Identification of rockfish (Sebastes spp.) by restriction site analysis of the mitochondrial ND-3/ND-4 and 12S/16S rRNA gene regions.
The species-rich genus of Sebastes rockfish has challenged both fisheries scientists and ichthyologists since they were first described from Alaskan waters by Tilesius (S. ciliatus; 1813, cited in Eschmeyer, 1998) and Richardson (S. caurinus; 1845). Both the large number of species, about 100 worldwide (Ishida, 1984; Kendall, 1991), and the metamorphoses that occur in larval and juvenile fish produce a confusing number of forms. The diversity of species and forms combine to limit our knowledge of the biology, including life histories, of rockfishes. To date, identification to species is not possible for many larvae and juveniles (e.g. Kendall, 1991; Moser, 1996), and distinguishing between some adult species may be difficult. For example, adult S. variegatus is similar to S. zacentrus and adult S. mystinus, S. melanops, and S. ciliatus are often misidentified (Love(1)). The inability to identify species constrains surveys of larval abundance and, consequently, ecological studies that are important for conservation and management of rockfish and other species. In addition, the questions facing biologists and fishery managers require tools that can resolve intraspecific population (stock) structure, as well as methods for identifying species.
The size of the genus and the paucity of information about some of the species have also contributed to a chaotic history of their systematics and many aspects of the phylogeny have not been resolved (Kendall(2) and reviewed in Cramer  and Phillips . Cuvier (1829, cited in Eschmeyer, 1998) first described the genus Sebastes for northern Atlantic specimens. The number of genera recognized for the species presently placed in Sebastes has expanded and contracted repeatedly, reaching a maximum of 15 (Jordan et al., 1930) and now these genera are generally considered subgenera. When combined with five northwestern Pacific Ocean (Matsubara, 1943) and one northern Atlantic Ocean subgenus, Sebastes comprises about 22 subgenera (Kendall, 1991).
Identification and systematics of fish depend largely on morphological characters; morphology alone, however, does not always provide sufficient criteria, especially for identification of larval and juvenile forms. Genetic information, obtained by using biochemical or molecular methods, has been used to address systematic problems. In some instances, genetic differences can be used to differentiate between species that have overlapping morphologies. For example, cryptic species of southern Atlantic Ocean Sebastes species were recognized from mtDNA analysis (Rocha-Olivares et al., 1999a). Genera and many species of rockfish can be distinguished from protein electrophoresis differences (e.g. Tsuyuki et al., 1968; Johnson et al., 1972). More recently, allozyme data (Seeb, 1986) and mtDNA variation (Johns and Avise, 1998; Rocha-Olivares, 1998a; Seeb, 1998; Rocha-Olivares et al. 1999b, 1999c) have been used to address questions about the evolution and systematics of Sebastes. Genetic differences may provide the means for identifying rockfish larvae and juveniles that cannot be identified from their morphology (Seeb and Kendall, 1991). Recently, Rocha-Olivares (1998b) devised a PCR-based approach for identification of Sebastes species. The advantage of his approach is that it is fast. The disadvantage is that failed PCR reactions are part of the identification scheme. However, failed reactions can also result from poor quality DNA or intraspecific variation and lead to misidentification of the specimens. Intraspecific genetic variation can also provide information about population structure (e.g. Wishard et al., 1980; Seeb et al., 1988; Rocha-Olivares and Vetter, 1999).
Vertebrate mitochondrial DNA (mtDNA) is compact (about 16,500 base pairs) and has been completely sequenced in a variety of organisms including carp (Cyprinus carpio; Chang et al., 1994) and rainbow trout (Oncorhynchus mykiss; Zardoya et al., 1995). Because mitochondria are transmitted primarily through maternal genes (Gyllensten et al., 1991), mtDNA is haploid and clonally inherited (Meyer, 1993). Restriction fragment analyses of PCR-amplified regions of mtDNA provide a rapid and practical method for detecting nucleotide sequence variation in mtDNA between individuals or species. Sequence variation detected by restriction enzymes produces binary character-state data that can be used in phylogenetic analyses (e.g. Dowling et al., 1992). An advantage of restriction site surveys over sequencing is that they are practical for detecting variation in large sequence spans. The number of nucleotides screened in restriction site surveys depends on the number of restriction enzymes used and their match with the DNA sequence.
We have developed primers that can be used to PCR amplify regions of Sebastes mtDNA. The amplified regions provide material for addressing species identification and stock identification questions about rockfish. In addition, the haplotypes observed provide information for addressing systematic relationships among Sebastes.
Our objective in this study was to examine the potential that restriction fragment analyses of PCR-amplified mtDNA regions have for the study of rockfish biology. We asked the following specific questions: 1) Is there interspecific haplotype variation? 2) Is there intraspecific haplotype variation? 3) Does intraspecific variability compromise the use of mtDNA restriction fragments in species identification? 4) Can a simple strategy for identifying species be devised? 5) If there is interspecies variation, how do similarities between species correlate with (presumed) systematic relationships? To answer these questions, we conducted restriction site analyses on five individuals from each of 15 different Sebastes species common in Alaskan waters and mapped the sites using double digests to determine individual-based haplotypes. From these data, we examined intra- and inter-specific divergences and used both phenetic and cladistic procedures to examine relationships among the haplotypes. We also mapped the sites for short-spine thornyhead (Sebastolobus alascanus) and Helicolenus hilfendorfi to facilitate analysis. Finally, we developed a mtDNA restriction fragment-based strategy for identifying Sebastes species.
Materials and methods
Adult specimens of 15 different species of Sebastes rockfish and Sebastolobus alascanus were collected from the eastern Gulf of Alaska (Table 1). These species are the most abundant of the approximately 25 species reported in the region. In the field, species identification was confirmed by using the pictoral guide of Kramer and O'Connell (1988) and the key and descriptions in Hart (1973). H. Ida (Kitasato University, Sanriku, Japan) provided samples of Helicolenus hilgendorfi from Japanese coastal waters. Samples of heart tissue from each specimen were preserved in 95% ethanol or a solution of 20% dimethyl sulfoxide (DMSO), 0.25M ethylenediaminetetraacetic acid (EDTA) at pH 8 and saturated with NaCl (Seutin et al., 1991).
Table 1 Rockfish and related species and subgenera of Sebastes spp. used in mitochondrial DNA haplotype comparisons. The number designates the species and the letter indicates the particular composite haplotype observed.
Designation Common name Species Subgenus 1, a and b Pacific ocean Sebastes Acutomentum perch alutus 2, a and b rosethorn Sebastes Sebastomus rockfish helvomaculatus 3 quillback Sebastes maliger Pteropodus rockfish 4, a and b redbanded Sebastes Rosicola rockfish babcocki 5, a and b black rockfish Sebastes Sebastosomus melanops 6 yellowtail Sebastes Sebastosomus rockfish flavidus 7, a-d sharpchin Sebastes Allosebastes rockfish zacentrus 8 harlequin Sebastes Allosebastes rockfish variegatus 9 redstripe Sebastes Allosebastes rockfish proriger 10, a and b rougheye Sebastes Zalopyr rockfish aleutianus 11, a and b yelloweye Sebastes Sebastopyr rockfish ruberrimus 12 shortraker Sebastes Zalopyr rockfish borealis 13 light dusky Sebastes Sebastosomus rockfish ciliatus 14 silvergray Sebastes Acutomentum rockfish brevispinis 15, a and b copper rockfish Sebastes Pteropodus caurinus 16 helicolenus Helicolenus hilgendorfi 17, a-d shortspine Sebastolobus thornyhead alascanus
Total cellular DNA was isolated by phenol-chloroform extraction (Wallace, 1987) or with Puregene DNA[TM] isolation kits (Gentra Systems, Inc., Minneapolis, MN). Two target regions were PCR-amplified from total cellular DNA with primers that we developed for coho salmon (Oncorhynchus kisutch) mtDNA studies. The ND3/ND4 region begins in the glycyl tRNA gene and spans the NADH-dehydrogenase subunit-3, arginyl tRNA, NADH-dehydrogenase subunit-4L, and NADH-dehydrogenase subunit-4 genes, ending in the histidyl tRNA gene. The 12S/16S region extends from near the phenylalanyl tRNA end of the 12S rRNA gene through the valyl tRNA gene to near the leucyl tRNA end of the 16S rRNA gene (Table 2). From restriction digests, we estimated that the ND3/ND4 and 12S/16S regions comprised 2385 and 2430 base pairs (bp), respectively, as compared with 2331 and 2402, respectively, for O. mykiss. Target sequences were amplified by heating to 94 [degrees] C for 5 min, followed by 30 cycles for 1 min at 94 [degrees] C, 1 min at 55 [degrees] C, and 3 min at 72 [degrees] C using Taq polymerase from Perkin Elmer (Norwalk, CT) according to manufacturer's directions. ND3/ND4 amplification required 3mM Mg[Cl.sub.2], whereas amplification of 12S/16S required 2mM Mg[Cl.sub.2].
Table 2 Primers used for polymerase chain reaction amplification of rockfish (Sebastes, Helicolenus, and Sebastolobus spp.) mtDNA regions. (a) = Thomas and Beckenbach (1989); (b) = Cronin et al. (1993); (c) = Gharrett(1); (d) = Anderson et al. (1981); (e) = Anderson et al. (1982); (f) = Roe et al. (1985); (g) = Chang et al., 1994; (h) = Zardoya et al. (1995).
Region Location in amplified Sequence O. mykiss(h) Source ND3/ND4 5' TAACGCGTATAAGT- bp 10574-10596 from a GACTTCCAA 3' (similar to b) 5' TTTTGGTTCCTAAG- bp 12881-12904 from a and c ACCAATGGAT 3' (similar to b) 12S/16S 5' AATTCAGCAGTGAT- bp 1234-1254 consensus: AAACATT 3' d, e, f, g 5' AGATAGAAACTGA- bp 3615-3635 consensus: CCTGGATT 3' d, e, f, g
(1) Gharrett, A. J. 2000. Unpubl. Oncorhynchus kisutch sequences. Fisheries Division, Univ. Alaska, Fairbanks, 11120 Glacier Hwy., Juneau, AK 99801.
Single digests of subsamples of the PCR-amplified mtDNA regions were made by using 10 restriction endonucleases. BstU I, Cfo I, Dde I, Hinf I, Mbo I, Msp I, and Rsa I have 4-nucleotide recognition sites; BstN I recognizes an ambiguous 5-nucleotide site; and Hind II and Sty I recognize ambiguous 6-nucleotide sites. Digestions were carried out under conditions recommended by the manufacturers. Fragments were separated by electrophoresis through 1.5% agarose (a mixture composed of one part Ultra Pure[TM] agarose [BRL Gibco, Grand Island, NY] and two parts Synergel[TM] [Diversified Biotech Inc., Boston, MA]) in 0.5xTBE buffer (TBE is 90 mM tris-boric acid, and 2 mM EDTA, pH 7.5). DNA in the gel was stained with ethidium bromide and photographed on an ultraviolet light transilluminator. Digests that produced small unresolvable fragments on agarose gels were subjected to electrophoresis on 8% polyacrylamide gels (29:1 acrylamide:bisacrylamide) in 2xTAE (TAE is 40 mM tris-acetic acid and 1 mM EDTA, pH 8.0). DNA in polyacrylamide was stained with SYBR Green 1 Nucleic Acid Stain[TM] (Molecular Probes, Eugene, OR). Molecular weight markers used to estimate restriction fragment sizes were 100 base pair (bp) or 25-bp ladders (BRL Gibco, Grand Island, NY). Restriction sites were mapped by using double digests. Double digests were examined both in agarose and polyacrylamide by using 100- and 25-bp ladders. Composite haplotypes for all 10 restriction enzymes and both mtDNA regions were determined for each individual.
Generalized (relaxed Dollo) parsimony trees (Swofford et al., 1996) were computed from shared restriction sites by a heuristic search with PAUP* 4.0 (Swofford, 1998), which assumed unordered states. Because the likelihood of the loss of a site is higher than the restoration of a lost site, we conducted analyses that assumed 1) no added cost, 2) twice the cost, and 3) four-times the cost for restoring a site. Multiple maximum parsimony trees from each analysis were combined to produce a majority consensus tree using PAUP* 4.0 (Swofford, 1998). A maximum-likelihood tree was estimated with the program RESTML in PHYLIP 3.57c (Felsenstein(3)), assuming that all restriction sites were 4 bp long (PHYLIP, Felsenstein(3)). Nucleotide divergences (proportion of nucleotide substitutions) and their standard errors were estimated according to Nei and Tajima (1983), Nei (1987), and Nei and Miller (1990) by using REAP (McElroy et al., 1990).
Restriction fragment patterns from double digests were used to construct restriction site maps for comparisons of species and detection of intraspecies variation (Appendix 1). The map includes 153 restriction sites, 36 of which were common in all haplotypes and 28 of which were cladistically uninformative because the presence or absence occurs only in a single haplotype. Many of the cladistically uninformative sites, however, were useful in species delineation. These data represent 153 restriction sites (79.3 on average) corresponding to 640 nucleotides (332.05 on average) per haplotype.
Among the 85 fish examined were 30 different composite haplotypes (Table 3); each species had haplotypes that were distinct from those of other species, although S. variegatus composite haplotype 8 differed at a single site from S. zacentrus composite haplotype 7c (Table 4). All other pairs of species differed by 5 or more sites. Intraspecific variation was observed in nine of the seventeen species even when only five specimens of each species were analyzed. The most variable species were S. zacentrus and Sebastolobus alascanus, each of which had four haplotypes. In the study, differences between haplotypes ranged from a single site difference or 0.0014 nucleotide substitutions per site to 65 restriction site differences and 0.120 nucleotide substitutions per site (Table 4). Nucleotide divergence within variable species averaged 0.0024 subsitutions (1.56 site changes), whereas divergences between Sebastes species averaged ten-fold higher, 0.0249 (15.4 site changes), ranging from 0.0015 (1 site change) to 0.0384 (25 site changes). Nucleotide divergences between Sebastes species and Sebastolobus alascanus averaged 0.1073 (59.2 site changes) and divergences between Sebastes species and H. hilgendorfi averaged 0.0805 (43.5 site changes).
[TABULAR DATA 3-4 NOT REPRODUCIBLE IN ASCII]
Distribution of the variation between the two different mtDNA regions (ND3/ND4 and 12S/16S) reflects their rates of evolution. In the 12S/16S region, which is more conservative, 27 of 58 restriction sites were shared by all haplotypes. Nucleotide diversities between Sebastes species averaged 0.0094 nucleotide changes per nucleotide (a total of 3.29 sites differences in the region), divergences between Sebastes and H. hilgendorfi averaged 0.0641 (12.67 site differences), and divergences between Sebastes and Sebastolobus alascanus averaged 0.0561 (18.03 site differences). In contrast, in the ND3/ND4 region only 9 of 95 sites were common to all haplotypes; and nucleotide divergences between Sebastes species averaged 0.0471 (12.11 site differences) and divergences between Sebastes and H. hilgendorfi and between Sebastes and Sebastolobus alascanus averaged 0.1373 (31.75 site differences) and 0.1929 (40.93 site differences), respectively. The maximum likelihood and majority consensus tree for the 60 maximum parsimony trees that imposed a cost of two for regained restriction sites had identical topologies (Fig. 1). The topologies of parsimony trees, which had either no additional cost or a cost of four, were somewhat different. Several groups of species were present in all three parsimony topologies. The S. zacentrus-S, variegatus pair, mentioned above, and each of four species pairs--S, melanops-S, flavidus, S. babcocki-S, helvomaculatus, S. proriger-S, brevispinis, and S. maliger-S, caurinus--clustered tightly at subterminal nodes. A more interior cluster of species included S. melanops, S. flavidus, S. babcocki, and S. helvomaculatus. In addition, S. maliger and S. caurinus clustered separately from all other Sebastes species and the Sebastes species were distinct from H. hilgendorfi and Sebastolobus alascanus.
[Figure 1 ILLUSTRATION OMITTED]
The mtDNA variation we observed among Sebastes species provides a tool for identifying species. From our data, numerous schemes could be devised that distinguish among the Sebastes species examined. We propose a simple scheme that minimizes the number of digests required and involves separation of restriction fragments from the ND3/ND4 PCR product on an agarose-Synergel[TM] gel using only four restriction enzymes. Mbo I digests produce 11 different haplotypes (haplotypes A-K; Figure 2A; Table 3); S. alutus (B), S. melanops (E), S. babcocki (G and H), S. ruberrimus (I), and S. caurinus (J) are species specific. If Mbo I haplotypes A (S. helvomaculatus or S. flavidus) or C (S. maliger or S. caurinus) are observed, digest the ND3/ND4 PCR product with Hind II; Hind II haplotype B is specific for S. helvomaculatus and Hind II haplotype C is specific for S. maliger (Fig. 2B; Table 3). If Mbo I haplotypes F (S. ciliatus or S. borealis) or K (S. aleutianus, S. proriger, or S. brevispinis) are observed, digest the ND3/ND4 PCR product with BstN I; BstN I haplotype A is specific for S. ciliatus and BstN I haplotype G is specific for S. brevispinis (Fig. 2C; Table 3). Mbo I and BstN I haplotypes do not distinguish between S. aleutianus and S. proriger, but Cfo I haplotype B is specific for S. aleutianus (Fig. 2D, Table 3). The combined haplotype of Mbo I, Hind II, BstN I, and Cfo I can be used to identify S. borealis (KAFD) and S. proriger (FAFD) (Fig. 2). The single difference between S. zacentrus and S. variegatus is the presence of a 123-bp fragment in Rsa I digests of S. zacentrus (Table 2; Appendix 1).
[Figure 2 ILLUSTRATION OMITTED]
This simple scheme takes advantage of unique single-site differences for several of the species. Although a neighbor-joining tree (Saitoh and Nei, 1987) appeared stable to intraspecific variation for increased sample sizes of three species (data not shown), a single site change that produces apparent convergence between taxa in our scheme is conceivable. Increased certainty can be achieved by conducting digests with all four enzymes. With this strategy there will be at least two site differences between every pair of species, except S. proriger and S. brevispinis, which can be resolved by using Msp I, and S. zacentrus and S. variegatus (see above). We do not recommend using Dde I because it has many sites, often produces small fragments requiring both agarose and polyacrylamide gels for resolution, and is, therefore, time consuming to analyze. However, the restriction patterns of Dde I are nearly species specific.
Sufficient interspecies restriction site variation occurred in the ND3/ND4 and 12S/16S mtDNA regions in Sebastolobus alascanus, Helicolenus hilgendorfi, and 15 Sebastes species to distinguish among them. Intraspecific variation was observed in nine of the seventeen species, but it did not interfere with our ability to distinguish between species. We used the interspecific variation to devise a strategy to identify the species we studied. Intraspecific variability can serve as a basis for stock identification.
A broader survey, particularly for S. zacentrus and S. variegatus, might reveal overlaps in haplotype compositions that compromise the ability to distinguish between some species pairs. This would be most likely if there were gene flow between the species or if the species had recently diverged. Otherwise, extending the analysis to other mtDNA regions and additional restriction endonucleases should increase resolution. Of course, additional intraspecific variation has the potential to obscure the topology of trees. To test this possibility, we examined trees that included the additional haplotypes observed in samples of 40 to 126 individuals each from S. caurinus (n=79), S. aleutianus (n=126), and S. borealis (n=40) (data not shown). The additional haplotypes (5, 13, and 5, respectively) increased the number of branches at the tip of the species limbs but did not influence or obscure relationships with other species. We are currently investigating the population structure of S. aleutianus, S. borealis, S. alutus, S. caurinus, and Sebastolobus alascanus by using mtDNA restriction site variation.
Because of the similarity of many Sebastes species, there is a chance that very similar species can be misidentified. In fact, a young dusky rockfish (S. ciliatus) and a young yellowtail rockfish (S. flavidus) were misidentified in the field as black rockfish (S. melanops) prior to our mtDNA analysis. Also, it is possible that closely related species may hybridize (e.g. Seeb, 1998). Because hybrids carry only the maternal lineage and because only the maternal contributor can be identified, mtDNA analysis is a poor tool for identifying hybrids.
In addition to providing a tool that can distinguish among a variety of rockfish species, the data appear to provide criteria that may prove useful in unraveling some questions about rockfish systematics. Both outgroups are distinct from Sebastes; H. hilgendorfi is more closely related than Sebastolobus alascanus. The 15 Sebastes species studied include eight subgenera, five of which were represented by two or more species. Despite the uncertainty in some of the subgenus assignments,(2) our analyses of mtDNA restriction sites show some concordance with subgeneric assignments. Unfortunately, the only recently reviewed subgenus is Sebastomus (Chen, 1971), for which we have only a single representative (S. helvomaculatus). A phylogeny of subgenera is unavailable.
Several species pairs were persistent in the analyses. Within Sebastes, S. maliger and S. caurinus (subgenus Pteropodus) were distinct from the other Sebastes species. None of the other subgenera were as coherent. The haplotypes of S. zacentrus and S. variegatus (subgenus Allosebastes) were very similar and the haplotype of a third member, S. proriger, generally clustered nearby. Similarly, the haplotypes of S. maliger and S. flavidus (subgenus Sebastosomus) were tightly clustered, but the branch for the haplotype of the third member, S. ciliatus, was distal; and different tree construction methods inconsistently placed S. ciliatus on the tree (not shown). Haplotypes of S. aleutianus and S. borealis (subgenus Zalopyr) were found in the same general region of the tree, but are not sister taxa. Likewise, the two representatives of Acutomentum, S. alutus and S. brevispinis, were not monophyletic sister taxa. Disparities, such as we observed between relationships of haplotype and assignments of subgenera, have also been reported for allozyme comparisons (Seeb, 1986) and mtDNA cytochrome b sequences (Johns and Avise, 1998; Rocha-Olivares, 1998a; Rocha-Olivares et al., 1999a, 1999b). The members of subgenera Acutomentum and Allosebastes, in particular, seem discordant with trees. It is important to recall that the systematics is not unequivocal and controversies date back more than a century (e.g. Cramer, 1895). Therefore, discrepancies between the molecular-based comparisons and current systematic placements do not necessarily discredit the validity of the molecular comparisons.
Use of restriction site data in mtDNA holds promise for the identification and systematics of Sebastes and suggests the possibility of applications for stock identification. Larval and juvenile rockfish carry mtDNA that is adequate for PCR amplification (e.g. see Seeb and Kendall, 1991; Rocha-Olivares 1998b). Combining molecular identification with morphometry may solve many of the problems of identification that accompany rockfish studies. The apparent coherence of closely related rockfish species that we observed in both cladistic and phenetic analyses suggests that we should focus our applications on groups of species that are presumed to be close relatives. The consensus tree depicting relationships among interior clades within the Sebastes parsimony tree did not unequivocally position those clades either in this study or analyses of the cytochrome b region (Johns and Avise, 1998; Rocha-Olivares, 2000). Consequently, determination of higher level relationships among Sebastes requires analysis of additional mtDNA regions. Moreover, because the divergence of mtDNA sequences provides only one perspective of the evolution of Sebastes divergence, the relationships inferred by mtDNA analyses must be corroborated by analysis of the interspecific divergence of nuclear genes.
We gratefully acknowledge the many crew members and scientists aboard the research vessels John N. Cobb and Miller Freeman who participated in collecting specimens for our study. L. Densmore and T. Dowling provided constructive comments on early drafts of this manuscript. Three anonymous reviewers provided constructive comments. A. W. Kendall Jr. and M. S. Love contributed advice and insight that helped us develop this paper. J. A. Gharrett and D. Churikov reviewed drafts of this paper. This work was supported by the National Marine Fisheries Service Auke Bay Laboratory and the U.S. Geological Services (Biological Resources Division) Western Regional Office in Seattle, WA (R.W.O. 32).
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Manuscript accepted 22 August 2000.
[APPENDIX 1 NOT REPRODUCIBLE IN ASCII]
Anthony J. Gharrett Andrew K. Gray Fisheries Division, School of Fisheries and Ocean Sciences University of Alaska Fairbanks 11120 Glacier Highway Juneau, Alaska 99801 E-mail address (for A. J. Gharrett): email@example.com
Jonathan Heifetz Auke Bay Laboratory Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 11305 Glacier Highway Juneau, Alaska 99801-8626
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|Author:||Gharrett, Anthony J.; Gray, Andrew K.; Heifetz, Jonathan|
|Article Type:||Statistical Data Included|
|Date:||Jan 1, 2001|
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