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Fishery Bulletin 93(1), 1995 



lecular data set that consists of DNA sequences from 

 the mitochondrial gene cytochrome b. This gene codes 

 for a functionally conserved protein that should fa- 

 cilitate sequence alignment over ancient divergences. 

 Additionally, it has been used to examine both in- 

 traspecific genealogy (Finnerty and Block, 1992) and 

 much deeper phylogenetic questions such as the ori- 

 gin of the mammalian orders (Irwin et al., 1991). The 

 initial scombroid radiation probably occurred in the 

 Paleocene epoch (Bannikov, 1985; Carroll, 1988). 

 Therefore, cytochrome b sequence should be phylo- 

 genetically informative about divergences within the 

 suborder. 



The analysis presented in this paper builds on our 

 earlier molecular study (Block et al., 1993). However, 

 we have improved on the previous study in several 

 ways which allow us to directly test the competing 

 hypotheses of billfish relationships. First, we have 

 obtained sequences from additional outgroups. The 

 inclusion of presumably more distant outgroups per- 

 mits us to address the question of scombroid mono- 

 phyly. This is important because the nonscombroid 

 hypothesis of billfish relationships argues that the 

 Scombroidei is not a monophyletic group. Second, we 

 include sequence information from the scombrid 

 Acanthocybium, a taxon which is integral to the 

 scombrid subgroup hypothesis. Third, we utilize sta- 

 tistical tests to directly compare different hypoth- 

 eses of billfish relationships. Finally, we emphasize 

 character-state changes that accrue relatively slowly 

 in order to minimize the effects of phylogenetic noise. 



Materials and methods 



Samples 



Partial cytochrome 6 sequences (590 base pairs) were 

 obtained from 75 individuals representing 34 spe- 

 cies of perciform fishes: 30 scombroid species and four 

 putative outgroup taxa (Sphyraena, Coryphaena, 

 Mycteroperca, and Morone; Table 1). We included 

 Sphyraena based on the placement by Johnson ( 1986) 

 of this taxon as the most primitive member of the 

 Scombroidei. Several percoid taxa (Coryphaena, 

 Mycteroperca, and Morone) were included because 

 of the suggestion by some authors that billfishes are 

 percoids (Gosline, 1968; Potthoff et al., 1980; 

 Nakamura, 1983; Potthoff et al., 1986). Published 

 cytochrome b sequences from two cypriniform fishes 

 obtained from Genbank were used to root the phylo- 

 genetic analysis (Crossostoma lacustre [Tzeng et al., 

 1990] and Cyprinus carpio [Chang, 1994]). We veri- 

 fied the outgroup status of the cyprinids by first con- 

 ducting a phylogenetic analysis using published se- 



quence from the sturgeon Acipenser transmontanus, 

 a holostean, to root a parsimony analysis. We at- 

 tempted but were unable to obtain full length se- 

 quences (590 base pairs) from two fixed and preserved 

 specimens of Scombrolabrax heterolepis possibly be- 

 cause of DNA degradation in these specimens. 



DNA extraction 



DNA was obtained from frozen tissue samples of the 

 mitochondria-rich "heater tissue" (found in Istio- 

 phoridae, Xiphiidae, and Gasterochisma melampus; 

 Block, 1986), red muscle, white muscle, or liver. Di- 

 gestion of 0.1-0.6 g tissue was performed in ten vol- 

 umes of extraction buffer containing 100 mM Tris CI 

 (pH 8.0), 10 mM EDTA, 100 mM NaCl, 0.1% SDS, 50 

 mM DTT, and 0.7 mg/mL proteinase K. Digestion 

 proceeded for 2-4 hours at 41°C. The homogenate 

 was extracted twice with equal volumes of phenol 

 (pH 8.0), once with 1:1 phenol/chloroform, and once 

 with chloroform. The final extract was precipitated 

 with 1/9 volume of 3M sodium acetate (pH 5.2) and 

 2.5 volumes of 100% ethanol. 



DNA amplification and sequencing 



The polymerase chain reaction (PCR) was used to am- 

 plify a 700 base pair region of cytochrome b. A 305 base 

 pair segment (not including primers) was generated 

 by using published oligonucleotide sequences (Kocher 

 et al., 1989). We amplified an overlapping, 425-bp 

 region farther downstream with primers L15079 (5- 

 GAGGCCTCTACTATGGCTCTTACC-3') or L15080 (5- 

 CGAGGCCTTTACTACGGCTCTTACCT-3) and 

 H15497 (5'-GCTAGGGTATAATT GTCTGGGTCGCC- 

 3). Double stranded amplification was performed in 

 a 100-uL volume containing 50 mM KC1, 10 mM Tris- 

 HC1 (pH 8.3), 1.5-3.0 mM MgCl, 200 ^M of each 

 dNTP, each primer at 1 mM, 1 |j,g of template DNA, 

 and 2 units of Amplitaq DNA polymerase (Perkin- 

 Elmer/Cetus). Most templates were amplified 

 through thirty cycles of PCR [1 minute denaturation 

 (92-95°C), 1 minute annealing (40-50°C), and 3 min- 

 utes extension (72°C)] on an Ericomp thermal cycler. 

 Alternatively, PCR was performed on a DNA Ther- 

 mal Cycler 480 (Perkin-Elmer) with the following 

 temperature cycling regime: 5 cycles of 1 minute de- 

 naturation at 95°C, 1 minute primer annealing at 

 40°C, 1:30 ramp to 72°C, and one minute extension 

 at 72°C, followed by 25-35 cycles with an annealing 

 temperature of 45°C. An 18-pL aliquot of the double 

 stranded product was run by means of electrophore- 

 sis through a IX TBE 1% agarose gel (Sea Plaque, 

 FMC) at 5 V/cm for 45 minutes. A single stranded 

 template was produced by asymmetric PCR (Gyl- 



