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Short communication
Population structure of albacore (Thunnus alalunga) in the Northwestern
Pacific Ocean inferred from mitochondrial DNA
Georgiana Cho-Chen Wu
a,1, Hsin-Chieh Chiang
b,1, Kuo-Shu Chen
b,
Chien-Chung Hsu
b,∗, Hsi-Yuan Yang
a,∗aInstitute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan, ROC bInstitute of Oceanography, National Taiwan University, Taipei, Taiwan, ROC
a r t i c l e i n f o
Article history:
Received 10 January 2008
Received in revised form 23 July 2008 Accepted 27 July 2008
Keywords:
Albacore (Thunnus alalunga) Mitochondrial DNA Control region Population structure Northwestern Pacific Ocean
a b s t r a c t
Albacore (Thunnus alalunga) is a highly migratory cosmopolitan fish commonly distributed throughout all oceans. It is an important commercial species in the world fisheries. In the present study, popula-tion structure of albacore in the Northwestern Pacific Ocean was investigated using mitochondrial DNA sequence data analysis. A total of 175 individuals were sampled from three regions in the Northwest-ern Pacific Ocean (Taiwan, Japan and North of Hawaii) and among them, 168 haplotypes were revealed. Nucleotide diversities and haplotypic diversities were high in all sampling regions. The reconstructed neighbor-joining tree based on the Kimura two-parameter model indicated that two clades of albacore coexisted in the Northwestern Pacific Ocean. Clade I is the main clade consisting of 98% of the total spec-imens and is further divided into two lineages (Lineages I and II). Hierarchical AMOVA tests and pairwise ˚STanalysis showed that albacore tuna in the Northwestern Pacific Ocean constituted a single stock with no significant differences in geographic distributions.
© 2008 Published by Elsevier B.V.
1. Introduction
Albacore (Thunnus alalunga), also known as longfin tuna, is rec-ognized by its remarkably long and slender pectoral fins about 30% of its fork length. It is a highly migratory pelagic fish inhabiting waters at 13.5–25.2◦C of all oceans including the Mediterranean but not the Gulf of Mexico (Collette and Nauen, 1983). Albacore has a high commercial value in world fisheries. In recent years, increasing concerns on the albacore stock status due to worldwide overfishing has been pointed out (Bard, 2001; Uozumi, 2004). Thus, a better understanding of its genetic structure would definitely aid to a more effective fishery management strategy.
In 1995, Chow and Ushiama detected highly significant het-erogeneity between the Pacific and Atlantic albacore but no heterogeneity within each ocean using restriction fragment length polymorphism (RFLP) analysis of the mitochondrial ATPase gene. However, later studies using the same sample lots inferred from microsatellite DNA analysis suggested much higher genetic struc-turing between the North and South stocks of each ocean (Takagi et al., 2001). Furthermore, blood groups and tag-recapture analyses
∗ Corresponding authors. Tel.: +886 2 33662479; fax: +886 2 33662478.
E-mail address:hyhy@ntu.edu.tw(H.-Y. Yang). 1These authors contributed equally to this work.
(Arrizabalaga et al., 2004) suggested no gene flow between these two oceans.
Regarding the Atlantic Ocean and the Mediterranean Sea, mito-chondrial DNA (mtDNA) sequence analysis performed by Vi ˜nas et al. (1999)and allozyme analysis conducted by Pujolar et al. (2003)had detected no genetic differentiation between the two populations. In contrast, genetic differentiation between the North Atlantic and Mediterranean albacores was detected by blood groups and tag-recapture analyses (Arrizabalaga et al., 2004), mtDNA con-trol region sequencing methods (Vi ˜nas et al., 2004; Nakadate et al., 2005), and nuclear glucose-6-phosphate dehydrogenase (G6PD) DNA RFLP analysis (Nakadate et al., 2005). Lastly, regarding the Indian Ocean, both morphometric and mtDNA sequencing analy-ses suggested the Indian Ocean albacore samples to be divided by the 90◦E longitude into two major groups (Yeh et al., 1996) In 2004, blood-group analysis also suggested that the Indian albacore pop-ulation is similar to those of the South Atlantic Ocean (Arrizabalaga et al., 2004).
The inconsistency of the above data may be due to different analysis methods used. Furthermore, albacore genetic structure of the Northwestern Pacific Ocean remains unexplored despite the fact that this area is also an important fishing ground. Prior to the present study, only one molecular sequencing investigation was performed on the Pacific albacore (Vi ˜nas et al., 2004). Over the years, mtDNA sequencing was shown to be one of the more 0165-7836/$ – see front matter © 2008 Published by Elsevier B.V.
effective approaches in predicting population structures among numeral other molecular techniques (Buonnacorsi et al., 2001). In this study, 175 albacore samples were collected from 3 different regions namely Taiwan, Japan and North of Hawaii of the North-western Pacific Ocean. Amplification and sequencing of the mtDNA control region were completed, and subsequent analyses were car-ried out specifically targeting its first hyper-variable region (HVR-1) to access stock structure and phylogenetic information of albacores in these waters.
2. Materials and methods
2.1. Sampling and DNA sequencing
Albacore samples were collected by commercial fishing vessels from three local regions of the Northwestern Pacific Ocean during January 2002 to July 2004: Taiwan (n = 95), Japan (n = 50) and North of Hawaii (n = 30) (Fig. 1andTable 1). Samples of muscle tissue were preserved in 95% ethanol and stored frozen at−20◦C until DNA
extraction.
DNA extraction, amplification and sequencing were performed as previously described (Chiang et al., 2006, 2008). The two primers used in this procedure were: the forward primer, TAF (5-TAC CCC AAA CTC CCA AAG CTA-3) located inside the proline tRNA gene which flanks the 5end of the control region and the reverse primer, TAR (5-GCG GAG GCT TGC ATG TGT A -3) located inside the pheny-lalanine tRNA gene which flanks the 3end of the control region. Amplification conditions are listed as followed: initial denaturation at 94◦C for 5 min, followed by 30 amplification cycles (denaturation at 94◦C for 45 s, annealing at 53◦C for 45 s and extension at 72◦C for 1 min) with a final extension at 72◦C for 5 min. The sequencing primers used were the PCR primers, TAF and TAR.
2.2. Data analyses
In addition to haplotypes derived from this study, several orthol-ogous mtDNA sequences of other species were added to the gene pool as out-groups for analysis. These included: two bluefin tuna (Thunnus thynnus) of Accession Nos. AF390438 and AF390439 (renamed to be NBF02 and NBF01, respectively) and three yellowfin tuna (Thunnus albacares) of Accession Nos. AF301203, AF301206 and AF301207 from GenBank (http://www.ncbi.nlm.nih.gov/), along with four previously published bigeye tuna (Thunnus obesus) mtDNA control region sequences from Chiang et al. (2006).
DNA sequences were aligned by ClustalX, version 1.83 (Thompson et al., 1997), then subsequently optimized by eye (e.g., gap-filling, nucleotide editing) in BIOEDIT, version 7.0.5.3 (Hall, 1999).
The phylogenetic trees were constructed by the Neighbor-joining (NJ) method (Saitou and Nei, 1987) using the Kimura-two-parameter (K2P) model (Kimura, 1980) in MEGA, version 3.1 (Kumar et al., 2004). The statistical robustness in the nodes of the result-ing tree was determined by 1000 bootstrap replicates (Felsenstein, 1985).
Arlequin, version 2.000 (Schneider et al., 2000), DnaSP, version 4.0 (Rozas et al., 2003) and MEGA, version 3.1 (Kumar et al., 2004) were used to calculate statistical values such as the nucleotide com-position, number of polymorphic sites (S), haplotype diversity (Hd; Nei, 1987), nucleotide diversity (;Lynch and Crease, 1990), aver-age number of pairwise nucleotide differences (k;Tajima, 1983) and the expected heterozygosity per site (;Watterson, 1975) were cal-culated for each geographic population. The isolation-by-distance effects on population genetic structure were estimated by pair-wise FST statistics (Wright, 1951, 1965), for which computations
of the correlation between pairwise geographic and genetic dis-tances between populations were statistically analyzed by means of the Mantel test (Mantel, 1967; Smouse et al., 1986). The amounts of genetic variability partitioned within and among populations were accessed by an analysis of molecular variance (AMOVA; Excoffier et al., 1992). Significance of pairwise population com-parison was tested by 20,000 permutations. Organization of the AMOVA tests was in a hierarchical manner and 1000 permuta-tion procedures were used to construct null distribupermuta-tions and to test the significance of variance components (Guo and Thompson, 1992).
The entire mitochondrial control region data set revealed from the phylogenetic analysis was tested against constant population size and sudden population expansion models using the mismatch distribution as implemented in Arlequin, version 2.000 (Schneider et al., 2000) and DnaSP 4.0 (Rozas et al., 2003). The fit between the observed and expected distributions was tested using the Harpend-ing raggedness index (Hri;Harpending, 1994) and sum of squared deviations (SSD) for the estimated stepwise expansion models (Schneider and Excoffier, 1999).
Tajima’s D (Tajima, 1989a,b) and Fu’s Fs (Fu, 1997) tests con-ducted through Arlequin, version 2.000 (Schneider et al., 2000) were carried out to examine for deviations from neutrality (as would be expected under population expansion). Tajima’s test is
Table 1
Descriptive statistics for the studied T. alalunga samples
Population Location Date n H S Hd k
Taiwan 2002 123◦–129◦E; 18◦–22◦N June 2002 30 29 87 0.998± 0.009 23.5± 10.7 0.069± 0.035 23.5± 11.9
Taiwan 2003 124◦–130◦E; 14◦–23◦N April 2003 38 38 84 1.000± 0.006 20.8± 9.4 0.061± 0.031 20.7± 6.4
Taiwan 2004 119◦–140◦E; 14◦–21◦N July 2004 27 26 76 0.997± 0.011 22.1± 10.1 0.065± 0.033 20.5± 6.8 Japan 153◦–175◦E; 27◦–41◦N January 2002 50 47 92 0.997± 0.005 23.6± 10.5 0.069± 0.034 21.0± 6.1 Hawaii 161◦–171◦W; 34◦–43◦N October 2002 30 28 68 0.991± 0.012 20.5± 9.3 0.060± 0.030 17.2± 5.6 N, sample size; H, number of haplotypes; S, number of polymorphic sites; Hd, gene diversity (Nei, 1987); k, mean pairwise nucleotide differences (Tajima, 1983);, nucleotide diversity (Nei, 1987);, expected heterozygosity per site (Watterson, 1975).
widely used as a test for neutrality, and Fu’s Fs test outperforms other tests in detecting population growth for large sample sizes (Ramos-Onsins and Rozas, 2002).
3. Results
3.1. Molecular characteristics
The entire mtDNA control region fragment approximately 860 bp in length immediately flanking the tRNAPro gene was
sequenced in 175 albacores. Analyses of the current study aimed solely at the 366 bp HVR-1 region for sequence comparisons since this fragment is the most highly variable and informative among the whole D-loop segment. In total, 130 variable sites were observed and 168 haplotypes defined. A/T base contents were higher than C/G base contents among the sequences examined (mean: A = 36.5%, T = 29.7%, C = 20.2% and G = 13.7%), consistent with previous studies showing the D-loop to be an A–T rich region of the mitochondrial genome (Brown et al., 1986; Saccone et al., 1987). Among the 130 polymorphic sites observed, 33 were singleton variable sites and 97 were parsimony-informative. Both nucleotide diversities (average = 0.065) and haplotype diversities (average h = 0.998) were high between samples. Regional population genetic statistics are listed inTable 1.
3.2. Phylogeny and patterns of population structure
The reconstructed Neighbor-joining phylogeny tree using the K2P model was presented inFig. 2. Albacore haplotypes from all three sampling regions were grouped into two divergent clades, Clades I and II. Clade I contained most (98%) of the specimens in each of the three sampling regions (Taiwan = 99%, Japan = 94%, North of Hawaii = 100%) and was weakly supported by a boot-strap value of 52%. Two lineages (Lineages I and II) were revealed among the haplotypes of Clade I. Lineage I was supported by a boot-strap value of <50% and lineage II by a value of 54%. On the other hand, Clade II, strongly supported by a bootstrap value of 99%, con-tained a fairly small amount (2%) of specimens, including none from North of Hawaii. Overall, there seemed to be no geographic structuring among haplotypes in Clade I and Clade II. However, one exception was observed in North of Hawaii where no Clade II was found.
Hierarchical AMOVA analysis was first performed using the K2P model for the Taiwan samples taken in three separate years (Table 1) and no structure was revealed (data not shown). Thus, these samples were grouped into one as the Taiwan sam-ple in the present study. Further AMOVA analysis revealed no structure (˚ST=−0.001; p = 0.40) among the entire albacore
col-lections. Moreover, no structure was revealed for either Lineage I (˚ST= 0.006; p = 0.22) or II (˚ST=−0.019; p = 0.98) or for the whole
Clade I population (˚ST=−0.00357; p = 0.58). Pairwise FSTvalues
ranging from −0.0125 to 0.004 with all the p-values over 0.05 showed no significant differentiation between any two of the three sampling regions.
3.3. Demographic patterns
The Harpending’s raggedness index (Hri;Harpending, 1994), sum of squared deviations (SSD) and other demographic param-eters of the expansion model for the entire HVR-1 data set and the phylogroups of albacores were listed inTable 2. Mismatch distri-bution analysis on the entire data set revealed a bimodal shape; one mode corresponded to the number of differences between the two lineages of Clade I, and the other to differences among indi-viduals within both lineages. In addition, a unimodal distribution was revealed by each of the separate analysis of the two lineages (figure not shown). According to the measured sum of squared devi-ation (SSD; p > 0.05), the observed data results were interpreted as of non-significant differences from that predicted by the growth expansion model. Furthermore, the low Harpending’s raggedness index values suggested a significant fit between the observed and the expected distributions, which further supported population expansion. Derived from the diversity index = 2Nf, where Nfis
the effective female population size and is the mutation rate per sequence per generation, the estimated effective female popula-tion size after expansion (1) was about 20 and 50 times higher
than before expansion (0) for Lineages I and II, respectively. Similar
estimated values (Li, 1977) suggested that the population expan-sion in both lineages may date back to about the same historical period.
4. Discussion
Prior to the present study, genetic analyses on the popula-tion structure of albacore were carried out using several different approaches such as morphometric comparisons (Yeh et al., 1996), mtDNA RFLP analysis (Chow and Ushiama, 1995), microsatellite analysis (Takagi et al., 2001), blood group and tag-recapture anal-ysis (Arrizabalaga et al., 2004), nuclear G6PD DNA RFLP analysis (Nakadate et al., 2005) and mtDNA sequence analysis (Yeh et al., 1996; Vi ˜nas et al., 1999, 2004; Nakadate et al., 2005). Among these studies, different methods have lead to different results. For exam-ple, studies on the Pacific albacores using mtDNA ATPase RFLP (Chow and Ushiama, 1995) and microsatellite DNA analyses on four loci (Takagi et al., 2001) have pointed towards opposing con-clusions regarding the north and south populations. Moreover, contradictory results were also obtained regarding albacore popu-lation structure between the Atlantic Ocean and the Mediterranean Sea (Vi ˜nas et al., 1999, 2004; Pujolar et al., 2003; Arrizabalaga et al., 2004; Nakadate et al., 2005). Selection of the most informa-tive analyzing approach is thus essential. In general, among all approaches, PCR sequence analysis was shown to be more effec-tive in predicting population genetics (Buonnacorsi et al., 2001). Regarding the genetic markers for population genetic studies, the HVR-1 region of the mtDNA control region appears to have a muta-tion rate paralleling that of populamuta-tion evolumuta-tion of a species (Page and Holmes, 1998). In particular, for animal species such as alba-core tuna which has a worldwide geographic distribution, complete sampling is usually extremely difficult. Data sets obtained from
Fig. 2. Neighbor-joining tree estimated with the Kimura-2-Parameter (K2P) model among mtDNA lineages of T. alalunga. Haplotypes were collected from the Northwestern Pacific Ocean (Taiwan, Japan and North of Hawaii). Numbers at nodes indicate the bootstrap values. Only values >50% are shown.
Table 2
Statistical tests of neutrality, and demographic parameters estimates for T. alalunga’s entire mitochondrial control region data set, and phylogroups
All samples Clade I Clade I–Lineage I Clade I–Lineage II Clade II
Goodness of fit tests Tajima D −0.25 −0.29 −1.12 −0.95 −0.33 Fu’s FS −23.75* −23.77* −24.28* −24.27* 0.12 Demographic parameters Hri 0.0007 0.0008 0.0015 0.0024 0.4444 SSD 0.0042 0.0071 0.0012 0.0016 0.1975 S 122 127 93 87 15 0 0.016 19.629 3.221 4.203 0.002 1 45.818 461.241 62.305 196.172 4682.500 29.674 9.024 11.773 9.554 10.438
Hri, Harpending raggedness index (Harpending, 1994); SSD, sum of squared deviations (Schneider and Excoffier, 1999);, theta (Rogers, 1995);, tau value (Li, 1977). *Significant values at p < 0.05
DNA sequence analysis can be deposited onto databases such as GenBank and accumulate for later comparisons. Thus, in this study, we investigated phylogenetic relationship and population structure of albacore in the Northwestern Pacific Ocean by sequencing and analyzing the HVR-1 region of the mitochondrial control region.
Samples from all three geographic regions in this study were characterized by high values of haplotype diversity with major-ity of the haplotypes appearing only once, falling into the typical pattern of Scombroid fishes (Zardoya et al., 2004). This is in con-cordance with previous albacore data (Chow and Ushiama, 1995; Vi ˜nas et al., 1999, 2004; Nakadate et al., 2005). High haplotypic diversity within regional populations can be maintained through large effective population sizes, environmental heterogeneity and life-history traits favoring rapid population increase (Nei, 1987). Nucleotide diversity values of the HVR-1 region were also high, corresponding to those reported for other highly migratory pelagic fishes (Alvarado-Bremer et al., 1997, 2005; Grant and Bowen, 1998; Carlsson et al., 2004; Chiang et al., 2006, 2008). Particularly, high genetic diversities within each regional population for marine fish species such as albacore may be best explained by its characteris-tics of large population sizes and its wide distribution throughout the world (reviewed inAvise, 1998; Chiang et al., 2006, 2008).
Differentiation among sub-populations of marine fish species is commonly lower than that of freshwater ones. Particularly, it has been shown that genetic differentiation is generally low among tuna populations within and between oceans (Alvarado-Bremer et al., 1998; Grewe and Hampton, 1998; Vi ˜nas et al., 1999; Chow et al., 2000; Appleyard et al., 2002; Durand et al., 2005; Chiang et al., 2006, 2008). The lack of genetic differentiation within an ocean demonstrated extensive gene flow at intra-oceanic scales. In this study, low FSTvalues indicated low genetic differentiation between
albacore populations from the three Northwestern Pacific regions. Moreover, low and non-significant˚ statistics (˚ST) from
hierar-chical AMOVA also revealed genetic homogeneity among the three regional albacore populations.
In this study, neighbor-joining tree revealed two divergent clades among the three regional albacore populations. Clade I, sup-ported by a weak bootstrap value of 52% contained 98% of the total individuals sampled and was further divided into two lin-eages, lineages I and II, supported by weak bootstrap values of <50% and 54%, respectively. Clade II contained 2% of the total indi-viduals sampled and was supported with a strong bootstrap value of 99%. Previously, two studies on albacore population structure were performed through DNA sequence analysis (Vi ˜nas et al., 2004; Nakadate et al., 2005). The two phylogroups revealed in the data of Vi ˜nas et al. (2004)seemed to correspond to the two Clade I lin-eages of this study. Regarding the data of Nakadate et al. (Fig. 4; 2005), two sequences from each of their Clade I (NEA21A/Accession Nos. AB181140, SWA19C/AB181142) and II (MEDL425-6B/AB181141, MED03-33D/AB181143) were deposited onto GenBank. When these
four sequences were added to our sequence pool, the reconstructed neighbor-joining tree revealed that sequences of their Clades I and II were grouped, respectively, with Lineages II and I of this study (data not shown). It is interesting to note that one sample was grouped outside of their Clade I and II (Fig. 4,Nakadate et al., 2005). This may correspond to the minor Clade II revealed in the present study. Sim-ilar phylogeny pattern of two major mitochondrial control region lineages was also revealed in other closely associated Scombroid fishes such as bigeye tuna (Alvarado-Bremer et al., 1998; Martínez et al., 2005; Chiang et al., 2006, 2008), Atlantic mackerel (Nesbø et al., 2000), swordfish (Alvarado-Bremer et al., 1995, 1996; Rosel and Block, 1996), blue marlin (Finnerty and Block, 1992) and sail-fish (Alvarado-Bremer, 1994; Graves and Mcdowell, 1995). In the present study, both lineages of Clade I contained haplotypes from all three sampling regions and no apparent differences were observed in frequencies of these two lineages between the three regional populations. The same condition was observed regarding Clade II, with no presence of haplotypes from North of Hawaii, which may be simply due to the small sample size. In conclusion, it seemed likely that the Northwestern Pacific albacore population shows no apparent phylogeographic distribution.
In the present study, hierarchical AMOVA revealed low and non-significant˚STfor all combinations, suggesting spatial genetic
homogeneity not only among albacore populations from different localities, but also within each of the two lineages of Clade I from all three sampling regions. This observation, consistent with the non-significant low pairwise FSTvalues, suggested a single stock of
alba-core in the Northwestern Pacific Ocean, given that the existence of two lineages in Clade I is considered. This further indicated possible gene flow between regions within the Northwestern Pacific Ocean. It has been proposed that based on different combinations of haplotype diversity (h) and nucleotide diversity () magnitudes of the mtDNA sequences, marine fishes can be classified into four categories (Grant and Bowen, 1998). In the present study, large values of both h and were observed for the three regional alba-core populations from the Northwestern Pacific Ocean (Table 1), which characterizes the fourth category of marine fishes defined byGrant and Bowen (1998). High level of divergence is usually associated with either a long evolutionary history in a large stable population or with secondary contacts between previously differentiated allopatric lineages (Grant and Bowen, 1998). To decide which explanation best describes the above albacore phylogroups, sequence data were tested by Tajima’s D statistical test. Negative but non-significant Tajima’s D values and significant negative Fu’s Fs values were revealed for both lineages of Clade I, suggesting possible population expansion. Regarding Clade II, positive non-significant Fu’s Fs value suggested that the Clade II population was under equilibrium. Mismatch distribution analysis displayed a unimodal pattern for each of the two lineages of Clade I, suggesting population expansion after genetic isolation (Fu, 1997;
Harpending, 1994). Clade II exhibited a discontinuous multimodal pattern, which prevented us to predict its demographic history. This may be caused by the small amount of haplotypes in Clade II. Further investigations revealed that the demographic expansion of Clade I and its respective lineages was dated about 400,000 years ago (data not shown). An alternative explanation is that the iso-lation of popuiso-lations occurred at the time of popuiso-lation reduction and was followed by the re-establishment of gene flow, since the recovery of populations was slow for lineage I as indicated by the small difference between the estimations of0and1, and much
faster for lineage II. For Clade II, population reduction seemed to be drastic and the recovery was very fast.
At present, the main albacore fisheries in the Pacific Ocean are roughly divided into North and South Pacific stocks (Nakamura, 1969; Kume, 1974; Bartoo and Foreman, 1994; Murray, 1994; Uosaki and Bayliff, 1999) and managed as distinct units by different asso-ciations. In the present study, the demonstration of a panmictic albacore stock throughout the Northwestern Pacific Ocean was supported through the observations of no significant genetic struc-turing, no apparent phylogeographic distribution, and points to homogeneity within this region. Thus, the continuation of the current management strategy within the Northwestern Pacific is recommended.
Acknowledgments
We would like to thank Dr. Chaolun Allen Chen and Mr. Ming-Che Yang for their helpful comments. The funding for this work is provided by National Science Council, Taiwan, ROC (Grant num-bers: NSC 92-2313-B-002-060, NSC 95-2611-M-002-013 and NSC 96-2611-M-002-008).
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