BULLETIN OF MARINE SCIENCE, 79(3): 505–519, 2006 Molecular Data SUPPort SEParate Scombroid and XIPHioid Clades Thomas M. Orrell, Bruce B. Collette, and G. David Johnson Abstract Molecular data (single copy nuclear DNA Tmo-4C4 and combined Tmo-4C4 and mitochondrial DNA cytochrome b) lead to the following conclusions about the inter- relationships of “scombroid” fishes. In both the single copy nuclear DNA (scnDNA) and combined molecular data sets, billfishes (Xiphioidei) and tunas and mackerels (Scombroidei) are separate clades. In the scnDNA parsimony clade, Pomatomus is basal to other scombroids. Sphyraena is excluded from the scombroids in all analy- ses, but only in the scnDNA analysis is Gasterochisma the basal scombrid. The sub- families Gasterochismatinae and Scombrinae and the tribe Scombrini (mackerels) are supported by both data sets, but the tribes Scomberomorini, Sardini, and Thun- nini do not have molecular support. Morphological data place Allothunnus at the base of the Thunnini but the molecular data do not support this. The first modern definition of the suborder Scombroidei was that of Regan (1909). Regan included the louvar, Luvarus imperialis Rafinesque, 1810, but it was not con- sidered a scombroid by either Collette et al. (1984) or Johnson (1986), and it was later shown to be a highly specialized oceanic member of the typically reef-associ- ated surgeonfishes (Acanthuroidei) by Tyler et al. (1989) and corroborated by Tang et al. (1999). The nearly 100 species of scombroids (mackerels, tunas, billfishes, and relatives) are circumglobally distributed, primarily in marine waters, and are among the most important of all fishes economically. Numerous studies of scombroid in- terrelationships have produced competing hypotheses that have resulted in mark- edly different and often unstable taxonomic classifications (Carpenter et al., 1995). The instability of these classifications can confound management of scombroids as a fisheries resource and the understanding of the diversity and complexity of marine communities. Cladistic parsimony analysis has been used to infer evolutionary history (phylog- eny) of the scombroids based on either morphological or molecular data. Despite numerous morphological studies, the taxonomic limits of the group remain problem- atic. Based on a survey of 40 morphological characters, Collette et al. (1984) defined the Scombroidei to include the Scombrolabracidae (longfin escolar), Trichiuridae (cutlassfishes), Gempylidae (snake mackerels), Xiphiidae (swordfish), Istiophoridae (billfishes), and Scombridae (mackerels, tunas, and bonitos). The monotypic Gas- terochisma was included as a member of the Scombridae, and Scombrolabrax was found to be the basal scombroid clade. Johnson (1986) reviewed and re-evaluated the 40 characters of Collette et al. (1984), and presented an alternative morphologi- cal hypothesis based on 49 characters, 24 of which were from Collette et al. (1984) and 25 of which were new. Johnson’s hypothesis excluded Scombrolabrax from the Scombroidei (placing it instead as the second outgroup with Pomatomus as the first) and included, for the first time,Sphyraena (barracudas) as the basal scombroid clade. In contrast to the phylogeny of Collette et al. (1984), Johnson’s hypothesis placed the swordfish and billfishes as sister to the scombrid Acanthocybium (wahoo) and thus embedded them within the family Scombridae. Carpenter et al. (1995) re-evalu- Bulletin of Marine Science 505 © 2006 Rosenstiel School of Marine and Atmospheric Science of the University of Miami 506 BULLETIN OF MARINE SCIENCE, VOL. 79, NO. 3, 2006 ated the characters of Collette et al. (1984) and Johnson (1986), and the resulting phylogenies suggested that Johnson’s placement of the swordfish and billfish within the Scombridae was unstable, although placement of these two families within the Scombroidei was shown to be stable and has not been questioned subsequently in any morphology-based parsimony analysis. As with morphological studies, molecular phylogenies have not resulted in a clear- ly defined Scombroidei. Finnerty and Block (1995) used a 590 base pair fragment of the mitochondrial (mtDNA) cytochrome b (cyt b) gene to infer phylogenies of the scombroids from both nucleotide sequences and from translated amino acid resi- dues. Neither the nucleotide or inferred amino acid residue phylogenies supported Johnson’s placement of Xiphiidae and Istiophoridae within the Scombridae and, also contrary to Johnson (1986), Sphyraena fell outside of the scombroids. Xiphiidae and Istiophoridae were sister to basal percoids in the amino-acid residue phylogeny. The latter suggests that billfishes were derived from within the perciforms but not from scombroids, a hypothesis not supported by the morphological data. In addi- tion, placement of Gasterochisma within the scombroids was unstable. Based on the results of Finnerty and Block (1995), cyt b was not able to resolve the limits of the Scombroidei. Collette et al. (2001) suggested that cyt b sequences were unable to resolve scombroid relationships because of homoplastic transitional saturation that occurs with this gene. Therefore, cyt b is not an appropriate molecular marker for family level relationships. In this regard, Meyer (1993) noted that cyt b is probably best suited for closely related taxa, where nucleotide sequence variation is less satu- rated by multiple substitutions. Despite the limitation of cyt b, mitochondrial DNA sequences have been used to address scombroid relationships on many taxonomic levels, but are best suited for generic or species level relationships (Collette, 1999). Scoles (1994) and Scoles et al. (1998) used cyt b sequences to examine relationships of the mackerels. Banford et al. (1999) used mtDNA from ATP synthase 8 and 6, and from the NADH ubiqui- none oxidoreductase subunit 2 genes to infer relationships of the Spanish mackerels (Scomberomorus). Higher level relationships among fishes may be resolved with a molecular marker that has low homoplasy, low saturation, and an adequate number of substitutions to resolve relationships. Mitochondrial DNA is clonally (maternally) inherited and most substitutions are point mutations. Of the 13 protein-coding genes in the fish mitochondrion, most evolve very quickly. Often these genes are uninformative or misinformative for family level relationships. The maternal mode of inheritance is not sensitive to introgression which can confound mtDNA-based phylogenies (Orrell et al., 2002). Nuclear DNA provides an alternative to mitochondrial DNA as a source for genes from which to infer independent phylogenies. Many loci are conserved and are appropriate for more distantly related teleost taxa. Streelman and Karl (1997) successfully amplified a nuclear locus-specific primer pair (Tmo-4C4) in 17 labroid fishes. This locus is an open-reading frame and extended to 511 nucleotides in all fishes sampled. Only a single copy of the locus was amplified in the study. A BLAST (Altschul et al., 1990) search of GenBank databases determined that no sequence matched the locus although low probability matches were made to muscle specific proteins titin and connectin (Streelman and Karl, 1997). Tmo-4C4 has been used to elucidate phylogenetic and biogeographic relationship of the labroids (Streelman and ORRELL ET AL.: NUCLEAR PHYLOGENY OF THE SCOMBROIDEI AND XIPHIOIDEI 507 Karl, 1997), cichlids (Streelman et al., 1998; Farias et al., 2000; Vences et al., 2001), scarids (Streelman et al., 2002), beloniforms (Lovejoy et al., 2004), and others. In this paper we use analyses of novel sequences of the single copy nuclear gene Tmo-4C4 alone and in combination with previously published sequences of the mtDNA cytochrome b gene to examine the following questions and discuss how it conflicts (or agrees) with certain aspects of the morphology: (1) Are billfishes scom- broid fishes or unrelated to them?; (2) What is the basal scombroid, Scombrolabrax, Pomatomus, or the barracudas?; (3) Is Gasterochisma the basal scombrid?; (4) Are the Scombrinae and Scombrini monophyletic?; (5) Are bonitos, tribe Sardini, a valid taxon?; (6) Are higher tunas, Thunnini, a valid taxon? Materials and Methods Collection Data.—Collection data (GenBank accession number for cyt b and sample number for Tmo-4C4) are given in Table 1. The primer sequences used forP CR amplification in this study were the Tmo-4C4 locus specific primers of Streelman and Karl (1997): Tmo- 4C4F CCT CCG GCC TTC CTA AAA CCT CTC Tmo-4C4R CAT CGT GCT CCT GGG TGA CAA AGT. Amplification.—A 35 µl PCR amplification of the Tmo-4C4 locus was prepared with 5–10 ng of each template DNA. The following reagents were used in each reaction: 3.5 µl 10× PCR Buffer (Invitrogen Corporation, 200 mM Tris-HCL (pH8.4), 500 mM KCL); 1.75 µl MgCl2 (Invitrogen Corporation, 50 mM MgCl2); 2.8 µl 2.5 mM dNTP mix (Promega, 1:10 dilution of 25 mM each dATP, dCTP, dGTP, dTTP); 07 µl (~0.5 pmols) each of primers; 0.28 µl Platinum Taq DNA polymerase (Invitorgen Corporation, [5U/µl]) and 23.87 µl diH2O. All amplifica- tions were performed on a MJ Research TETRAD thermocycler (Watertown, MA) with the following cycle parameters: initial denaturation (97 °C for 3.0 min); 34 cycles of (denaturation 95 °C for 1.0 min, annealing 54–55.5 °C (depending on sample) for 1.0 min; extension 72 °C for 30 s); final extension (72 °C for 7 min); icebox (4 °C indefinitely). Sequencing.—PCR purification was performed using MinElute PCR Purification Columns Qiagen: Valencia, CA). PurifiedP CR product (6 µl) was used in the cycle sequencing reactions with both the original PCR primers. Each 20 µl cycle sequencing reaction contained: 6 ul of DNA template; 3.21 µl of primer; 2 µl of BigDye v3.1 (Applied Biosystems, Inc (ABI); Foster City, CA); 6 µl of BigDye dilution buffer (2.5×), 0.4 µl of 0.5% DMSO, and 2.4 µl of water. Cycle sequencing profile consisted of 35 cycles of denaturation (94 °C for 30 s), annealing (55 °C for 15 s) extension (60 °C for 4 min). Cycle sequencing products were purified via Sephadex© G- 50 column filtration in Multiscreen plates (Millipore: Billerica, MA) and run on an ABI 3100 DNA sequencer (50 cm or 80 cm array as per manufacturer’s instructions).