Conservation genetics of harbour porpoises ... - Springer Link

Conservation Genetics 2: 309–324, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

309

Conservation genetics of harbour porpoises, Phocoena phocoena, in eastern and central North Atlantic Liselotte Wesley Andersen1,b,∗ , Daniel E. Ruzzante1 , Michael Walton2, Per Berggren3 , Arne Bjørge4 & Christina Lockyer5 1 Danish

Institute for Fisheries Research, Dept. of Inland fisheries, Vejlsøvej 39, 8600 Silkeborg, Denmark; address: National Environmental Research Institute, Dept. of Coastal Zone Ecology, Kalø, Grenåvej 12, DK-8410 Rønde, Denmark; 2 Sea Mammal Research Unit, Gatty Marine Laboratory, University of St. Andrews, St. Andrews, Fife KY 168LB United Kingdom; 3 Dept. of Zoology, University of Stockholm, S-106 91 Stockholm, Sweden; 4 Institute of Marine Research, Bergen, Norway; 5 Danish Institute for Fisheries Research, Charlottenlund Slot, 2920 Charlottenlund, Denmark (∗ Author for correspondance: [email protected])

b Present

Received 5 October 2000; accepted 12 December 2000

Key words: conservation genetics, Harbour porpoise, microsatellites, North Atlantic, Phocoena phocoena, population structure

Abstract We examined polymorphism at 12 microsatellite loci in 807 harbour porpoises, Phocoena phocoena, collected from throughout the central and eastern North Atlantic to the Baltic Sea. Multilocus tests for allele frequency differences, assignment tests, population structure estimates (FST ) and genetic distance measures (DLR and DC ) all indicate six genetically differentiated populations/sub-populations after pooling sub-samples within regions. Harbour porpoises from West Greenland, the Norwegian Westcoast, Ireland, the British North Sea, the Danish North Sea and the inland waters of Denmark (IDW) are all genetically distinguishable from each other. A sample of harbour porpoises collected off the Dutch coast (mainly during winter) was genetically heterogeneous and likely comprised a mixture of individuals of diverse origin. A mixed stock analysis indicated that most of the individuals in this sample (∼77%) were likely migrants from the British and Danish North Sea.

Introduction Knowledge of the population and social structure of marine mammals is essential for a proper evaluation of their present and potential future distribution and abundance. For species like the harbour porpoise, which in some areas (e.g. eastern North Atlantic) are likely to be caught incidentally by fishermen, such knowledge is fundamental for understanding the impact of by-catch (Berggren 1994; Vinther 1995, 1999). The biological importance of by-catches is likely to be underestimated if the existence of subpopulations is not properly recognised. The harbour porpoise is distributed throughout the North Atlantic and the North Sea and several populations and sub-populations are thought to exist in these areas (Hammond et al. 1995; Heide-Jørgensen

et al. 1993; Lockyer 1999). Current understanding of population structure of harbour porpoises in the central and eastern North Atlantic is largely based on the model suggested by Gaskin (1984) and revised by the IWC (1996). Apart from a division of the eastern and central North Atlantic into four populations (Irish Sea/west England, English Channel, North Sea and Baltic Sea) (Gaskin 1984; Yurick and Gaskin 1987), further subdivided the North Sea into three local subpopulations (east English, a Dutch to Danish North Sea and a southern Norway sub-population; equivalent to the regions in the present study Table 1). This model is, however, based mainly on observations of gaps in suitable habitats and a presumed limited migration (Figure 1) rather than on knowledge of the distribution of genetic variance.

310 Table 1. List of the total number of individuals in the six sampling regions, IDW (Belts, Swedish Baltic and Kattegat (ICES square IIIas)), Danish part of the North Sea (Skagerrak (ICES square IIIan), Danish North Sea 1980 and 1997 (ICES IVb)), British North Sea (Shetland, east Scotland, east England), Ireland (Cornwall, Ireland/Wales, Irish Sea), Norway and West Greenland and the Dutch sample, when divided into the variables sex and season Summer

Winter

Females

Males

Total

59 14 38

24 6 12

44 10 24

37 22 28

85 32 52

31 48 35

10 25 1

19 29 11

22 40 16

41 74 36

16 42 23

12 23 15

15 30 15

13 35 23

28 65 38

11 39 5

10 22 18

11 27 11

10 30 9

21 61 23

49

0

20

29

49

106 29 15 14

0 0 0 34

42 15 7 27

54 13 8 21

106 29 15 52

Region 1: Inner Danish Waters Belts Sweden Kattegat Region 2: Danish North Sea Skagerrak Danish North Sea 1980 Danish North Sea 1997 Region 3: British North Sea Shetland East Scotland East England Region 4: Ireland Cornwall Ireland/Wales Irish Sea Region 5: Norway Norway Region 6: West Greenland Maniitsoq Nuuk Paamiut Netherlands

Not all information on season and sex was obtained in some of the sampling areas.

The social structure of the species is poorly understood. Seasonal changes in the sex ratio suggest sexual segregation, especially in winter (Lockyer and Kinze 2000). Earlier population genetic studies provide evidence of female related philopatry in the breeding season in certain areas (Tiedemann et al. 1996; Andersen et al. 1997; Walton 1997). Seasonal movements have been reported both in Northwestern and Northeastern Atlantic porpoise populations (Northwest: Read and Kraus 1991; Rosel et al. 1999; Northeast: Møhl-Hansen 1954), and are probably a general phenomenon in the species. There are a number of studies on the genetic structure of harbour porpoises, all of which point to some level of subdivision among populations (Table 2). These studies are, however, difficult to compare because of differences in the genetic method and marker types employed and because of incomplete overlap in the geographic areas examined (Andersen

1993; Andersen et al. 1995; Tiedemann et al. 1996; Andersen et al. 1997; Walton 1997; Wang and Berggren 1997; Tolley at al. 1999). In addition, little information exists on whether the genetic structure in this species differs between sexes and/or seasons as a consequence of differential migration patterns. The objectives of the present study were twofold. We first examined the genetic structure of harbour porpoises throughout the species’ distributional range in the central and eastern North Atlantic using 12 highly polymorphic microsatellite DNA loci. We included harbour porpoises from (1) West Greenland, (2) the west coast of Norway, (3) the Inner Danish Waters including Swedish Baltic Sea samples [Kattegat (ICES IIIas), Belts (ICES IIIc), Øresund (ICES IIIb), Swedish Baltic (ICES IIId)], (4) the Danish North Sea [Skagerrak (ICESIIIan), ICES IVb], (5) the British North Sea [Shetland (ICES IVa), east Scotland and east England (ICES IVb, western

311

Figure 1. Map showing ICES and NAFO Fisheries Statistical Areas indicating the sampling locations. Region 1:Inner Danish Waters (IDW consisting of Kattegat (IIIas), Øresund (IIIb), Belts (IIIc), Swedish Baltic (IIId)); Region 2: Danish North Sea (1980, 1997(IVb) and Skagerrak (IIIan)); Region 3: British North Sea (Shetland (IVa), east Scotland and east England (IVb western sector)); Region 4: Ireland(Cornwall and Ireland/Wales (VIIe, f, g, h), Irish Sea (VIIa)); Region 5: Norwegian Westcoast (IIa); Region 6: West Greenland (NOFA 1C, 1D, 1E); Netherlands (IVc).

sector)], (6) Ireland [Cornwall, Ireland/Wales (ICES VIIe, f, g, h), Irish Sea (ICES VIIa)], and (7) Netherlands (ICES IVc). We tested the population structure model hypothesised by the IWC (1996; see also Gaskin 1984; Kinze 1985; Yurick and Gaskin 1987) (Figure 1) for harbour porpoises in the eastern North Atlantic. We then examined the influence of social structure on the genetic structure by comparing levels of population subdivision between sexes and among seasons.

Materials and methods Sample collection and laboratory analysis We collected and examined genetically a total of 807 porpoise individuals distributed among the seven sampling locations or hypothesised sub-populations mentioned in Table 1. The majority of the samples in the present study were stranded individuals. There were four exceptions, these were (1) The sample Danish North Sea 1980, which was a mixture of

by-caught and stranded porpoises collected in 1980– 1981. (2) The sample Danish North Sea 1997, which consisted of by-caught porpoises, collected in 1997 (Table 1). Both, 1980 and 1997 Danish North Sea porpoises were thought to belong to the same subpopulation (Danish North Sea). (3) The samples from the Swedish part of the Baltic Sea which consisted of porpoises by-caught mainly in 1985–1998, and (4) the samples from the West Greenland, which were obtained from the subsistence hunt conducted by Inuit. The vast majority of individuals in the present study are examined at microsatellite DNA loci for the first time. Exceptions are 35 of the 150 West Greenland porpoises, 33 of the 48 Danish North Sea 1980 summer individuals, and 53 of the 97 individuals in the Belts + Kattegat summer samples. These 121 porpoises were analysed with just three microsatellites Igf-I, 415/416, and 417/418 in Andersen et al. (1997). The summer season April to September includes the breeding season centred in June (Sørensen and Kinze 1994; Lockyer and Kinze 2000) and individuals caught or stranded in this period are presumed to be in or close to their breeding grounds. DNA was

312 Table 2. Genetic studies on the population structure of harbour porpoises in the eastern North Atlantic Authors

Population hypothesis

Method

Results

Andersen 1993

1) West Greenland 2) Inner Danish Waters 3) Danish North Sea 4) Netherlands 5) Gulf of St. Lawrence

Isozyme electrophoresis (2 loci)

Separate West Greenland, Danish North Sea and Gulf of St. Lawrence populations, Dutch not separate from the Danish North Sea but from the other three. IDW not separate from West Greenland. (allele frequency differences)

Andersen et al. 1997

1) West Greenland 2) IDW-summer 3) Danish North Sea-summer

Isozyme electrophoresis and DNA microsatellites (5 loci)

Separate West Greenland, IDW-summer and Danish North Sea-summer populations (allele frequency diff. and Fst)

Tiedemann et al. 1996

1) German North Sea 2) German Baltic Sea

Seq. of D-loop in mtDNA (1 locus)

Separate German Baltic Sea and German North Sea sub-populations. (haplotype frequency differences)

Walton 1997

1) Northern North Sea (Shetland + east Scotland) 2) Southern North Sea (east England + Netherlands) 3) Celtic Shelf/Irish Sea 4) English Channel


Separate Northern North Sea, Southern North Sea and Celtic Shelf/Irish Sea subpopulations. (haplotype frequency diff. and PhiST )

Wang and Berggren 1997

1) Norwegian waters 2) Kattegat-Skagerrak 3) Swedish Baltic

RFLP on mtDNA (1 locus)

Separate Norwegian waters, KattegatSkagerrak and Swedish Baltic sub-populations. (haplotype frequency differences)

Tolley et al. 1999

1) Barents Sea-females (BSF) 2) Norwegian North Sea-females (NNSF) 3) British northern North Sea-females (BNNF)


Separate BSF and BNNF sub-populations and NNSF and BNNF sub-populations. (haplotype frequency differences)

extracted according to Andersen et al. (1997) and 686 individuals were analysed with a total of 12 DNAmicrosatellite loci while a further 121 individuals were analysed with a subset of 9 DNA microsatellite loci (Table 3). The amplification conditions are given in Table 3. The microsatellites were detected and scored as described in Andersen et al. (1998). Data analysis Population genetics We estimated observed and expected heterozygosity and number of alleles per locus (Nei 1987) for the samples representing the six main regions and the Netherlands. To test the proposed population structure model it was necessary to address the hypothesis of sub-structuring within regions as well as among regions. This was conducted by testing for departures from Hardy-Weinberg expectations (HWE), and for homogeneity of allele-frequencies, by assignment

tests, by conventional F-statistics and by RST statistics. Tests were performed among all possible combinations of sampling localities, season and sex within regions. Samples within regions showing no substantial evidence of heterogeneity were pooled in the subsequent tests among regions. The tests for goodness of fit to the HWE and for homogeneity of allele frequencies were performed with GENEPOP (Raymond and Rousset 1995a). The significance values of the parameters tested in GENEPOP were computed using Fisher’s exact test and the Markov chain method to avoid biases caused by rare alleles or low sample size (Gou and Thompson 1992; Raymond and Rousset 1995b). The tests for conventional F- and RST -statistics were performed with ARLEQUIN version 1.1 (Schneider et al. 1997), and here significance was estimated with 10,000 permutations over loci. The FST estimator used is identical to Weir and Cockerham’s (1984) weighted average, ∧ θ . The estimator of RST used is described by

313 Table 3. The 12 polymorphic DNA microsatellite-loci used in the study, the nucleotide repeats and annealing temperature (C◦ ) Locus

Repeat No. Annealing temp. Allele-sizes

1 Igf-Ia,b 2 415/416b,c 3 417/418b,c 4 GT011d 5 GT015e 6 GT101e 7 GT136e 8 EV104f 9 EV94f 10 EV96f 11 TAA031g 12 GATA053g

2 2 2 2 2 2 2 2 2 2 3 4

50◦ 40◦ 46◦ 59◦ 59◦ 56◦ 52◦ 48◦ /56◦ 48◦ /56◦ 48◦ /56◦ 53◦ 55◦

132–168 201–225 161–189 95–129 118–180 93–113 83–113 124–168 196–224 161–203 211–244 201–213

a Kirkpatrick, B.W. 1992; b Andersen et al. 1997; c Amos et al. 1993; d Bérubé, M. at al. 1998,; e by courtesy of P. Palsbøll, Bérubé, M. and Jørgensen, H; f Valsecchi, E. and Amos, W. 1996; g Palsbøll et al. 1997.

PCR-conditions for 1, 2, 3, 4, 5, 6, 7, 11, 12: Denaturation at 95 ◦ C for 3 min, 35 cycles of denaturation at 94 ◦ C for 1 min, annealing temperature for 30 s, extension at 72 ◦ C for 10s. PCR-conditions for 8, 9 and 10 see Valsecchi and Amos (1996) except for annealing temperature which is given in the table.

Michalakis and Excoffier (1996) and Rousset (1996). The FST -statistic is based on the assumption that observed differences can be attributed mainly to drift and gene flow (infinite mutation model) while RST statistic is based on the assumption that mainly mutation (stepwise) and drift are responsible for the variation (Slatkin 1995; Michalakis and Excoffier 1996). Mixed stock analysis One of our samples exhibited consistent heterozygote deficiencies regardless of whether the whole sample or only the male, female, summer or winter subsets were analysed (the Dutch sample, see Results). This sample is therefore, likely to consist of a mixture of porpoises of heterogeneous origin. We used the maximum likelihood method of mixed stock analysis (MSA, Pella and Milner 1987; Millar 1987) to examine whether the porpoises in this sample originate from neighbouring sub-populations. Briefly, the method estimates the proportions of individuals from some predetermined reference (baseline) sub-populations that most likely explain the genotype distribution in the presumed mixed sample. We considered as baseline samples those from regions that could potentially contribute migrants to the Dutch waters. These were the samples from Ireland, the British

North Sea, the Danish North Sea, the Inner Danish Waters, and Norway (Table 1). For this analysis we used the software SPAM version 3.2 developed by the Alaska Department of Fish and Game (Anchorage, Alaska) and available at http://www.cf.adfg.state.ak. us/geninfo/research/genetics/software/SpamPage.htm. Symmetrical confidence intervals around the expected estimates were obtained by bootstrapping (1000 replicates) both the mixed sample and each of the baseline or reference collections. Mixed stock analysis has been applied to a variety of fish species most notably, Pacific salmon (reviewed in Utter and Ryman 1993). In the vast majority of cases the studies have used allozyme of mtDNA markers. More recently, the technique has been applied to Atlantic cod (Gadus morhua) using highly polymorphic microsatellite loci as markers (Ruzzante et al. 2000, with a brief review of the method’s assumptions and limitations and with references to other fish examples). Assignment tests Assignment tests (Paetkau et al. 1995) were used to determine the population of most likely origin of individuals given their multilocus genotypes and the allelic frequencies in baseline populations. Assignment tests and mixed stock analysis are closely related maximum likelihood methods. Here we performed the tests using the calculator at HTTP://www.biology. ualberta.ca/jbrzusto/. The expected multilocus genotype based on the 12 loci was calculated for each individual in each population. To deal with zerofrequencies (i.e. an allele not present in a given population) we chose the option where allele frequencies in every population are adjusted according to p = (f + 1/a)/(n + 1) (Titterington et al. 1981; Cornuet et al. 1999), (p = adjusted probability estimate for a given allele at a locus in a population, f = number of allele copies of a given type, a = number of different alleles for that locus, n = number of gene copies for that locus in the given population). This is equivalent to adding the allele to all populations. In the assignment test we tested the hypothesis: H0 Populations are actually one well-mixed population in HWE using a randomisation process where a population of new individuals is formed by drawing individuals from the combined gene pool. For each population every individual’s genotype was re-drawn and sampled with replacement from the combined gene pool of all populations assuming HWE. We then observed the probability (after 1000 randomisations) of cross-assignments.

314 If there were at least as many cross assignments as in the original assignment test, the null hypothesis is true. The assignment test was also used to estimate the genotype likelihood ratio distance, DLR , described by Paetkau et al. (1997) as a spatial distance measure between the samples also when partitioning according to sex and season. The DLR topology was compared to the topology obtained by DC , the spatial distance measure of Cavalli-Sforza and Edward’s (1967). Bootstrap values (2000 randomisations) for the tree topologies were obtained with the program SEQBOOT in PHYLIP (Felsenstein 1993), DC was estimated in GENDIST, and Fitch and Margoliash trees were constructed from the genetic distance data in the PHYLIP-package (Felsenstein 1993). The sequential Bonferroni procedure was applied using a significance level of 5% whenever multiple tests were performed to give table-wide significance levels (Rice 1989). Results Genetic Diversity and test for HW-proportions The mean expected heterozygosity and mean allele number based on the 12 microsatellites (Table 4) ranged from 0.672 ± 0.228 and 9.0 ± 5.1 in the Dutch sample to a maximum mean heterozygosity of 0.712 ± 0.194 in the combined British North Sea sample (Shetland + east Scotland + east England) and maximum mean number of alleles of 11.8 ± 5.7 in the combined Danish North Sea sample (Skagerrak + Danish North Sea 1980 + Danish North Sea 1997). Some departures from HWE within regions were found but for any one regional sample these deviations were only observed in two or less of the 12 loci examined. In general, deviations occurred in the female or summer subsets, they always involved deficiencies of heterozygotes and the loci involved (EV104, 415/416, EV94, GT011, TAA031) varied with the sample examined. Together these results suggest that deviations from HWE are likely minor and they likely result from a Wahlund effect or non-random mating, though we cannot exclude the possibility that they may reflect inbreeding or the presence of null alleles.

Structure within regions Tests of Homogeneity: allele frequency differences between samples within regions We compared allele frequency distributions and estimated population subdivision (FST ) between locations within regions with data from single locations pooled, separated by sex, and separated by sampling season (Table 5a, only allele frequencies). In the Inner Danish Water region (region 1, Figure 1) different allele frequencies were observed between the Belt and Kattegat samples in the multi-locus test for allele frequency differences. Among the summer samples there were differences between porpoises from the Belts and those from the Kattegat (2 loci) and between porpoises from the Kattegat and those from the Belt and Swedish Baltic combined (3 loci). The other 27 comparisons indicated no differences in allele frequencies (Table 5a). In the Danish North Sea region (region 2) none of the multilocus tests for allele frequencies were significant (after Bonferroni, α = 0.05, Table 5a). A significant (P < 0.005) multi-locus FST estimate was, however, observed between males from the Danish North Sea (1980) and males from the Skagerrak and Danish North Sea (1997) (data not shown). In the British North Sea region (region 3) differences in allele frequencies were observed (multilocus test) between the totals (i.e. sexes and sampling seasons pooled) from east Scotland and those from Shetland and between the former and the pooled porpoises from east England and Shetland. The summer samples from east Scotland differed in allele frequencies (multilocus test) from the summer sample from Shetland. Both for the totals and when stratifying according to season only two and three of the 12 loci showed significant allele frequency differences. The other 27 tests were not significant (Table 5a). In the Irish region (region 4), only 5 comparisons were made, as the sample sizes became very small upon further stratification (See Table 1). No differences were observed in any of the five comparisons (Table 5a). For Norway (our region 5), Bjørge and Øien (1995) have suggested the existence of two sub-populations according to whether the porpoises originate north or south of latitude 66◦ N. We, however, detected no significant differences in allele

FIS 0.03 0.14 –0.06 –0.04 0.02 –0.03 –0.01

GT136 He A 0.822 10 0.823 14 0.852 12 0.801 12 0.852 13 0.852 14 0.817 11

Ho Inner Danish Waters 0.799 Danish North Sea 0.821 British North Sea 0.901 Ireland 0.833 Norway 0.837 West Greenland 0.874 Netherlands 0.827

Ho 0.953 0.960 0.954 0.895 0.959 0.947 0.942

Ho 0.775 0.768 0.817 0.810 0.735 0.762 0.808 GT015 He A 0.947 26 0.948 26 0.948 26 0.942 24 0.945 25 0.947 27 0.920 22 FIS –0.01 0.01 –0.01 0.05 –0.01 0.004 0.02

EV104 He A FIS 0.856 12 0.09∗ 0.866 14 0.04 0.831 10 0.02 0.854 12 0.05 0.854 9 0.14∗ 0.860 11 0.113 0.859 10 0.06

Ho 0.130 0.152 0.237 0.352 0.204 0.219 0.154

Ho 0.503 0.510 0.626 0.543 0.531 0.470 0.365

∗ Significant at the 5% level after application of the Bonferroni procedure.

FIS –0.07 0.11 0.09 0.08 –0.001 0.07 –0.06

GT101 He A 0.754 9 0.767 9 0.734 9 0.772 9 0.734 7 0.727 9 0.783 10

Ho Inner Danish Waters 0.805 Danish North Sea 0.762 British North Sea 0.672 Ireland 0.714 Norway 0.735 West Greenland 0.675 Netherlands 0.827 GATA 053 He A 0.144 3 0.144 4 0.254 4 0.338 4 0.187 3 0.223 4 0.180 3 FIS 0.10 –0.03 0.07 –0.04 –0.09 0.02 0.14

415/416 He A FIS 0.584 8 0.14∗ 0.553 11 0.03 0.643 6 0.03 0.507 7 –0.07 0.527 6 –0.01 0.522 8 0.09 0.474 5 0.23

Ho 0.799 0.847 0.802 0.800 0.816 0.801 0.827

Ho 0.716 0.788 0.792 0.790 0.735 0.874 0.827 Igf-I He A FIS 0.864 15 0.08 0.868 16 –0.04 0.871 13 0.08 0.842 14 0.05 0.873 11 0.07 0.853 15 0.06 0.882 12 0.06

GT011 He A FIS 0.732 12 0.02 0.823 13 0.02 0.813 11 0.03 0.812 15 0.03 0.846 11 0.13 0.868 14 –0.09∗ 0.823 11 –0.01∗

Ho 0.432 0.490 0.489 0.476 0.531 0.417 0.404

Ho 0.627 0.583 0.608 0.600 0.531 0.636 0.654

FIS 0.05 0.01 –0.05 0.01 –0.01 –0.07 –0.21 EV96 He A FIS 0.444 4 0.03 0.503 6 0.06 0.487 3 –0.004 0.510 4 0.07 0.464 3 –0.14 0.469 3 0.103 0.458 3 0.12

417/418 He A 0.732 8 0.589 9 0.581 6 0.605 8 0.528 5 0.593 10 0.539 7

Ho 0.657 0.702 0.771 0.686 0.694 0.695 0.365

Ho 0.757 0.722 0.725 0.810 0.714 0.768 0.769

TAA 031 He A FIS 0.671 10 0.02 0.719 11 0.08 0.733 10 –0.05 0.740 11 0.07 0.803 10 0.14 0.707 11 0.02 0.542 7 0.33∗

EV94 He A FIS 0.766 8 –0.05 0.814 8 0.08 0.799 9 0.09∗ 0.796 8 –0.02 0.803 8 0.11 0.805 8 0.04 0.791 7 0.03

169 151 131 105 49 150 52

N

Table 4. Observed (Ho ) and expected (He ) heterozygosity, number of alleles (A), and tests for goodness of fit to the Hardy-Weinberg expectation (FIS ) performed by testing specifically for heterozygote deficiency in GENEPOP (Raymond and Rousset 1995a) for harbour porpoise samples constituting Inner Danish Waters including the Swedish part of the Baltic Sea, Danish North Sea, British North Sea, Netherlands, Norway, and West Greenland

315

316 Table 5a. Observed number of differences in allele frequency distributions between sensible pair-wise multilocus comparisons within the regions of Inner Danish Waters, Danish North Sea, British North Sea, Ireland, Norway and West Greenland without stratification and when stratifying according to season and sex (after application of Bonferroni procedure). N = total number of pair-wise comparisons after all possible combinations of locations within the region. This included pair-wise pooling of locations which explain the number N (i.e. three locations and pair-wise sensible poolings = 6 for each variable total, summer, winter, female and male)

Totals Region 1: Inner Danish Waters Region 2: Danish North Sea Region 3: British North Sea Region 4: Ireland Region 5: Norway∗∗ Region 6: West Greenland∗∗

No. of significant different allele dist. Summer Winter Female Male

1∗ 0∗ 2∗ 0∗ 0∗ 1∗

frequencies or FST between Norwegian porpoises (region 5) from north and south of this latitude. In the West Greenland sample (region 6), which consisted of porpoises collected during 1988, 1989 and 1995, a significant difference in allele frequencies was observed between the Maniitsoq sample from 1989 and 1995 in the multi-locus test (Table 5a). In general, however, the multi-locus tests based on allele frequency differences and the FST estimates rejected the hypothesis of further sub-structuring among subsamples within regions. Population differentiation between regions Allele frequency differences Allele frequencies differed between all pairs of the six regions (multi-locus tests for homogeneity of allele frequencies, Table 5b). These differences generally persisted when stratifying according to season or sex. The few between-region comparisons that were not significant included as one member of the pair the Dutch porpoises when summer, winter, male or female samples were considered separately. We also examined the spatial relationship among samples using the genetic distances DLR (Paetkau et al. 1997) and DC (Cavalli-Sforza and Edward’s 1967) (Figure 2a, b, DLR tree not shown). In the stratification according to sex the various British samples were not pooled. The clusters showed an overall grouping of male-female samples from the same sampling area especially in the DC based tree. Another feature observed was a tendency of females and males from the British North Sea to cluster together. This cluster also includes the Dutch samples.

2∗ 0∗ 1∗ 0∗ – –

0∗ 0∗ 0∗ 0∗ – –

0∗ 0∗ 0∗ 0∗ – 0∗

0∗ 0∗ 0∗ 0∗ – 0∗

N

30 25 30 5 1 9

Table 5b. Observed number of significant different allele distributions obtained from the pair-wise multilocus comparison of the allele distribution amongst the seven samples Inner Danish Waters, Danish North Sea, British North Sea, Ireland, Norway, West Greenland and Netherlands without stratification and when stratifying according to season and sex (after application of Bonferroni procedure). N = total number of pair-wise comparisons without and with stratification according to season and sex

No stratification Summer-samples Winter-samples Female-samples Male-samples

No. of significant different allele dist.

N

21 14 9 17 17

21 21 11∗∗ 21 21

∗ After application of the Bonferroni procedure to each region separately. ∗∗ The West Greenland and Norway samples are only summer samples.

These results suggest that, in general, female and male porpoises collected in the same region are genetically more closely related to each other than they are to female or male porpoises collected elsewhere and the same applies to seasonal samples: winter and summer samples from a given region are more closely related to each other than they are to winter and summer samples from elsewhere (Figure 2b). Mixed stock analysis The mixed stock analysis (Figure 3) indicates that the majority of porpoises in the Dutch sample may be migrants from the British and Danish North Sea

317

Figure 2. Fitch-Margoliash consensus trees based on Cavalli-Sforza and Edward’s (1967) chord distance, DC , when stratifying according to sex (a) and according to season (b). For data pooled into six regions: West Greenland, Norway, British North Sea, Ireland, Danish North Sea and Inner Danish Waters, both the DLR distance (Paetkau et al. 1997) (c) and the chord distance, DC , (d) are depicted.

318

Figure 3. Box and whisker plots of the expected maximum likelihood (bootstrap) estimates of contributions (proportions) by porpoise populations from 5 regions in the Northeast Atlantic (Ireland, UK, Denmark, Norway, and Inner Danish Waters) to the porpoise population found off the coast of the Netherlands. Estimates reflect the proportions of porpoises from each of the 5 regions that can explain the allele frequency distribution found in the Dutch sample. The figure is based on 1000 bootstrapped samples, with each of the 5 reference samples and the Dutch sample bootstrapped. The boxes indicate the limits of the middle half of the data (i.e. from the 25% to the 75% quartiles). The white horizontal bars in each box represent the medians; skewness is reflected by the position of the median within each box. “Whiskers” (lines extending from the top and bottom of each box) are drawn to the nearest value not beyond 1.5 times the interquartile range. Straight horizontal lines beyond whiskers’ range represent extreme bootstrapped estimates.

(44.9% and 31.9%, bootstrap means, respectively). Only a minority of the porpoises is likely of Norwegian (6.5%), Inner Danish Water (7.8%) or Irish (8.9%) origin (Figure 3). Six of the 52 porpoises in the Dutch sample possessed an allele not present in any of the baseline British North Sea, Danish North Sea, Norway, Inner Danish Waters, or Ireland samples. We chose to eliminate them from the mixed stock analysis because of the possibility that they may belong to a resident porpoise sub-population or they may have originated from some other location not represented among our baseline samples (e.g. Iberia). Their inclusion in the analysis increased the contribution by British North Sea porpoises slightly. Hence, our results suggest that a mixture of harbour porpoises from regions surrounding the Dutch waters (i.e. mainly the British and the Danish North Sea), possibly mixed with a few resident porpoises could explain the allele frequency distribution in the Dutch sample. Assignment tests The assignment tests were carried out according to Paetkau et al. (1995, 1997) (Table 6) and were based on the 12 microsatellite loci. These tests were performed to further examine the genetic relationship

among porpoises from the six regions. Table 6 shows, for each of the six regions (columns), the number of individuals assigned to their original sampling region (diagonal elements) as well as to each of the remaining 5 regions (off diagonal). In all cases more individuals were assigned to their region of origin than to any other region. Miss-assignments occurred mainly to the nearest region indicating isolation by distance effect. The hypothesis that all porpoises originated from a well-mixed population is therefore rejected (Table 6). Phylogeographical reconstruction The population structure estimate FST was used together with the DLR to quantify the genetic relationship among the six regions (Table 7). The results of the pair-wise multilocus FST tests (Table 7 above diagonal) were all significantly different from zero. The genetic distance measures based on the individual multilocus genotype, DLR , (Table 7, below diagonal) were in general agreement with the FST tests with a corresponding low genetic distance between West Greenland and Ireland, and between the Danish and British North Sea. The topology obtained from the Fitch and Margoliash trees based on individual multilocus genotypes (DLR ) and the genetic distance DC (Figure 2c, d, respectively) separate the Norwegian

319 Table 6. Assignment test based on the total number of individuals sampled in the six main regions the Danish NS (DKNS) (Skagerrak (ICES IIIan) + Danish North Sea 1980 + Danish North Sea 1997 (ICES IVb)), British NS (BRNS) (east England + east Scotland + Shetland), IDW (Belts (ICES IIIc) + Kattegat (ICES IIIas) + Swedish Baltic (ICES IIId) + Øresund (ICES IIIb)), Ireland, Norway and West Greenland (W. Gr). The hypothesis H0 : Populations are actually one well-mixed population in Hardy-Weinberg proportions (p-values) was tested after a randomisation process where new individuals were drawn from the total gene pool for all populations and assigned to the different populations. See text for further information

DKNS IDW Norway Ireland BRNS West Greenland

DKNS

IDW

Norway

Ireland

BRNS

W. Gr

N

H0

45 17 7 19 11 25

34 92 3 16 18 11

15 16 24 5 6 13

20 13 1 28 19 29

17 19 6 19 51 20

20 12 8 18 26 52

151 169 49 105 131 150

0.005∗ 0∗ 0∗ 0.03∗ 0∗ 0∗

Table 7. Genetic distances in terms of FST (above diagonal) and DLR (below diagonal) between the six regions (IDW, Inner Danish Waters (Belts (ICES IIIc) + Kattegat (ICES IIIas) + Swedish Baltic (ICES IIId) + Øresund (ICES IIIb)), DKNS, Danish North Sea (Skagerrak (ICES IIIan) + Danish North Sea 1980 + Danish North Sea 1997 (ICES IVb)), BRNS, British North Sea (east England + eastScotland + Shetland), Ireland, Norway and West Greenland (W. Gr). The FST measure is based on allelic variance equivalent to Weir and Cockerham’s (1984) unbiased estimator, θ , and tested in ARLEQUIN (10,000 permutations) (Schneider et al. 1997) while DLR is the mean genotype log likelihood ratio across individuals from the two populations in question (Paetkau et al. 1997) IDW IDW DKNS BRNS Ireland Norway W. Gr

0.58 0.87 0.95 1.42 0.92

DKNS

BRNS

Ireland

Norway

W Gr

0.005∗

0.009∗ 0.003∗

0.012∗ 0.004∗ 0.004∗

0.014∗ 0.007∗ 0.011∗ 0.014∗

0.01∗ 0.002∗ 0.002∗ 0.004∗ 0.009∗

0.53 0.44 0.65 0.32

0.54 1.00 0.41

1.22 0.43

1.00

∗ P < 0.05 Significant after application of the Bonferroni procedure.

from the remaining porpoises. Among the remaining groups there seems to be a division between east and west sub-populations: the West Greenland, Ireland and British North Sea porpoises seem to be more closely related to each other than they are to porpoises in Inner Danish Waters and in the Danish North Sea. None of the RST estimates were significant (RST (total samples) ranged from –0.00084 to 0.00485 between IDW-British North Sea and IDW-Norway, respectively).

Discussion Harbour porpoise populations, fisheries by-catch and conservation Based on evidence from sightings and stranding data gathered during the 20th century, the abundance of harbour porpoises is thought to have declined significantly in several European areas especially in the English Channel, southern North Sea and Baltic Sea

320 (Klinowska 1991). One of the current major causes of concern for the conservation of the species is the extent of the by-catch of porpoises by fisheries. For example, the seasonal migration of porpoises out of the Baltic in winter (Møhl-Hansen 1954) appear to have ceased (Gaskin 1984) suggesting a decline in the size of the Baltic Sea population. The growing concerns about population numbers led to the formation of ASCOBANS (Agreement on the Conservation of Small Cetaceans in the Baltic and North Seas) under the UN Bonn Convention in 1994. The International Whaling Commission also recommended (IWC 1996) that further research be performed into ascertaining porpoise population size and structure and obtaining accurate by-catch rates both in the North Sea and adjacent waters. The question of population size was addressed by a census study of cetacean numbers in North European waters (North Sea: 263,000) performed in 1994 (Hammond et al. 1995) and it is hoped to repeat this in the near future. By-catch rates in the North Sea by Danish and UK fishing fleets have been estimated at around 8,100 porpoises per year (Clausen and Andersen 1988; Vinther 1995, 1999; BYCARE 1999). This figure exceeds the generally agreed guideline that if any by-catch exceeds 2% of the population size then the population is not-sustainable (ASCOBANS 2000). However, to determine the true impact of by-catch rates knowledge of population structure is important. The impact is likely to be overestimated for a group of porpoises mistakenly considered to be a separate population when in reality they are a part of a larger population complex. Conversely the impact is likely to be underestimated, if in reality a separate population existed when it was thought to interbreed with other groups. As listed in the introduction a number of studies have already been published which have used isoenzyme or mtDNA variation to study the population structure of various porpoise ‘populations’ in European waters, but they are difficult to compare because of differences in the techniques employed and in geographic areas examined. The present study used 12 microsatellite loci to perform a comprehensive assessment of population structure of porpoises in Greenland and European waters. We test the model of population distribution first proposed by Gaskin (1984) and refined by the IWC (1996), which was based on available sightings and stranding data. We also examined the distribution of genetic variance

within each of the 6 main regions. It is highly likely that there is some overlap in distributions between neighbouring regions (Heide-Jørgensen et al. 1993; Hammond et al. 1995). Social structure The mean heterozygosities (0.67–0.71) found in our study were slightly lower than those reported (0.80– 0.86) in a recent study of 4 presumed populations of harbour porpoises off NE America and West Greenland based on 7 microsatellite loci (Rosel et al. 1999). For porpoises in Inner Danish Waters the high genetic diversity at the nuclear level contrasted with the low haplotypic and nucleotide diversities obtained in mtDNA studies (Wang and Berggren 1997; Tiedemann et al. 1996). This difference likely results from differences in the degree of male and female philopatry (gene flow), and probably also from differences in mutation rates between the two markers in relation to the time since population divergence (See Structure among regions, below). We generally detected more cases of heterozygote deficiencies in the female or female-summer samples (see Results text) compared to the pooled samples from each region (Table 4). We attribute this observation as well as the occasional sub-structure between groups from neighbouring areas within regions to female philopatry (Tiedemann et al. 1996; Walton 1997; Andersen et al. 1997). Hence, this kind of sub-structure between small neighbouring areas within the regions may reflect family structure within regions. Sub-structure caused by the social structure or breeding system has been detected in other mammals, e.g. the black-tailed prairie dog (Cynomys ludovicianus), where the breeding system (typical polygyny and male dispersal) caused a significant coancestry locally without extreme inbreeding costs or loss of genetic variance (Sugg et al. 1996). The influence of social structure on population structure may also be inferred from a comparison of male and female summer and winter samples in the Danish North Sea. Males in this region exhibit heterozygote deficiencies in both summer and winter collections likely as a result of mixing with porpoises from neighbouring regions in the Danish North Sea (Andersen 1993; Andersen et al. 1997). Females in the present study instead, exhibit heterozygote deficiencies only in summer samples, suggesting differences in migration patterns between sexes.

321 Structure within regions North Sea, Dutch sample Overall, with one exception, no significant deviations from the Hardy-Weinberg expectations were observed. The exception was the Dutch sample. The heterozygote deficiency in this last sample was found in the pooled sample as well as in the summer, male and female-winter subsets. The consistent heterozygote deficiency in the various subsets suggests that the pooled Dutch sample consists of porpoises of diverse origin or non-random mating. We therefore examined whether its composition could be explained as a mixture of porpoises from neighbouring regions using mixed stock analysis. This analysis indicates that the Dutch sample may indeed largely consist of a mixture of British and Danish North Sea porpoises, though we cannot exclude the possibility that some of the porpoises in this sample may be local in origin. Previous studies based on mtDNA sequencing could not detect a difference between Dutch and east English porpoises (Walton 1997). Northern and southern North Sea samples Walton (1997) and Lockyer (1999, based on teeth ultrastructure analysis) suggested that porpoises in the North Sea are divided into a northern and a southern North Sea sub-population. This division was not borne out by our genetic analysis: We detected no difference in allele frequencies nor a significant FST estimate between the presumed southern and northern North Sea porpoise samples. North Sea, Danish sample Male porpoises in the Danish North Sea 1980 and in the Skagerrak-Danish North Sea 1997 samples are likely a mixture of males from nearby sub-populations (Andersen 1993; Andersen et al. 1997; Walton 1997) as suggested by the small but significant FST estimate. The porpoises from 1980 were mainly stranded individuals whereas those from 1997 were by-caught only. Stranded and by-caught individuals did, however, not differ in allele frequencies, thus the significant FST cannot be explained by method of capture. Structure among regions When analysing the population structure between regions the sub-populations samples within the regions were pooled. The pairwise multilocus FST estimates among the six regions were very low (0.002– 0.014) suggesting high-levels of contemporary or

historic gene flow. FST assumes that drift and gene flow are the main mechanisms of population differentiation. The method is an appropriate measure of population differentiation as long as the time that populations/sub-populations have been separated is short (tens to hundreds of generations; Slatkin 1995; Takezaki and Nei 1996). RST instead, assumes that mutation and drift are the main mechanisms of population differentiation. Population structure among the six regions in our study is slight for FST , but it is statistically significant when tested at the 5% level. No structure was detected with RST . The difference between the two estimates can most likely be attributed to the fact that the current population structure has arisen since the last ice-age about 10,000 years ago. Pairwise FST estimates (Table 7) support the hypothesis of genetically different harbour porpoise sub-populations in Irish waters, British North Sea, West Greenland, Danish North Sea, Norwegian and Inner Danish Water. The consistent relatively low number of miss-assigned individuals in the assignment test (Table 6) also suggests the existence of six genetically differentiated sub-populations. The genetic distance DC (Cavalli-Sforza and Edwards 1967) has been suggested to depict the most correct topology for the analysis of population relationships (Takezaki and Nei 1996). Assuming the population structure of harbour porpoises is as indicated in our analysis we would expect males and females, and summer and winter samples from the same sampling area to cluster together. Our expectations were borne out (Figure 2a, b). The stratification according to sex and season (Figure 2a, b) supported a division of North Sea porpoises into separate British and Danish sub-populations. This east/west structuring of the North Sea porpoises is consistent with Tolley et al.’s (1999) observation of different mtDNA haplotype frequencies between the British northern North Sea and the Norwegian North Sea. The differences we found in the present study among porpoises from Inner Danish Waters and the Danish North Sea are consistent with a general German Baltic Sea/IDW – North Sea differentiation (Tiedemann et al. 1996 based on mtDNA; Andersen et al. 1997 based on isozymes and microsatellite). These results are also consistent with Wang and Berggren (1997, based on mtDNA) under the assumption that their Kattegat-Skagerrak sample is actually a Skagerrak and thereby, a North Sea sample. Morphological differences between Baltic Sea and North Sea (Kattegat-Skagerrak) porpoises (Kinze

322 1985; Børjesson and Berggren 1997) also indicate population differences. Porpoises caught along the west coast of Norway seem to be the most genetically differentiated among those examined in the present study (Figure 2d). This observation confirms the results of a very recent study by Tolley et al. 2001 (this issue) based on mtDNA. In the genetic comparison of porpoises representing six locations across the North Atlantic (Gulf of Maine, Gulf of S. Lawrence, Newfoundland, West Greenland, Iceland and Norway) the Norwegian porpoises were genetically distinct from all the other 5 regions (Tolley et al. 2001). The remaining porpoises in the present study cluster into an eastern and western group regardless of the distance measure used, FST , DLR or DC (only DLR and DC are shown). The relationship among porpoises from the IDW, Danish North Sea (DKNS) and the Norwegian samples appear to indicate common migration routes among these porpoises, going from Inner Danish Waters northwest to Skagerrak and the Danish North Sea, then northeast to Norske Rennen, and finally north along the Norwegian westcoast and vice versa. The topology or spatial organisation in the west group obtained with the multilocus genotype measure (DLR ) is consistent with that obtained with DC . For the more easterly group the Norwegian sample was again more distinct while the Danish North Sea sample had a multi-locus genotype composition more closely related to the Norwegian sample than to the Inner Danish Water sample. This slight difference in the structures of the DLR and DC trees probably arise from the fact that DLR is based on multi-locus genotypes and DC is based on allele frequencies (Figure 2c, d). In conclusion our study largely supports the population structure model first proposed by Gaskin (1984) and modified by the IWC (1996). There are, however, two major disagreements: (1) We distinguish North Sea porpoises into separate British and Danish North Sea sub-populations and (2) Our Skagerrak porpoises appear to belong to the Danish North Sea subpopulation and not to the Inner Danish Water subpopulation. We detected at least six genetically differentiated harbour porpoise sub-populations as follows: an Irish Sea/Wales, British North Sea, Danish North Sea, IDW, Norwegian and West Greenland. The heterozygote deficiency detected in the Dutch sample together with the mixed stock analysis both indicate this sample may consist, at least in part, of migrant individuals coming from the British North Sea and the Danish North Sea.

Acknowledgement This study was 50%-funded by EU FAIR contract CT05-0523-BYCARE. The tissue-samples were kindly provided by T. Kuiken and P. Jepson, Institute of Zoology, UK, E. Rogan and S. Berrow, Aquaculture Development Centre, Dept. of Zoology and Animal Ecology, Cork, Ireland, N. Tregenza, Cornish Wildlife Trust, UK, M. Addink, University of Leiden, Netherlands, C.C. Kinze, Zoological Museum, University of Copenhagen, Denmark, B. Clausen and S. Andersen, G. Désportes, The Fjord and Belt centre, Kerteminde, Denmark, and Mads-Peter Heide Jørgensen, Greenland Institute of Natural Resources, Nuuk, Greenland. A special thanks to M. Bérubé, P. Palsbøll, School of Biological Sciences, University of Bangor, Wales, UK and H. Jørgensen, Dept. of Population Biology, University of Copenhagen, Denmark who generously made primers available for this study and to D. Meldrup for her invaluable help and assistance in the lab. Finally, we want to thank two anonymous referees and A. R. Hoelzel, Dept. of Biological Sciences University of Durham, UK for commenting and reviewing the paper and Ib Kragh Petersen, National Environmental Research Institute, Department of Coastal Zone Ecology, Denmark for his great effort concerning the production of the map. References Amos B, Schlötterer C, Tautz D (1993) Social structure of pilot whales revealed by analytical DNA-profiling. Science, 260, 670–672. Andersen LW (1993) The population structure of the harbour porpoise, Phocoena phocoena, in Danish waters and part of the North Atlantic. Mar. Biol., 116, 1–7. Andersen LW, Holm LE, Clausen B, Kinze CC (1995) Preliminary results of a DNA-microsatellite study of the population and social structure of the harbour porpoise. In: Whales Seals, Fish and Men (eds. Blix AS, Walløe L, Ulltang Ø), pp. 117–127. Elsevier Science. Andersen LW, Holm LE, Siegismund H, Clausen B, Kinze CC, Loeschcke V (1997) A combined DNA-microsatellite and isozyme study of the population structure of the harbour porpoise in Danish waters and West Greenland. Heredity, 78, 270–276. Andersen LW, Born EW, Gjertz I, Wiig Ø, Holm LE, Bendixen C (1998) Population structure of the Atlantic walrus (Odobenus rosmarus rosmarus) in the eastern Atlantic Arctic based on mitochondrial DNA and microsatellite variation. Mol. Ecol., 7, 1323–1336. ASCOBANS (2000) Incidental Take of Small Cetaceans. Proceedings of the 3. meeting of Parties to ASCOBANS, Bristol, UK 26–28 of July. Resolution, 3, 93–96. Bérubé M, Aguilar A, Dendanto D, Larsen F, Notarbartolo Di Sciara G, Sears R, Sigurjónsson J, Urban R, Palsbøll, PJ (1998) Population genetic structure of North Atlantic, Mediterranean Sea

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