Orr et al.: Systematics of North Pacific sand lances of the genu s Ammodytes and description of a new species 
131 
none of these groups were genetically similar to the 
Kodiak population (Okamoto, 1989). In recent studies 
where the mitochondrial DNA (mtDNA) control re- 
gion and 16s ribosomal RNA (rRNA) were examined 
(Kim et al., 2006) and where mtDNA cytochrome ox- 
idase c subunit 1 (COI) was used to identify larvae, 
Kim et al. (2006, 2008, 2010) proposed that at least 2 
species of sand lances were present in material from 
around Korea and Japan. Han et al. (2012) examined 
the population structure of Ammodytes in waters off 
Japan and determined that 2 lineages resulted from 
the past isolation of the Sea of Japan from neighbor- 
ing seas — a separation that was facilitated by cur- 
rent flows and temperature barriers. Mecklenburg et 
al. (2011) used evidence from COI to suggest that 2 
species were among specimens collected in Alaska: 
1 species (A. hexapterus) in the Bering and Chukchi 
seas and 1 species in the Gulf of Alaska (“likely ... 
A. personatus”). Most recently, Turanov and Kartavt- 
sev (2014) combined sequence data from 7 specimens 
collected in the Bering and Okhotsk seas with 34 
samples taken from the online data source GenBank 
(www.ncbi.nlm.nih.gov) and concluded that 4 species 
are present in the North Pacific region. 
Two names are now considered valid in the region 
of the North Pacific Ocean, with A. hexapterus applied 
to populations in the eastern North Pacific Ocean, 
Bering Sea, Chukchi Sea, Sea of Okhotsk, and Sea of 
Japan (Miller and Lea, 1972; Hart, 1973; Mecklenburg 
et al., 2002; Hatooka, 2013) and A. personatus recog- 
nized in the western North Pacific Ocean, southern 
Sea of Okhotsk, and Sea of Japan (e.g. Ida, 1984; Ha- 
tooka, 2000, 2002, 2013). As a result of this study, we 
revise the genus Ammodytes in the North Pacific re- 
gion, on the basis of an examination of genetic varia- 
tion at the COI gene and morphological variation 
in specimens collected throughout nearly the entire 
range of the genus in the North Pacific region: from 
California to Puget Sound, the Gulf of Alaska, the 
Bering and Chukchi seas, Sea of Okhotsk, Sea of Ja- 
pan, Seto Inland Sea, and the Pacific coast of Japan. 
Materials and methods 
Whole samples of Ammodytes were obtained from ar- 
chival collections and collected over the 13 years from 
2000 to 2013, from the eastern and western coastlines 
of the North Pacific Ocean and its fringing seas from 
latitudes of 40°N to 70°N (Table 1; Fig. 1; Appdx. 1). 
Additional samples of Atlantic species were obtained 
from the eastern and western North Atlantic Ocean for 
comparison. Specimens with known preservation his- 
tories from archival collections had been fixed in 10% 
formalin and preserved in 70% ethanol or 45% isopro- 
panol. For freshly collected specimens obtained for ge- 
netic analyses, tissues were fixed and preserved in 95% 
ethanol, and whole specimens were then fixed in 10% 
formalin and preserved in 70% ethanol. Institutional 
abbreviations are listed in Leviton et al. (1985). 
Genetics 
DNA was extracted and purified from more than 498 
tissue samples with a Qiagen 2 (Valencia, CA) DNeasy 
Blood & Tissue Kit. Primers were developed for the 
flanking regions of the COI gene of the mitochondrial 
DNA from a consensus of 21 DNA sequences obtained 
from GenBank (FJ666901-FJ666921). Primers used to 
sequence DNA were F: 5'CTCCTGCAGGGTCAAAGAAG 
and R: 5'GGCACCCTTTATCTAGTATT, resulting in a 
638 base-pair (bp) fragment of the COI region. The 
fragment was amplified by polymerase chain reaction 
(PCR) in a 50-pL reaction volume containing IX GoTaq 
Buffer (Promega Corp., Madison, WI), 2.5 mM MgCl 2 , 
0.2 mM of each dNTP, 0.2 pM each of forward and re- 
verse primer, 1 unit of GoTaq, 10 ng DNA, and deion- 
ized water. Thermal cycling conditions were as follows: 
95°C for 2 min; 30 cycles of 95°C for 40 s, 55°C for 40 
s, and 72°C for 1 min; 72°C for 7 min; and 4°C indefi- 
nitely. Forward sequences were obtained from the PCR 
product by the Sanger sequencing method. Sequences 
were aligned and edited with the software program 
CodonCode Aligner, vers. 3.7.1 (CodonCode Corp., Ded- 
ham, MA). Ambiguous end regions, particularly on the 
3' end of the sequences, were aggressively trimmed to 
560 bp in length to achieve the maximum number of 
high-quality sequences. 
The following genetic indices were calculated in Ar- 
lequin, vers. 3.5 (Excoffier and Lischer, 2010): number 
of haplotypes ( H ), number of polymorphic sites ( S ), 
haplotype diversity (h), and average nucleotide diver- 
sity (jt). For the purpose of examining phylogenetic re- 
lationships within the genus Ammodytes, the data were 
supplemented with 15 samples representing 4 species 
of Ammodytes from the Atlantic Ocean and a DNA 
sequence from Uranoscopus oligolepis from GenBank 
(FJ237962) to use as an out-group for phylogenetic 
analysis. 
The number of nucleotide differences between se- 
quences was visually represented by a haplotype net- 
work (minimum spanning tree) created with the pro- 
gram Sneato, vers. 2 (Wooding, 2004; http://user.xmis- 
sion.com/~wooding/Sneato/). Phylogenetic relationships 
among unique mtDNA haplotypes were evaluated with 
the program MEGA 6 (Tamura et al., 2013), wherein 
trees were constructed with maximum parsimony (MP) 
(Nei and Kumar, 2000), maximum likelihood (ML) 
(Tamura and Nei, 1993), and neighbor-joining (NJ) 
methods (Saitou and Nei, 1987; by using the distance 
algorithm of Tamura and Nei, 1993). Support for nodes 
of the 3 trees was evaluated with 1000 bootstrap rep- 
licates (Felsenstein, 1985). Branches with >95% boot- 
strap support were collapsed into consensus sequences, 
which were used to create summary trees. 
Analyses of the number of substitutions per site be- 
tween all unique sequences were examined with the 
2 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
