Orr et al.: Systematics of North Pacific sand lances of the genu s Ammodytes and description of a new species 
135 
Table 2 
Genetic distance calculated in MEGA 6 (Tamura et al., 2013) with the Tamura and Nei (1993) model for species of Ammo- 
dytes. On the diagonal, which is indicated with a gray background, is the range of distances among haplotypes within each 
species. Mean distance between each species pair is below the diagonal. Above the diagonal is the range of distances among 
all haplotypes between each species pair. Ammodytes dubius and A. americanus are reported together because the samples 
exhibited identical sequence patterns. 
dubius! 
hexapterus 
personatus 
japonicus 
heian 
marinus 
americanus 
tobianus 
Uranoscopus 
hexapterus 
0-0.004 
0.029-0.043 
0.059-0.074 
0.044-0.052 
0.004-0.005 
0.033-0.047 
0.044-0.051 
0.274-0.280 
personatus 
0.031 
0-0.013 
0.056-0.077 
0.043-0.056 
0.022-0.032 
0.034-0.049 
0.052-0.066 
0.276-0.290 
japonicus 
0.065 
0.065 
0-0.013 
0.063-0.076 
0.059-0.070 
0.059-0.082 
0.070-0.087 
0.274-0.282 
heian 
0.047 
0.049 
0.068 
0-0.007 
0.040-0.046 
0.051-0.063 
0.057-0.068 
0.265-0.273 
marinus 
0.005 
0.027 
0.063 
0.042 
0 
0.032-0.041 
0.042-0.045 
0.282 
dubius! americanus 
0.04 
0.043 
0.068 
0.057 
0.036 
0-0.009 
0.049-0.059 
0.281-0.289 
tobianus 
0.048 
0.058 
0.079 
0.062 
0.044 
0.055 
0.004 
0.276-0.282 
Uranoscopus 
0.276 
0.282 
0.277 
0.268 
0.282 
0.286 
0.279 
0 
fornia to the Aleutian Islands and the southeastern 
Bering Sea), A. hexapterus (range: Chukchi Sea, north- 
ern and western Bering Sea, and Sea of Okhotsk), A. 
japonicus (range: Sea of Japan and Seto Inland Sea), 
and a new species (range: Pacific Ocean side of north- 
ern Japan and Soya Strait) (Fig. 1). Only 3 clades of 
Atlantic species emerged from the analyses; specimens 
identified morphologically as A. dubius or A. america- 
nus as defined by Nizinski et al. (1990) were homog- 
enous at the COI gene (as also noted by McCusker et 
al., 2013). Ammodytes marinus was closely related to A. 
hexapterus, differing by up to 10 bp. At 8 of these sites, 
however, A. marinus appeared to be heteroplastic or 
otherwise ambiguous. These results indicate that fur- 
ther investigation of Ammodytes of the North Atlantic 
Ocean is warranted. 
Differentiation between clades within the North Pa- 
cific region ranged from 15 to 32 nucleotide polymor- 
phisms. Ammodytes personatus differed from A. hexa- 
pterus by a minimum of 2.7% (15 of 560 bp), from A. 
japonicus by 5.7%, and from the new species by 4.3%. 
Percent divergence between A. hexapterus and A. ja- 
ponicus was 6.4% and between A. hexapterus and the 
new species, 4.8%. The percent divergence between the 
2 species in Japan, A. japonicus and the new species, 
was 6.4%. Sequence divergences, corrected for satura- 
tion, between each species pair were similar and are 
reported in Table 2. The index of substitution satura- 
tion was significantly below both the symmetrical and 
asymmetrical critical values (P=0.000) for all tests, 
indicating little evidence for substitution saturation 
within the COI DNA sequences among Ammodytes or 
between out-groups. 
In all genetic analyses, 4 monophyletic and highly 
divergent groupings among Pacific specimens were ob- 
served, representing 4 distinct species in the region 
of the North Pacific Ocean. Bootstrap support of the 
phylogenetic results was high (95-99%) for all spe- 
cies clades (Fig. 3). Summaries of the variation in the 
groupings within the phylogenetic trees, by method, 
are represented by trees of consensus sequences of 
each highly supported species clade (Fig. 4). These phy- 
logenetic resolutions differed only in the position of A. 
personatus as sister of the A. dubius and A. america- 
nus clade or sister of the A. hexapterus and A. marinus 
clade. The low bootstrap values indicate that neither of 
these resolutions is well supported by these data. In- 
traspecific variation differed within the 4 clades (Table 
3). The 2 clades representing A. japonicus and the new 
species displayed the highest levels of gene diversity 
(0.50 and 0.53), followed by a high level of diversity 
within A. personatus (0.31), which exhibited 3 second- 
ary nodes of haplotypes. Specimens within these sec- 
ondary branches were not geographically related (Fig. 
3). Ammodytes hexapterus was nearly monomorphic, 
with a low level of genetic diversity (0.04): only 6 single 
transitions were detected in 143 individuals. 
All COI sequences reported in GenBank and the 
Barcode of Life Database (BOLD) for Ammodytes of 
the North Pacific region were aligned in CodonCode 
Aligner. Each sequence clearly corresponded with 1 of 
the 4 species described in this article (Table 4). 
Morphology 
Meristic characters Among meristic characters of speci- 
mens identified genetically and with a complete suite 
of morphological characters, only counts of total gill 
rakers, lateral-line pores, dermal plicae, and pectoral- 
fin rays met variance assumptions necessary to conduct 
an ANCOVA (Table 5). Counts of gill rakers differed 
between A. japonicus, A. hexapterus, and A. personatus', 
counts in the new species and A. personatus did not 
differ significantly from each other, but each had sig- 
