Scoles et al.: Global phylogeography of Scomber 
827 
(5’GAGGCCTCTACTATGGCTCTTACC3') and H15497 
( 5’GCTAGGGTATAATTGTCTGGGTC GC C 3 ’ ) devel- 
oped by Finnerty and Block (1992) for blue marlin 
(Makaira nigricans). Double-stranded DNA amplifi- 
cations were accomplished in 100 pi of 10 mM Tris- 
HC1 pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% (w/v) 
gelatin, 0.2 mM of each dNTP, 50 pmol of each primer, 
50 ng genomic DNA, and 2.5 units Taq polymerase 
(Perkin-Elmer/Cetus). Cycling parameters were pre- 
ceded by a 4-min initial denaturation at 94°C and 
included 38 cycles of 94°C for 1 min, 50°C for 1 min, 
and 72°C for 1.5 min, followed by a 5-min final ex- 
tension at 72°C. Primers L15079 and H15497 would 
not amplify some haplotypes of S. australasicus. 
Specific primers with conserved 3' ends were de- 
signed from other sequences: CYT2A 
(5TACCTTTTCATGGAAACATG3') and CYT2B 
( 5’ AAG AGGTTGGG AG AG AAG A3 ' ) . Both strands 
were fully sequenced by using the Sequenase kit 
(United States Biochemical) or the CircumVent Cycle 
Sequencing Kit (New England BioLabs) following the 
vendors’ protocols. 
Sequences were aligned by eye to the human mi- 
tochondrial cytochrome b sequence 1 (Anderson et al., 
1981). Nucleotide sequence divergences ( <7 ) among 
cytochrome b sequences were generated with the 
program K of REAP. Cytochrome b sequences were 
analyzed by parsimony analysis with rooting to 
Rastrelliger kanagurta by using ALLTREES in PAUP. 
Results 
MtDNA restriction site analysis of 40 Scomber 
scombrus from two locations revealed a total of 56 
restriction sites, of which 16 were polymorphic, de- 
fining 13 haplotypes. Nucleotide sequence diver- 
gences among the haplotypes of S. scombrus ranged 
from <7=0.17% to 0.86%. An average of 294 bp was 
surveyed, representing 1.75% of the mitochondrial 
genome. The average size of the S. scombrus mtDNA 
genome, determined from several restriction frag- 
ment profiles from each of the 12 restriction enzymes, 
was 16,784 ±213 bp (SD). 
Restriction site analysis of 246 S. japonicus and 
S. australasicus revealed a total of 93 unique 
haplotypes, including information from 86 restric- 
tion sites, of which 58 were polymorphic. Among 
haplotypes of S. japonicus and S. australasicus , 
nucleotide sequence divergences ranged from 
<7=0.15% to 2.9%. On average, 310 bp were surveyed 
per individual, representing 1.85% of the mtDNA 
genome. The average size of the mtDNA genome of 
1 GenBank accession no. V00662. 
S. japonicus or S. australasicus was estimated to be 
16,781 ±195 bp (SD), not statistically different than 
that of S. scombrus. 
Genetic diversities 
MtDNA restriction site analysis revealed that the 
genetic variability was lower in S. scombrus than the 
other two species (Table 1). S-ENG was the least 
variable sample in the study (h= 0.28), consisting of 
four haplotypes of which three occurred only once 
(Table 2). Ten haplotypes were revealed in S-MAS, 
with three at elevated frequencies, resulting in a 
haplotype diversity of h= 0.85. The overall haplotype 
diversity for S. scombrus was /? =0.58, and nucleotide 
sequence diversity was 71=0.18%. Three restriction 
enzymes, Pauli, Bgll, and Bsu36l, were invariant in 
S. scombrus, whereas the remaining nine restriction 
enzymes each revealed from two to five fragment 
patterns. 
A wide range of diversities was observed among 
samples of S. australasicus (h- 0.59-0.95) and S. 
japonicus (77=0.64-0.93) based on the restriction site 
data. Eastern Pacific samples of S', australasicus and 
S. japonicus from Mexico (A-MEX, h =0.59, 71=0.13%) 
and California (J-CAL, /7=0.64, 71=0.14%) had lower 
diversities than other samples, possessing only four 
and six haplotypes, respectively (Tables 1 and 2). The 
highest haplotype diversity occurred in A-RED 
(h- 0.95), with five of 10 haplotypes represented 
twice; the highest nucleotide sequence diversities 
occurred in A-NZL (ti= 0.77%) and A-AUS (71=0.75%) 
owing to the presence of two divergent mtDNA 
matrilines within each sample (Tables 1 and 2). With 
the exception of Bgll in S. australasicus, all restriction 
enzymes were variable in both species, revealing two 
to seven fragment patterns in S. australasicus, and two 
to 15 in S. japonicus. The enzyme Apa I revealed the 
greatest number of variants in both species. 
Genetic divergences 
Restriction fragment analysis showed S. scombrus 
to be highly divergent from S. australasicus and S. 
japonicus , with a net nucleotide sequence divergence 
of 8=11.9% between S. scombrus and the pooled data 
of S. japonicus and S. australasicus . In comparison, 
the mean net nucleotide sequence divergence be- 
tween S. japonicus and S. australasicus based on the 
restriction site data was only 8=1.17%. Probabilities 
of Roff and Bentzen chi-square tests among samples 
that shared haplotypes are given in Table 3. 
Scomber scombrus Considerable intraspecific di- 
vergence was evident in S. scombrus from the mtDNA 
