Are the Subgenera of Sebastes Monophyletic?

We examined genetic relationships among Sebastes rockfishes to evaluate the subgeneric relationships within Sebastes. We analyzed restriction site variation (12S and 16S rRNA and NADH dehydrogenase-3 and -4 genes) by using parsimony and distance analyses. Seventy-one Sebastes species representing 16 subgenera were included. Thirteen subgenera were represented by more than one species, and three subgenera were monotypic. We also evaluated three currently unassigned species. The only monophyletic subgenus was Sebastomus, although some consistent groups were formed by species from different subgenera. The northeastern Pacific species of Pteropodus clustered with one northeastern Pacific species of the subgenus Mebarus (S. atrovirens) and two northeastern Pacific species of the subgenus Auctospina (S. auriculatus and S. dalli) forming a monophyletic group distinct from northwestern Pacific 1Present address: National Marine Fisheries Service, Auke Bay Laboratory, 11305 Glacier Highway, Juneau, Alaska 99801 186 Li et al.—Are the Subgenera of Sebastes Monophyletic? Pteropodus species. The subgenera Acutomentum and Allosebastes were polyphyletic, although subsets of each were monophyletic. Sebastes polyspinis and S. reedi, which have not yet been assigned to subgenera, are closely related to two other northern species, S. crameri (subgenus Eosebastes) and the S. ciliatus/variabilis complex (subgenus Sebastosomus), which differed from other species assigned to their subgenera. These and other molecular studies show promise in determining the phylogenetic relationships among Sebastes species. Introduction The genus Sebastes is a species-rich and ecologically diverse group. Currently about 100 species are recognized; members of this genus are currently assigned to 21 subgenera, including six that are monotypic (Kendall 2000). Historically, morphological, meristic, and morphometric characters have been used for species identification and subgeneric determination. Abundant variation in many characters has provided means to separate most morphologically similar species. However, some characters that are subjective and possibly adaptive, are inappropriate for cladistic analysis (Li et al. 2006a). Consequently, the current subgeneric groupings need to be reevaluated using alternative methods that consider characters more suitable for cladistic analysis. Molecular methods have been used to test the validity of some subgenera of Sebastes. Comparison of sequences of a mitochondrial cytochrome b gene led to the conclusion that the subgenus Sebastomus as defined by Chen (1971) was monophyletic (Rocha-Olivares et al. 1999). All members of a monophyletic group descend from a common ancestor, i.e., they are part of the same genetic lineage. Relationships among species of another subgenus, Pteropodus, have also been evaluated (Kai et al. 2003, Li et al. 2006a). Our analysis of restriction site variation in the NADH-dehydrogenase subunit -3 and -4 genes (ND3/ND4 region) and the 12S and 16S rRNA genes (12S/16S region) of the mitochondrial DNA (mtDNA) showed that the northeastern Pacific members of Pteropodus, along with three northeastern Pacific species from two other subgenera, form a monophyletic group (Li et al. 2006a). However, the northwestern Pacific members of Pteropodus were distinct from the northeastern Pacific Pteropodus species and need to be evaluated further and reassigned (Li et al. 2006a). In this paper, we extended our evaluation of Sebastes subgenera validity to 71 species and 16 subgenera. We used the subgeneric assignments summarized in Kendall (2000), except for S. gilli, which is unassigned (A.W. Kendall, pers. comm.). In addition, we examined the extent to which intraspecific variation interferes with phylogenetic determination by surveying variation in large samples of individuals from five of 187 Biology, Assessment, and Management of North Pacific Rockfishes the 71 species. Restriction site variation was used to generate maximum parsimony and neighbor-joining trees, and to estimate the extent of nucleotide divergences within and among subgenera. Materials and methods Species studied Seventy-one species from 16 Sebastes subgenera were examined (Table 1). Sebastolobus alascanus and Helicolenus hilgendorfi were included as outgroup species. The northeastern Pacific Sebastes species and Sebastolobus alascanus were captured in the Gulf of Alaska and along the coast of California. The northwestern Pacific Sebastes species and H. hilgendorfi were collected along the coasts of Japan. Generally, five individuals were used to represent each species. In several instances, fewer individuals were used because of limited availability. Many additional individuals of S. alutus, S. aleutianus, S. borealis, S. caurinus, and the S. carnatus/S. chrysomelas complex were included to evaluate the influence of intraspecific variation on the analysis. DNA amplification A sample of heart tissue of each specimen was preserved in either 95% ethanol or a DNA preservation solution (Seutin et al. 1991). Total genomic DNA was extracted using PuregeneTM DNA isolation kits (Gentra Systems Inc., Minneapolis). The mitochondrial ND3/ND4 and 12S/16S regions were amplified by polymerase chain reaction (Gharrett et al. 2001, Li et al. 2006b). Restriction site analysis A restriction site map was developed for both the ND3/ND4 and 12S/16S regions for endonucleases: BstN I, BstU I, Cfo I, Dde I, Hind II, Hinf I, Mbo I, Msp I, Rsa I, and Sty I. All restriction sites were mapped from fragment sizes observed in double digests (Li et al. 2006b). We attempted to find species-specific variation to separate S. emphaeus, S. variegatus, S. wilsoni, and S. zacentrus by identifying restriction site differences in published cytochrome b sequences (Rocha-Olivares et al. 1999). Although we expected to observe species-specific restriction digest patterns, the digests failed to delineate the species, probably because the nucleotide differences to which restriction enzymes were applied are the result of intraspecific variation shared by the four species. We also examined variation in the NADH-dehydrogenase subunit-5 and -6 genes for these four species. Although additional intraspecific variation was observed, there was little useful interspecific variation. 188 Li et al.—Are the Subgenera of Sebastes Monophyletic? aGenus Sebastes unless otherwise noted. Table 1. Names, abbreviation, subgenus assignments, and range of species included. Common name Speciesa Abbr. Subgenus Range Rougheye rockfish aleutianus ale Zalopyr NE-NW Pacific Pacific ocean perch alutus alu Acutomentum NE-NW Pacific Kelp rockfish atrovirens atr Mebarus NE Pacific Brown rockfish auriculatus ari Auctospina NE Pacific Aurora rockfish aurora aro Eosebastes NE Pacific Redbanded rockfish babcocki bab Rosicola NE Pacific Shortraker rockfish borealis bor Zalopyr NE-NW Pacific Silvergray rockfish brevispinis bre Acutomentum NE Pacific False jacopever capensis cap Sebastomus NE Pacific/ S. Hemisphere Gopher rockfish carnatus car Pteropodus NE Pacific Copper rockfish caurinus cau Pteropodus NE Pacific Greenspotted rockfish chlorosticus dhl Sebastomus NE Pacific Black-and-yellow rockfish chrysomelas chr Pteropodus NE Pacific Dusky rockfish ciliatus/ variabilis cil Sebastosomus NE Pacific Starry rockfish constellatus con Sebastomus NE Pacific Darkblotched rockfish crameri cra Eosebastes NE Pacific Calico rockfish dalli dal Auctospina NE Pacific Splitnose rockfish diploproa dip Allosebastes NE Pacific Greenstriped rockfish elongatus elo Hispaniscus NE Pacific Puget Sound rockfish emphaeus emp Allosebastes NE Pacific Swordspine rockfish ensifer ens Sebastomus NE Pacific Widow rockfish entomelas ent Acutomentum NE Pacific Pink rockfish eos eos Sebastomus NE Pacific Gulf rockfish exsul exs Sebastomus NE Pacific Yellowtail rockfish flavidus fla Sebastosomus NE Pacific Bronzespotted rockfish gilli gil ? NE Pacific Chilipepper goodei goo Sebastodes NE Pacific Rosethorn rockfish helvomaculatus hel Sebastomus NE Pacific Squarespot rockfish hopkinsi hop Acutomentum NE Pacific Yoroi-mebaru hubbsi hub Pteropodus NW Pacific Mebaru inermis ine Mebarus NW Pacific Shortbelly rockfish jordani jor Sebastodes NE Pacific Togotto-mebaru joyneri joy Mebarus NW Pacific Freckled rockfish lentiginosus Len Sebastomus NE Pacific Cowcod levis Lev Hispaniscus NE Pacific Mexican rockfish macdonaldi mac Acutomentum NE Pacific Table 1. (Continued.) Common name Speciesa Abbr. Subgenus Range Quillback rockfish maliger mal Pteropodus NE Pacific Black rockfish melanops mep Sebastosomus NE Pacific Blackgill rockfish melanostomus mes Eosebastes NE Pacific Vermillion rockfish miniatus min Rosicola NE Pacific Blue rockfish mystinus mys Sebastosomus NE Pacific China rockfish nebulosus neb Pteropodus NE Pacific Tiger rockfish nigrocinctus nig Sebastichthys NE Pacific Goma-soi nivosus niv Pteropodus NW Pacific Speckled rockfish ovalis ova Acutomentum NE Pacific Bocaccio paucispinis pau Sebastodes NE Pacific Chameleon rockfish phillipsi phi ? NE Pacific Canary rockfish pinniger pin Rosicola NE Pacific Northern rockfish polyspinis pol ? NE Pacific Redstripe rockfish proriger pro Allosebastes NE Pacific Grass rockfish rastrelliger ras Pteropodus NE Pacific Yellowmouth rockfish reedi ree ? NE Pacific Rosy rockfish rosaceus rsa Sebastomus NE Pacific Greenblotched rockfish rosenblatti rsb Sebastomus NE Pacific Yelloweye rockfish ruberrimus rbr Sebastopyr NE Pacific Flag rockfish rubrivinctus rbv Hispaniscus NE Pacific Bank rockfish rufus ruf Acutomentum NE Pacific Stripetail rockfish saxicola sax Allosebastes NE Pacific Halfbanded rockfish semicinctus sem Allosebastes NE Pacific Olive rockfish serranoides srd Sebastomus NE Pacific Treefish serriceps srp Sebastocarus NE Pacific Pinkrose rockfish simulator sim Sebastomus NE Pacific Spiny-eye rockfish spinorbis spi Sebastomus NE Pacific Ezo-mebaru taczanowski tac Mebarus NW Pacific Usu-mebaru thompsoni tho Mebarus NW Pacific Shima-zoi trivitattus tri Pteropodus NW Pacific Honeycomb rockfish umbrosus umv Sebastomus NE Pacific Harlequin rockfish variegatus var Allosebastes NE Pacific Kitsune-mebaru vulpes vul Neohispaniscus NW Pacific Pygmy rockfish wilsoni wil Allosebastes NE Pacific Sharpchin rockfish zacentrus zac Allosebastes NE Pacific Helicolenus Helicolenus hilgendorfi Hh NW Pacific Shortspine thornyhead Sebastolobus alascanus Sa NE Pacific 189 Biology, Assessment, and Management of North Pacific Rockfishes aGenus Sebastes unless otherwise noted. Table 1. Names, abbreviation, subgenus assignments, and range of species included. Common name Speciesa Abbr. Subgenus Range Rougheye rockfish aleutianus ale Zalopyr NE-NW Pacific Pacific ocean perch alutus alu Acutomentum NE-NW Pacific Kelp rockfish atrovirens atr Mebarus NE Pacific Brown rockfish auriculatus ari Auctospina NE Pacific Aurora rockfish aurora aro Eosebastes NE Pacific Redbanded rockfish babcocki bab Rosicola NE Pacific Shortraker rockfish borealis bor Zalopyr NE-NW Pacific Silvergray rockfish brevispinis bre Acutomentum NE Pacific False jacopever capensis cap Sebastomus NE Pacific/ S. Hemisphere Gopher rockfish carnatus car Pteropodus NE Pacific Copper rockfish caurinus cau Pteropodus NE Pacific Greenspotted rockfish chlorosticus dhl Sebastomus NE Pacific Black-and-yellow rockfish chrysomelas chr Pteropodus NE Pacific Dusky rockfish ciliatus/ variabilis cil Sebastosomus NE Pacific Starry rockfish constellatus con Sebastomus NE Pacific Darkblotched rockfish crameri cra Eosebastes NE Pacific Calico rockfish dalli dal Auctospina NE Pacific Splitnose rockfish diploproa dip Allosebastes NE Pacific Greenstriped rockfish elongatus elo Hispaniscus NE Pacific Puget Sound rockfish emphaeus emp Allosebastes NE Pacific Swordspine rockfish ensifer ens Sebastomus NE Pacific Widow rockfish entomelas ent Acutomentum NE Pacific Pink rockfish eos eos Sebastomus NE Pacific Gulf rockfish exsul exs Sebastomus NE Pacific Yellowtail rockfish flavidus fla Sebastosomus NE Pacific Bronzespotted rockfish gilli gil ? NE Pacific Chilipepper goodei goo Sebastodes NE Pacific Rosethorn rockfish helvomaculatus hel Sebastomus NE Pacific Squarespot rockfish hopkinsi hop Acutomentum NE Pacific Yoroi-mebaru hubbsi hub Pteropodus NW Pacific Mebaru inermis ine Mebarus NW Pacific Shortbelly rockfish jordani jor Sebastodes NE Pacific Togotto-mebaru joyneri joy Mebarus NW Pacific Freckled rockfish lentiginosus Len Sebastomus NE Pacific Cowcod levis Lev Hispaniscus NE Pacific Mexican rockfish macdonaldi mac Acutomentum NE Pacific Table 1. (Continued.) Common name Speciesa Abbr. Subgenus Range Quillback rockfish maliger mal Pteropodus NE Pacific Black rockfish melanops mep Sebastosomus NE Pacific Blackgill rockfish melanostomus mes Eosebastes NE Pacific Vermillion rockfish miniatus min Rosicola NE Pacific Blue rockfish mystinus mys Sebastosomus NE Pacific China rockfish nebulosus neb Pteropodus NE Pacific Tiger rockfish nigrocinctus nig Sebastichthys NE Pacific Goma-soi nivosus niv Pteropodus NW Pacific Speckled rockfish ovalis ova Acutomentum NE Pacific Bocaccio paucispinis pau Sebastodes NE Pacific Chameleon rockfish phillipsi phi ? NE Pacific Canary rockfish pinniger pin Rosicola NE Pacific Northern rockfish polyspinis pol ? NE Pacific Redstripe rockfish proriger pro Allosebastes NE Pacific Grass rockfish rastrelliger ras Pteropodus NE Pacific Yellowmouth rockfish reedi ree ? NE Pacific Rosy rockfish rosaceus rsa Sebastomus NE Pacific Greenblotched rockfish rosenblatti rsb Sebastomus NE Pacific Yelloweye rockfish ruberrimus rbr Sebastopyr NE Pacific Flag rockfish rubrivinctus rbv Hispaniscus NE Pacific Bank rockfish rufus ruf Acutomentum NE Pacific Stripetail rockfish saxicola sax Allosebastes NE Pacific Halfbanded rockfish semicinctus sem Allosebastes NE Pacific Olive rockfish serranoides srd Sebastomus NE Pacific Treefish serriceps srp Sebastocarus NE Pacific Pinkrose rockfish simulator sim Sebastomus NE Pacific Spiny-eye rockfish spinorbis spi Sebastomus NE Pacific Ezo-mebaru taczanowski tac Mebarus NW Pacific Usu-mebaru thompsoni tho Mebarus NW Pacific Shima-zoi trivitattus tri Pteropodus NW Pacific Honeycomb rockfish umbrosus umv Sebastomus NE Pacific Harlequin rockfish variegatus var Allosebastes NE Pacific Kitsune-mebaru vulpes vul Neohispaniscus NW Pacific Pygmy rockfish wilsoni wil Allosebastes NE Pacific Sharpchin rockfish zacentrus zac Allosebastes NE Pacific Helicolenus Helicolenus hilgendorfi Hh NW Pacific Shortspine thornyhead Sebastolobus alascanus Sa NE Pacific 190 Li et al.—Are the Subgenera of Sebastes Monophyletic? Phylogenetic analysis The rate of nucleotide substitution per nucleotide (dr) was calculated for all pairs of haplotypes following Nei and Tajima (1981) and Nei and Miller (1990, eq. 4) using REAP (McElroy et al. 1990). Nucleotide substitutions per nucleotide were calculated for all pairs of Sebastes spp., between Sebastes spp. and Helicolenus hilgendorfi, between Sebastes spp. and Sebastolobus alascanus, and between H. hilgendorfi and Sebastolobus alascanus. Pairwise restriction site differences were calculated using Arlequin 2.0 (Schneider et al. 2000). One hundred neighbor-joining trees (Saitou and Nei 1987) using PHYLIP 3.57c (Felsenstein 1993) were estimated by using randomized orders of the taxa. Maximum parsimony analyses were performed using heuristic searches with PAUP 4.0b10 (Swofford 1998). Because the likelihood of the loss of a site is higher than the gain of a site, three character-weighting schemes were used. The weight of gaining a site was analyzed as (1) equal to that of losing a site, (2) twice that of losing a site, and (3) four times that of losing a site. The following search parameters were used: exclude uninformative characters, retain minimal tree from each replicate, collapse zerolength branches, tree-bisection-reconnection branch swapping in effect, steepest descent not enforced, and save all optimal trees. One hundred replicates were performed for each of the three weighting schemes, and the multiple maximum parsimony trees generated for each scheme were combined to produce a 50% majority consensus tree. Results Restriction site analysis A total of 215 restriction sites were detected in the ND3/ND4 and 12S/16S regions (Li et al. 2006b). The faster evolving ND3/ND4 region had 141 sites, and 74 sites were in the more conserved 12S/16S region. Of the total 215 sites, 97 were unique to Sebastes species, 21 were unique to Sebastolobus alascanus, seven were unique to Helicolenus hilgendorfi, and one was shared only by Sebastolobus alascanus and H. hilgendorfi. Site differences in the two mtDNA regions yielded 132 composite haplotypes (Li et al. 2006b). Individuals of the Sebastes species had 127 haplotypes, Sebastolobus alascanus had four haplotypes, and H. hilgendorfi had a single haplotype. Thirty-four of the 71 species displayed intraspecific variation and were represented by more than one composite haplotype. In several instances, haplotypes were identical among species. These were (1) a variant of S. carnatus and a variant of S. chrysomelas were identical; (2) S. chlorostictus, S. eos, and S. rosenblatti shared a haplotype; (3) haplotypes of S. ciliatus/variabilis, S. crameri, and a vari191 Biology, Assessment, and Management of North Pacific Rockfishes ant of S. polyspinis were the same; and (4) S. emphaeus, a variant of S. variegatus, and a variant of S. wilsoni were the same. Also, haplotypes of several species differed by a single restriction site. Single site differences were observed in four instances: (1) between S. entomelas and S. mystinus; (2) between S. hopkinsi and S. ovalis; (3) between S. zacentrus and the S. emphaeus/S. variegatus/S. wilsoni complex; and (4) between S. reedi and the S. ciliatus/variabilis/S. crameri/S. polyspinis complex. Differences in restriction sites between haplotypes ranged from 0 between some pairs of species (e.g., S. carnatus and S. chrysomelas) to 71 sites between Sebastolobus alascanus and S. inermis, and Sebastolobus alascanus and S. joyneri. Nucleotide divergence within variable Sebastes species averaged 0.0027 substitutions per nucleotide. Nucleotide divergence between Sebastes species averaged 0.0285 substitutions per nucleotide, ranging from 0 (as mentioned above for several pairs of species) to 0.0664 per nucleotide between S. hubbsi and S. jordani. Nucleotide divergence between Sebastes species and H. hilgendorfi averaged 0.0767 substitutions per nucleotide. Nucleotide divergence between Sebastes species and Sebastolobus alascanus averaged 0.1047 substitutions per nucleotide, and the largest was observed between S. hubbsi and Sebastolobus alascanus, at 0.1226 substitutions per nucleotide. The average nucleotide divergence within subgenera ranged from 0.0089 substitutions per nucleotide for the subgenus Sebastomus to 0.0370 substitutions per nucleotide for the subgenus Sebastodes (Table 2). Six new restriction sites in the ND3/ND4 region were detected for the individuals included for investigation of intraspecific variation, which resulted in 10 additional haplotypes for S. aleutianus (n = 39), four for S. alutus (n = 60), nine for S. borealis (n = 78), 10 for S. carnatus and S. chrysomelas (n = 98), and five for S. caurinus (n = 78). Phylogenetic analysis A neighbor-joining tree (NJ) (Fig. 1) was constructed from all haplotypes, including those observed for the additional specimens, to evaluate the influence of intraspecific variation on the tree. Multiple haplotypes for each species formed many short terminal branches, but did not obscure interspecific differences. Each additional haplotype of S. aleutianus, S. alutus, S. borealis, S. carnatus, and S. chrysomelas clustered with the original haplotypes observed for those species. Haplotypes of S. aleutianus separated into two clusters at the tip of a branch. Subsequent analyses included only the original five specimens of each species so that all species would be represented by about the same number of individuals. In the ND3/ND4 region, 109 sites were polymorphic, six were monomorphic, and 26 were autapomorphic (variation was observed in only a single haplotype). In the 12S/16S region, 45 sites were 192 Li et al.—Are the Subgenera of Sebastes Monophyletic? Table 2. Number of Sebastes species analyzed in each subgenus, total number (Kendall 2000), and average nucleotide divergence (substitutions per nucleotide) within each subgenus. The subgenera are abbreviated in Figs. 1-4. Subgenus Abbr. No. species analyzed in subgenus No. species in subgenus, Kendall 2000 Average divergence Acutomentum Acuto 7 7 0.0308 3 speciesa 0.0144 Allosebastes Allo 8 13 0.0203 4 speciesb 0.0028 Auctospina Aucto 2 2 0.0129 Eosebastes Eoseb 3 3 0.0184 Hispaniscus Hispan 3 3 0.0272 Mebarus Mebar 5 7 0.0270 NWP cladec 0.0226 Neohispaniscus Nhispan 1 2 N/A Pteropodus Ptero 9 10 0.0291 NEP claded 0.0124 Rosicola Rosi 3 3 0.0179 Sebastichthys Sich 1 1 N/A Sebastocarus Scar 1 1 N/A Sebastodes Sode 3 5 0.0370 Sebastomus Stom 14 15 0.0089 Sebastopyr Spyr 1 1 N/A Sebastosomus Stoso 5 5 0.0224 3 speciese 0.0101 Zalopyr Zpyr 2 3 0.0171 Unassigned 3 5 N. Pacific speciesf 0.0084 aS. hopkinsi, S. ovalis, S. rufus. bS. emphaeus, S. variegatus, S. wilsoni, S. zacentrus. cw/o S. atrovirens. dNEP Pteropodus w/S. atrovirens, S. auriculatus, S. dalli. eS. flavidus, S. melanops, S. serranoides. fS. alutus, S. ciliatus/variabilis, S. crameri, S. polyspinis, S. reedi. NEP = northeastern Pacific; NWP = Northwestern Pacific. 193 Biology, Assessment, and Management of North Pacific Rockfishes polymorphic, 25 were monomorphic, and four were autapomorphic. A total of 154 sites provided information for the parsimony analysis. Three schemes assigned different weights to loss and gain of a restriction site in the parsimony analyses. The 1:1 loss/gain scheme produced 18,529 equally parsimonious trees, each with a total length of 536 steps. The 1:2 scheme produced 11,279 trees, each with a total length of 782 steps. The 1:4 scheme produced 1,324 trees, each with a total length of 1,082 steps. For each scheme, a 50% majority consensus tree was produced (Figs. 2-4). There were many areas of congruence among the three consensus trees and the NJ tree. The NJ tree and the consensus trees were similar in that members of the subgenus Sebastomus formed a tight cluster, as did the northeastern Pacific members of Pteropodus, whereas many of the other species did not cluster according to their subgeneric assignments. Rather, species from different subgenera often formed small, separate clusters, and the relationships among the clusters were not always clear. The species of the subgenus Sebastomus generally clustered together except for S. rosaceus, which often clustered with the group of species that phenotypically have conspicuous vertical bands: S. babcocki, S. nigrocinctus, S. rubrivinctus, and S. serriceps. This group of species formed a cluster near the Sebastomus cluster. The northeastern Pacific Pteropodus species, including S. chrysomelas, S. carnatus, S. caurinus, S. maliger, S. nebulosus, and S. rastrelliger, clustered consistently with S. auriculatus and S. dalli of the subgenus Auctospina, and S. atrovirens of Mebarus. Three species clustered near but not consistently with the Pteropodus group: S. saxicola and S. semicinctus (both subgenus Allosebastes), and S. elongatus (subgenus Hispaniscus). The northwestern Pacific members of Pteropodus, S. hubbsi, S. nivosus, and S. trivittatus, as well as the northwestern Pacific members of Mebarus, S. inermis, S. joyneri, S. taczanowski, and S. thompsoni, did not cluster with their northeastern Pacific counterparts. Instead, they generally clustered with other northwestern Pacific species in the analysis. These results are in agreement with earlier observations (Kai et al 2003, Li et al. 2006a). As for the species in other subgenera, four small consistent clusters of species representing three subgenera occurred in all phylogenetic trees. They were (1) S. hopkinsi, S. ovalis, and S. rufus (subgenus Acutomentum); (2) S. emphaeus, S. variegatus, S. wilsoni, and S. zacentrus (subgenus Allosebastes); (3) S. inermis, S. joyneri, and S. thompsoni (subgenus Mebarus); and (4) S. saxicola and S. semicinctus (subgenus Allosebastes). Each of these clusters was distinct from other species belonging to their subgenera. Two other groups formed with a little less consistency. Sebastes miniatus and S. pinniger (subgenus Rosicola) clustered together in all but the 1:1 consensus tree. Sebastes flavidus, 194 Li et al.—Are the Subgenera of Sebastes Monophyletic? Figure 1. Neighbor-joining tree (Saitou and Nei 1987) based on restriction site variation in Sebastes. Haplotypes of additional samples were included for five species to examine the influence of intraspecific variation. Vertical lines reflect multiple haplotypes for a species. Abbreviations are in Tables 1 and 2. car/chr