly east of Japan and several
validated O stock P-O pairs connected subareas close to Japan with areas far offshore (Figure 1).

DISCUSSION
Using kinship analysis to inform stock structure discussions has been repeatedly advocated when large
genetic data sets are at hand (e.g., Skaug et al., 2010; IWC, 2010; IWC, 2017b). Inference is often basedonassigning the likelihood of a given pairwise relationship (most frequently, Parent-Offspring)
relationship to pairs of individuals, based on genotypes and population-wide allele frequencies (Skaug etal., 2010; Benónísdóttir, 2012; Tiedemann et al., 2014).
Given a particular data set, there is a trade-off between the False Discovery Rate (FDR) and the Power (P)to detect dyads of related individuals (Skaug et al., 2010; Benónísdóttir, 2012; Tiedemann et al., 2014).
Both FDR and P are typically estimated by simulation based on population-wide allele frequencies (see,e.g., Tiedemann et al. 2014). However, such simulations implicitly assume random mating and lack of
population/stock structure, while real data sets frequently do not meet this assumption.
In most cases, estimated values of FDR and P (hereafter, FDRE and PE) are not explicitly validated. Here,
we use the rather unique data set of North Pacific common minke whales (4,554 specimens with fullgenotype information at 16 loci, mitochondrial DNA, and associated biological information) for avalidation of these values. Specifically, Parent-Offspring (P-O pairs) inferred at a given estimated False
Discovery Rate (FDRE) were genotyped at 10 additional microsatellite loci for validation, both bylikelihood inference and locus-wise inspection for fit with a P-0 relationship. Furthermore, biologicalinformation (length, sexual maturity, mtDNA type) was utilized to confirm or contradict inferred P-Opairs in order to arrive at validated “True Positives” of P-O relationships, as well as an observed value for
the False Discovery Rate (FDRO).
We utilized 53 Mother/Fetus pairs in the data set as biologically validated P-O pairs. Observed Power(PO) could be calculated as the proportion of these Mother/Fetus pairs detected under the various False
Discovery Rates and data partitions applied.
Observed vs. Estimated values of False Discovery Rate and Power
Our analysis clearly shows that estimations of False Discovery Rates based on random individuals
substantially underestimates the rate by which False Positives are actually detected (i.e., FDRE<<FDRO).
This is perhaps not surprising when considering that real populations do not comprise random
assemblages of alleles, but are constituted of individuals many of which related to one another to various
degrees. It remains to be inferred whether “False Positives” for P-O relationship may constitute, at least inpart, relatives of a less close relationship (e.g., half sibs or cousins). This pattern of “False Discovery” isparticularly apparent, if specimens of different populations/stocks are lumped into a single data set andsubsequently treated as if they would constitute a single panmictic population (here, J and O stock).
Indeed in our analysis, FDRO was substantially reduced, if stocks were analyzed separately.
This effect can be asymmetric, as – in our analysis – there was a much higher FDRO for the J than for the
O stock, when analyzing them jointly. The O stock is genetically considerably more diverse than the J
stock (e.g., average microsatellite allele number per locus: 11.94 vs. 8.44) and less inbred (inbreeding
coefficient FIS 0.002 vs. 0.008) (Goto et al., in prep). Apparently, the (relatively larger) genetic
homogeneity in the J stock leads to an increased rate of erroneously inferred P-O pairs, when jointly
analysed with the more diverse O stock. Interestingly, FDRO drops from 0.804 (combined data set) to
0.000, when the J stock is analysed separately.

Observed Power (PO) was in most data partitions lower than estimated Power (PE) (except for the full dataset), but the differences were not as large as for the FDR: Generally, PO and PE values were in the sameorder of magnitude. It remains to be further evaluated whether nominal differences between PO and PE
just constitute stochastic variation or whether there is any systematic bias in these estimates.
The dataset with 26 microsatellites clearly outperformed the 16 microsatellite data set. Even at
FDRE=0.001, Power was very high (PE=0.989 and PO=1.000) and observed False Discovery Rate wasrelatively low (FDRO=0.128). Because this data set is considerably smaller (271 vs. 4,554 specimens) andhas a doubled internal likelihood for occurrence of P-O pairs, these measures may contain some bias andare likely not as precise as those for the full data set. Nonetheless, these data provide strong experimental
support for the benefit of additional loci in kinship analyses.
Stock affinity and geographical position of validated P-O pairs
Not surprisingly, the data strongly support the distinctiveness of the J and the O stock in North Pacificcommon minke whales. It should be noted that – for the 16 microsatellite data set – this is notindependent evidence, as the same 16 microsatellites had been utilized to infer the stock assignment(Pastene et al., 2016). However, the addition of 10 further microsatellite loci did not change this pattern.
In essence, the J stock appears to be genetically distinct with lower variability and a geographic restriction
to the waters around the coasts of Japan. The O stock is genetically more diverse and geographicallywidely distributed from the East coast of Japan to far offshore. The cohort of “unassigned” specimens
shows a significant affinity to the O stock: P-O pairs across O stock appeared as often as expected under
the assumption of a single population, while there were no validated P-O pairs across J stock and“unassigned” specimens.
Conclusive remarks
The large North Pacific common minke whale genetic data set, the available biological information, theabundance of Mother-Fetus pairs in the data set, and the additional typing of 10 microsatellites for
inferred Parent-Offspring pairs offered the rare opportunity of a validation of kinship inferences and acalculation of observed values for False Discovery Rate and Power, as has been recommended in the
JARPNII final review (IWC 2017b) and the IWC Scientific Committee. This analysis shows that:•••

With the standard set of 16 microsatellites, high observed power is associated with quite highobserved False Discovery Rates.
Joint analysis of different stocks considerably increases the False Discovery Rate, in particular if
one of the stocks is genetically less variable/more inbred.
With the addition of 10 additional microsatellite loci, an observed Power of 1.000 could be
combined with an observed False Discovery Rate of around 0.1.

If applied for other studies or other microsatellite loci, these measures may evidently vary depending on
stock structure and locus characteristics. Nonetheless, we argue that our study provides experimental
evidence that a modest increase in the number of loci investigated (here, from 16 to 26 loci) may alreadysubstantially improve kinship inference under Maximum Likelihood.
With regard to stock structure in North Pacific common minke whales, the distinctiveness of J and O
stock is clearly seen in the analysis. Hitherto “unassigned” specimens show more affinity to the O stock.
The J stock seems to appear on both sides of Japan closer to the coast, while the O stock occurs mostly
east of Japan, both close to the coast and far offshore. This analysis provides no evidence for further stock
structure in the area covered by this data set.

ACKNOWLEDGEMENTS
We thank researchers that collected the genetic samples during the JARPN/JARPNII surveys. We are also
thankful to Helga Dröfn Högnadóttir for assistance with the compilation of tables.

REFERENCES

Benónísdóttir S (2012) Statistical approach to relatedness analysis in large collections of genetic profiles– an application to a DNA registry of Fin whales. M.Sc. thesis, University of Bergen.
Goto, M., Yoshida, H. and Pastene, L.A. 2016. A note with an estimate of microsatellite genotyping error
rate for common minke whales. Paper SC/66b/DNA/01 presented to the IWC Scientific Committee, June
2016 (unpublished). 3pp.
International Whaling Commission. 2010. Report of the expert workshop to review the ongoing JARPNII
programme. J. Cetacean Res. Manage. 11 (Suppl. 2): 405-449.
International Whaling Commission. 2017a. Report of the Expert Panel of the final review on the western
North Pacific Japanese Special Permit programme (JARPNII). J. Cetacean Res. Manage. 18 (Suppl.): inpress.
International Whaling Commission. 2017b. Report of the Scientific Committee. J. Cetacean Res. Manage.
18 (Suppl.): in press.
Pastene, L.A., Goto, M., Taguchi, M. and Kitakado, T. 2016. Temporal and spatial distribution of the ‘J’and ‘O’ stocks of common minke whale in waters around Japan based on microsatellite DNA. Paper
SC/F16/JR38 presented to the IWC Scientific Committee, February 2016 (unpublished). 14pp.
Pastene, L.A. 2017. Annex I, Appendix 3. A summary of the new information on stock structure in thewestern North Pacific common minke whale presented to the JARPNII final review. J. Cetacean Res.
Manage. 18 (Suppl.): in press.
Skaug HJ, Bérubé M, Palsbøll P (2010) Detecting dyads of related individuals in large collections ofDNA-profiles by controlling the false discovery rate. Molecular Ecology Resources 10, 693-700.
Tiedemann, R., Tiedemann, M.R., Gunnlaugsson, D., Pampoulie, C. and Vikingsson, G.A. 2014. Finding
relatives among North Atlantic common minke whales (Balaenoptera acutorostrata) based on
microsatellite data: the relationship between false discovery rate (FDR) and detection power. Paper
SC/65b/RMP05 presented to the IWC Scientific Committee, May 2014 (unpublished). 8pp.

Table 1: Sample details for used data sets. P-O inferences are from the full data set (in parentheses inferences from separate stock-specific data sets). Stockassignment according to Pastene et al. (2016)n- thereof fetuses
n with full genotypes for 16 loci- thereof fetuses% fetuses in dataset
Tested pairs
Inferred P-O pairs at FDR 0.1- thereof full genotypes at 26 locinumber of confirmed P-O pairs% confirmed P-O pairs of all pairs
Detected Mother-Fetus pairs at FDR 0.1- thereof full genotypes at 26 loci

Full dataset
4582
54
4554
53
0.012
10367181145
141
75
0.00000723
27
26

Only J stock
1779
2
1767
2
0.001
156026147 (5)
46 (4)9 (4)0.00000577
0 (0)
0 (0)

Only O stock
2376
50
2366
49
0.021
2797795
86 (67)83 (62)57 (46)0.00002037
23 (17)22 (15)

J stock + unassigned
2206
4
2188
4
0.002
2392578
48 (7)
47 (5)9 (4)0.00000376
0 (0)
0 (0)

O stock + unassigned
2803
52
2787
51
0.018
3882291
97 (81)94 (72)66 (55)0.00001700
27 (21)26 (19)

Table 2: Inference of Parent-Offspring (P-O) relationships. P-O Inferences of the full data set (16 microsatellite loci; n=4,554; FDR≤0.1) were evaluated by thefollowing means: All specimens of the inferred pairs were typed at 10 additional microsatellite loci; Inference of P-O pairs was repeated for this subset (26microsatellites; n=271). Further, at the 10 additional loci, a fit with a P-O relationship (i.e., at least one matched allele at any locus) was evaluated. Full fit meansthat all 10 loci are compatible with a P-O relationship. Biological data was used to support (##matching mtDNA in Mother-Offspring pairs) or contradict(#mismatch in mtDNA in Mother-Offspring pairs or both individuals of immature status or length) P-O relationships (see methods for details). For confirmed P-Opairs, the type of relationship as well as the area of origin are provided.
FP=False Positive; FDRE=False Discovery Rate; PE=Power (estimated by simulations); (*) inferred at FDR≤0.1; * inferred at FDR≤0.05; ** inferred at FDR≤0.01;*** inferred at FDR≤0.001; ### multiple offspring inferred for same parent. Stock assignment: O=O stock; J=J stock; U=unassigned; cf. Pastene et al., (2016)
Stock
FDRE
FDRE
FDRE
FDRE
FDRE
FDRE
Individual_1 (I1)
NP94MI017
NP94MI019
NP95MI025
NP95MI032
NP96MI051
NP97MI002
NP97MI028
NP97MI036
NP97MI041
NP97MI059
NP98MI029
NP98MI043
NP99MI013

0.3
0.2
0.1
0.05
0.01
0.001
Individual_2 (I2)
NP05MI019
NP01MI079
NP96MI067
NP99MI004
CS08MI017
NP01MI052
CK05MI042
NP08MI011
NP98MI050
NP98MI040
CK14MI011
NP00MI016
NP02MI004

I1I2
OO
OO
OU
OO
OO
OO
OO
OO
OO
OO
OO
OO
OO

Full data set 16 Loci
LOD Threshold
PE

Subset 26 Loci
LOD Threshold

3.673063241
4.331695124
4.921379986
5.112659013
5.690162171
5.690162171
LOD Value
4.96150295
4.990618879
5.388497561
5.224221146
5.55281185
6.2682621
5.238267556
6.361714917
5.420573721
5.153040633
7.103259317
7.227285909
5.047657352-5.049196083-4.046957537-2.37039593-0.985059046
1.347098689
3.708856137
LOD Value-2.539504471
6.231414457
6.590064324-0.51393265-7.432857429
8.985446939-0.276541245
2.583408749
6.543548357-0.012606712
9.316438867
8.428513014-2.706846076

0.796
0.617
0.437
0.383
0.240
0.240
(*)
(*)
*
*
*
***
*
***
*
*
***
***
(*)

Subset 10 Loci
Power
1.000
1.000
1.000
1.000
1.000
0.989
ns
***
***
*
ns
***
*
**
***
*
***
***
ns

Full fit
no
yes
yes
no
no
yes
no
no
yes
no
yes
yes
no

Interpretation
FP
Father-Son
Father-Son
FP
FP#
Daughter-Father
FP#
FP
Father-Son
FP
Son-Father
Father-Son
FP

Subarea I1

Subarea I2

9
9

8
7CN

9

9

9

7E

7WR
7E

7CN
9

NP99MI013
NP99MI018
NP99MI023
NP99MI032
NP99MI093
NP99MI098
NP00MI030
NP01MI034
NP01MI092
NP01MI094
NP01MI100
NP02MI005
NP02MI010
NP02MI028
NP02MI060
NP02MI079
NP02MI087
NP03MI009
NP03MI011
NP03MI038
NP03MI045
NP03MI054
NP03MI073
NP03MI073
NP03MI075
NP03MI098
NP03MI099
NP04MI025
NP04MI029

14BC058
NP02MI030
NP06MI018
NP10MI009
14BC030
03BC088
NP12MI025
CK09MI003
14BC054
CK04MI041
CK02MI035
NP02MI016
08BC065
NP04MI026
03BC051
NP10MI002
NP02MI087F
NP03MI009F
NP07MI100
CK13MI039
NP03MI045F
NP03MI054F
CS03MI012
CK15MI022
06BC073
02BC052
NP03MI099F
CS11MI013
CS06MI052

OO
OO
OO
OO
JJ
JJ
OO
UO
OO
OO
OO
OO
OO
OO
OO
OO
OO
OO
OU
OO
OO
UO
OU
OO
OO
OO
OO
OO
OO

5.18774905
5.05663181
4.985257975
5.078096243
5.772674741
5.301571497
6.376023688
4.978835842
6.733999537
5.008975645
5.428208345
5.099718419
4.98034817
6.966459578
5.269387838
4.952452294
6.363904788
6.29765356
5.403248091
6.626260945
5.040166394
5.388751508
5.944545833
6.50994481
5.948027705
7.951766683
6.070856519
6.068881686
5.536189841

*
(*)
(*)
(*)
***
*
***
(*)
***
(*)
*
(*)
(*)
***
*
(*)
***
***
*
***
(*)
*
***
***
***
***
***
***
*

-3.159217003
7.487393501
0.354713363-13.56208246
5.019246683
1.849251568-0.802177126-1.570407376
8.397905477-7.546925608
9.493898933
0.047976082-4.954903156
9.38298552
7.888491571-5.362461564
10.49397717
9.156212598-6.903202723
7.001652874
9.441137662
7.830643859
8.558190489
7.695996221
7.924329903
10.90533317
7.722216955
8.281565364-3.778967714

ns
***
*
ns
***
**
*
(*)
***
ns
***
*
ns
***
***
ns
***
***
ns
***
***
***
***
***
***
***
***
***
ns

no
yes
no
no
no
no
no
no
yes
no
yes
no
no
yes
yes
no
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
no

FP#
Father-Son
FP#
FP
FP
FP
FP#
FP#
Son-Mother##FP
Father-Daughter
FP#
FP
Father-Son
Son-Mother##FP
Mother-Fetus
Mother-Fetus
FP
Father-Son
Mother-Fetus
Mother-Fetus
Father-Son###Father-Son###Father-Son
Father-Daughter
Mother-Fetus
Father-Daughter
FP

7CN

8

9

7CS

9

7CN

8
7CN

9
7CS

7CN
7WR
8
8
8
9
9
9
9
9
9

7CN

7CS
7CN
7CS
7CS
7CN

NP04MI051
NP04MI068
NP04MI075
NP04MI077
NP05MI046
NP05MI084
NP05MI084
NP06MI026
NP06MI051
NP06MI052
NP06MI063
NP06MI069
NP06MI077
NP07MI033
NP07MI037
NP07MI037
NP07MI039
NP07MI040
NP07MI042
NP07MI051
NP07MI060
NP07MI079
NP08MI001
NP08MI011
NP08MI022
NP09MI011
NP09MI013
NP11MI040
NP11MI044

NP04MI051F
NP04MI068F
NP12MI013
NP07MI083
CK07MI029
CS07MI022
NP05MI084F
NP06MI026F
NP06MI051F
CK07MI035
CK14MI023
CS03MI013
NP06MI077F
NP07MI033F
NP07MI037FA
NP07MI037FB
NP07MI039F
01BC019
CS08MI026
NP07MI051F
NP08MI011
CK09MI011
NP07MI048F
NP11MI018
CK09MI028
CK15MI024
CS08MI054
09BC065
05BC039

OO
OO
OO
OO
OO
OO
OO
OO
UO
OO
OO
OO
OO
OO
OO
OO
UO
JJ
OU
OO
OO
OO
UO
OO
OU
OO
OO
JJ
OO

5.823847066
6.380774719
5.080019419
7.507904896
4.994978692
8.216023522
7.734071389
5.290664836
5.569264277
5.921581717
5.628770847
5.112659013
5.517589271
5.318944549
5.968106303
7.337656291
7.233658428
5.592389658
6.685440103
5.37463325
6.360368114
5.516177718
6.389589647
6.354500944
5.481248436
7.172404249
5.036313933
5.483783644
6.164243401

***
***
(*)
***
(*)
***
***
*
*
***
*
*
*
*
***
***
***
*
***
*
***
*
***
***
*
***
(*)
*
***

8.742311144
8.670532719-2.226150339
8.546866706-10.21854182
1.417784428
10.07059881
7.611251886
4.109219643
8.177587514-2.579521145
0.157192115
6.685760793
5.041122174
8.803691126
9.44685602
9.124357001-2.033399991
8.713431863
6.873540775
8.008819632-1.137735308
10.12457409
7.486557406
8.549818499
10.7163976
7.783421948-0.539037746
8.430359054

***
***
(*)
***
ns
**
***
***
***
***
ns
*
***
***
***
***
***
(*)
***
***
***
(*)
***
***
***
***
***
*
***

yes
yes
no
no
no
no
yes
yes
no
yes
no
no
yes
no
yes
yes
yes
no
yes
yes
yes
no
yes
yes
yes
yes
yes
no
yes

Mother-Fetus
Mother-Fetus
FP#
Father-Son
FP
FP#
Mother-Fetus
Mother-Fetus
Mother-Fetus
Father-Son
FP#
FP
Mother-Fetus
Mother-Fetus
Mother-Fetus###Mother-Fetus###Mother-Fetus
FP#
Father-Daughter
Mother-Fetus
Father-Son
FP
Father-Fetus
Father-Daughter
Father-Daughter
Son-Father
Father-Daughter
FP#
Mother-Son#.##

9
9
9

7CN

9
7E
8
8

7CN

7CN
7CS
7CS
7CS
7CS
7CS
7CS
7CS

7CS

8
9
9
9
7WR

7CS
7CS
7CN
7CN
7CS

7CN

2

9

CS03MI039
CS03MI043
CS05MI001
CS05MI015
CS05MI030
CS05MI030
CS06MI051
CS06MI053
CS06MI053
CS07MI008
CS07MI037
CS07MI052
CS07MI053
CS08MI030
CS08MI047
CS09MI019
CS09MI029
CS10MI007
CS10MI038
CS12MI029
CS12MI048
CS13MI017
CK04MI008
CK05MI002
CK05MI015
CK05MI025
CK07MI016
CK08MI017
CK08MI017

15BC031
CS06MI039
05BC071
13BC080
CS05MI050
06BC071
10BC055
06BC075
14BC007
CS07MI008F
CK05MI002
CS07MI052F
CS07MI053F
CS08MI030F
CS08MI047F
CK05MI059
CK08MI030
03BC019
08BC128
CS13MI025
CK11MI059
CK11MI005
14BC073
07BC085
06BC003
04BC059
12BC027
12BC035
13BC035

JJ
OO
OO
OO
OO
OO
JJ
JJ
JJ
OO
OO
OO
OO
OO
UO
OO
OO
JJ
JJ
JJ
OO
JJ
OO
OO
JJ
OO
JJ
JJ
JJ

5.830596569
5.425884102
5.529056427
4.988290596
5.033179244
5.691057503
5.361876013
5.514984404
5.681854884
4.989352893
6.104401408
6.127618962
7.617080593
5.187757237
6.320423521
8.064239622
5.510954467
5.158823191
5.508855758
5.005715691
6.977507196
5.950196149
5.156473253
6.337566516
5.300289974
5.585343699
5.373983062
5.320138268
5.32359816

***
*
*
(*)
(*)
***
*
*
*
(*)
***
***
***
*
***
***
*
*
*
(*)
***
***
*
***
*
*
*
*
*

-6.471802267
8.079097617
7.156328658
4.28114123
6.837755643
7.603225165-1.082572773
8.624029545
3.248027112
7.817658405
7.892061975
8.761445473
11.16567979
6.947101413
9.702462631
7.035748071
6.383277712
7.888686742
2.129123897
7.311716349
9.769846118-2.083597469-8.639407134
10.48917176-4.936920476
9.188445429
3.38823831
4.454807983
3.736657547

ns
***
***
***
***
***
(*)
***
**
***
***
***
***
***
***
***
***
***
**
***
***
(*)
ns
***
ns
***
**
***
***

no
yes
yes
no
yes
yes
no
yes
no
yes
yes
yes
yes
yes
yes
no
yes
yes
no
yes
yes
no
no
yes
no
yes
no
no
no

FP#
Daughter-Father
Mother-Daughter##FP#
Father-Son###Father-Daughter###FP#
Mother-Daughter##FP#
Mother-Fetus
Daughter-Father
Mother-Fetus
Mother-Fetus
Mother-Fetus
Mother-Fetus
FP#
Daughter-Father
Father-Daughter
FP#
Mother-Son##
Daughter-Father
FP#
FP#
Father-Daughter
FP
Father-Daughter
FP#
FP#
FP

7CS
7CS

7CS
7CS

7CS
7CS

7CS
7CS

7CS

6

7CS
7CS
7CS
7CS
7CS
7CS

7CN

7CS
7CS

7CN
6

7CS
7CS

7CS
7CN

7CN

11

7CN

7CS

CK08MI022
CK08MI031
CK09MI001
CK09MI042
CK11MI053
CK11MI054
CK11MI057
CK12MI026
CK13MI023
CK13MI029
CK13MI053
CK14MI001
CK14MI013
CK14MI041
CK15MI002
CK15MI007
CK15MI023
01BC04102BC014
02BC019
02BC099
03BC01203BC039
03BC097
03BC107
04BC008
06BC032
06BC032
06BC146

CK10MI006
CK10MI057
12BC082
05BC043
09BC100
CK15MI016
07BC086
02BC096
CK13MI023F
09BC045
CK13MI053F
CK14MI001F
14BC076
12BC079
CK15MI002F
CK15MI007F
06BC011
04BC100
15BC011
09BC027
15BC148
04BC001
09BC046
15BC157
07BC059
14BC101
07BC059
11BC049
13BC083

JJ
JJ
JJ
OO
JJ
OO
JJ
JJ
OO
OO
OO
OO
OO
OO
OO
OO
JJ
JJ
JJ
JU
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ

5.399412647
4.99864055
5.150179042
6.620542272
5.326476124
5.60875643
5.505400506
5.380758881
6.183902815
6.899047818
6.143216575
5.010673554
5.128969356
4.972944433
5.281425223
6.197143623
4.96592835
6.051870998
4.921379986
4.935135272
5,757088534
4.946890553
5.238494504
5.77080971
5.607899925
5.074361833
6.320384276
5.97018077
5.069104285

*
(*)
*
***
*
*
*
*
***
***
***
(*)
*
(*)
*
***
(*)
***
(*)
(*)
***
(*)
*
***
*
(*)
***
***
(*)

8.560511909-1.70527731-1.534344706
10.08875535-0.714138344
2.780626215-1.2705611-2.338407243
9.48600141
10.27031846
7.987463375
8.455671291
7.193052062
6.966744977
7.05822394
9.179321399
6.496122718-4.261839023
3.708856137
2.570335446
1,907859346-5.950002936-1.323994729
6.06239283
1.641527957
4.550839454
5.719751379
6.152264307
6.105701592

***
(*)
(*)
***
*
**
(*)
(*)
***
***
***
***
***
***
***
***
***
ns
***
**
**
ns
(*)
***
**
***
***
***
***

yes
no
no
yes
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
no
no
no
no
no
yes

Mother-Son##FP
FP#
Son-Father#
FP#
FP
FP#
FP#
Mother-Fetus
Father-Daughter
Mother-Fetus
Mother-Fetus
Mother-Daughter#.##Son-Mother#.##Mother-Fetus
Mother-Fetus
FP#
FP#
FP#
FP#
FP#
FP
FP#
Mother-Son##
FP#
FP#
Mother-Daughter##.###
Mother-Daughter##.###
FP#

7CN

7CN

7CN

2

7CN
7CN
7CN
7CN
7CN
7CN
7CN
7CN

7CS

7CS
7CN

6

6

6
6

6
6

07BC021
07BC02107BC059
07BC071
07BC104
07BC129
07BC14209BC024
10BC027
11BC085
12BC116
14BC112

07BC111
10BC123
11BC049
12BC068
08BC019
09BC074
08BC103
09BC050
10BC109
15BC049
15BC041
15BC059

JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ
JJ

5.253989451
6.312472248
6.263725782
5.626624701
4.979005062
5.022002254
4.92493136
4.961277638
5.690162171
4.990099531
5.048218178
5.619125037

*
***
***
*
(*)
(*)
(*)
(*)
***
(*)
(*)
*

1.413567849
6.94809719
9.42126446
4.319459213
6.581763676-2.49142979
2.845874829-1.885973737
1.124295205
3.020892717
3.045667593
1.105274626

**
***
***
***
***
ns
**
(*)
*
**
**
*

no
yes
yes
no
yes
no
no
no
no
no
no
no

FP
Mother-Son##
Daughter-Mother##
FP
FP#
FP#
FP
FP
FP
FP#
FP#
FP

7CS
6

7CS
6

Table 3: Comparison of estimated and observed values of False Discovery Rate (FDR) and Power (16 microsatellite loci; full data set n=4,554 nf=53; only J stockn=1767 nf=2; only O stock n=2366 nf=49; J stock + unassigned n=2188 nf=4; O stock + unassigned n=2787 nf=51). FDRO is calculated under the assumption thatpairs’ assignment as False Positives (FP) based on evaluation of 26 loci and biological data is correct. PO is the percentage of Mother-Fetus pairs in the data set
inferred to be P-O pairs by the Likelihood analysis.
P=power; E=estimated/expected; O=observed; nf=number of fetusses in data set; #no mother-fetus pair detected

FDRE
FDRE
FDRE
FDRE

0.1
0.05
0.01
0.001

Full dataset
FDRO PE
0.468 0.437
0.383 0.383
0.179 0.240
0.179 0.240

PO
0.500
0.444
0.277
0.277

Only J stock
FDRO PE
0.000 0.117
0.000 0.102
0.000 0.102
0.000 0.102

P O#
0.000
0.000
0.000
0.000

Only O stock
FDRO PE
0.258 0.555
0.133 0.413
0.103 0.272
0.103 0.272

PO
0.347
0.306
0.163
0.163

J stock + unassigned
FDRO PE
P O#
0.200 0.177
0.000
0.000 0.146
0.000
0.000 0.146
0.000
0.000 0.146
0.000

O stock + unassigned
FDRO PEPO
0.236 0.530
0.373
0.133 0.395
0,333
0.065 0.264
0.176
0.065 0.264
0.176

Table 4: Observed Parent-Offspring pairs within and among stocks compared to expected values under a random distribution (significance evaluated with Χ2 test)

Within J stock
Within O stock
Within unassigned
Among J stock and O stock
Among J stock + unassigned
Among O stock + unassigned

Expected P-O pairs
11.29
20.24
0.64
30.24
5.38
7.21

Observed P-O pairs
9
57
0
0
0
9

p of Χ 2-Test
0.460<0.001
0.422<0.001
0.016
0.482

ns
***
ns
***
*
ns

Figure 1: Geographical representation of the validated 49 Parent-Offspring pairs (excluding
Mother-Fetus pairs). The endpoints of any line represent the positions of the two individuals
inferred to form a P-O pair.
A) All 49 pairs
125E
50N

135E

145E

155E

165E

175E
50N

45N

45N

40N

40N

35N

35N

30N
125E

135E

145E

155E

165E

135E

145E

155E

165E

30N
175E

(B) O stock pairs
125E
50N

175E
50N

45N

45N

40N

40N

35N

35N

30N
125E

135E

145E

155E

165E

30N
175E

(C) J stock pairs

125E
50N

135E

145E

155E

165E

175E
50N

45N

45N

40N

40N

35N

35N

30N
125E

135E

145E

155E

165E

30N
175E