North Pacific Research Board Project Final Report
Mitochondrial DNA-based identification of eggs, larvae and dietary components of commercially and
ecologically important fish species and selected invertebrates in the northeast Pacific Ocean
NPRB Project 1220 Final Report
Michael Canino, Melanie Paquin and David Drumm
National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Alaska Fisheries
Science Center, 7600 Sand Point Way NE, Seattle, WA 98115. (206) 526-4108 mike.canino@noaa.gov
January, 2015
1
ABSTRACT
We compiled mitochondrial DNA (mtDNA) sequence databases (through direct sequencing and mining
public databases) for selected fish and shrimp species in the Gulf of Alaska and Bering Sea. Restriction
fragment length polymorphism (RFLP) protocols were then developed and tested for accurate
identification of eggs and/or larvae for six flatfish species that were historically difficult or impossible to
identify by conventional taxonomic approaches. RFLP protocols screening variation in the mitochondrial
cytochrome c oxidase (COI) gene were diagnostic for two pleuronectid species producing ‘large’ eggs
(Pacific halibut and Greenland halibut) and three species producing ‘small’ eggs (yellowfin sole, Sakhalin
sole and longhead dab). Protocols for two flatfishes producing ‘intermediate’ sized eggs could not be
developed due to a lack of fixed restriction sites between species in the mitochondrial COI, cytochrome b,
NADH dehydrogenase subunit 1 (ND1), 12S rRNA, 16S rRNA or control region sequences. Results from
this study demonstrated the potential to fill important knowledge gaps for commercially and ecologically
important species routinely studied at the Alaska Fisheries Science Center (AFSC), with particular regard
to species composition in fish diets and in ichthyoplantkon assemblages. The database provided the
foundation for development of rapid, cost-effective, and accurate molecular protocols to identify species
under circumstances where traditional taxonomic approaches founder or fail.
KEY WORDS
Mitochondrial DNA, cytochrome c oxidase, cytochrome b, ND1, 12S, 16S, species identification,
restriction fragment length polymorphism, RFLP, fish eggs, fish larvae, caridean shrimp
CITATION
Canino, M., M. Paquin, and D. Drumm. Mitochondrial DNA-based identification of eggs, larvae and
dietary components of commercially and ecologically important fish species and selected invertebrates in
the northeast Pacific Ocean. North Pacific Research Board Final Report Project 924. 26 p.
2
1
Table of Contents
2
3
Abstract ......................................................................................................................................................... 2
4
Key Words .................................................................................................................................................... 2
5
Citation.......................................................................................................................................................... 2
6
List of Figures ............................................................................................................................................... 3
7
List of Tables ................................................................................................................................................ 4
8
Study Chronology ......................................................................................................................................... 4
9
Introduction ................................................................................................................................................. 5
10
Overall Objectives ........................................................................................................................................ 7
11
Methods........................................................................................................................................................ 8
12
Database mining and sample collection ....................................................................................................... 8
13
DNA extraction, PCR and sequencing ......................................................................................................... 9
14
flatfish PCR-RFLP development ................................................................................................................ 11
15
flatfish PCR-RFLP protocol validation...................................................................................................... 12
16
Results ........................................................................................................................................................ 12
17
flatfish DNA sequencing and PCR-RFLP protocol development ........................................................... 12
18
flatfish PCR-RFLP protocol validation.................................................................................................. 12
19
Barcoding sculpins ................................................................................................................................. 13
20
Barcdoding caridean shrimps ................................................................................................................. 16
21
Discussion .................................................................................................................................................. 19
22
Conclusions ................................................................................................................................................ 21
23
Management Implications ........................................................................................................................ 22
24
Publications ................................................................................................................................................. 22
25
Outreach ...................................................................................................................................................... 24
26
Acknowledgements ..................................................................................................................................... 22
27
Literature Cited ........................................................................................................................................... 23
28
List of Figures
29
Figure 1. Hypothetical distributions of intraspecific variation and interspecific divergence in barcoding .. 6
30
Figure 2. Collection locations for caridean shrimps ..................................................................................... 9
31
Figure 3. PCR-RFLP profiles for two halibut species ................................................................................ 12
32
Figure 4. Neighbor-joining phylogenetic tree for caridean shrimps ........................................................... 17
33
Figure 5. Haplotype accumulation curve for caridean shrimps .................................................................. 17
3
34
List of Tables
35
Table 1. Target flatfish species and egg size distributions............................................................................ 7
36
Table 2. Mitochondrial DNA gene regions analyzed in two Hippoglossoides species .............................. 10
37
Table 3. Oligonucleotide primers and annealing temperatures for COI in shrimp ..................................... 11
38
Table 4. PCR-RFLP profiles for halibut species......................................................................................... 12
39
Table 5. Distribution of ND1 haplotypes in two Hippoglossoides species................................................. 13
40
Table 6. Distribution of COI haplotypes in two Hippoglossoides species.................................................. 14
41
Table 7. Sequence similarity scores of voucher specimens to GenBank sequences ................................... 15
42
Table 8. Intra- and interspecific genetic distances for caridean shrimps .................................................... 18
43
44
45
STUDY CHRONOLOGY
46
Bering Sea skates (Spies et al. 2006) and fishes (Canino et al. 2011) which showed that accurate species
47
identification through sequencing a fragment of the mtDNA cytochrome c oxidase subunit I (COI) gene
48
was a feasible approach for most species. This NPRB-sponsored project was initiated in January 2013.
49
Mining of public databases for relevant COI sequences and design PCR-RFLP protocols began
50
immediately. DNA extraction and sequencing for specimens of interest that were already available at the
51
UW Fish collection began in spring 2013 and continued throughout the year through February 2014 in
52
order to process samples collected during summer 2013 field surveys.
Two earlier studies leading to this work included a study of DNA barcoding in North Pacific Ocean and
53
4
54
Introduction
55
Accurate identification of various life history stages and prey items of marine fishes and invertebrates is
56
central for understanding their distribution, abundance, trophic ecology, and biodiversity. While
57
conventional taxonomic approaches have been successfully applied in ichthyoplankton (fish eggs and
58
larvae) identification and diet analysis efforts for many years, identification to the species level requires
59
varying degrees of taxonomic expertise and is sometimes limited by the lack of diagnostic characters in
60
many marine species.
61
Our approach used fixed nucleotide differences in the mitochondrial DNA (mtDNA) genome for species
62
identification. MtDNA accumulates mutational changes that are more likely be fixed than in nuclear
63
genomic DNA, largely due to the accelerated effects of neutral genetic drift on the strictly asexually-
64
transmitted (maternal), nonrecombinant haploid mtDNA genome. This inheritance mode results in more
65
rapid DNA sequence divergence of mtDNA lineages compared to nuclear genes, often revealing
66
discontinuities among currently recognized plant and animal species. Avise and Walker (1999) reviewed
67
mtDNA variation in 252 vertebrate species (mostly fishes, birds, and mammals) and reported a general
68
concordance of both topology and species numbers between mtDNA gene trees and taxonomic trees,
69
accurately detecting approximately 90% of congeneric sister species. Additional advantages to using
70
mtDNA markers are that mtDNA is present in many copies per cell depending upon tissue type (103 - 104
71
copies in mammalian cells; Brown and Clayton 2002), and it is relatively robust to degradation. Both
72
properties increase the likelihood for recovering usable DNA from archival (museum specimens, scales,
73
etc.) and other types of degraded samples (e.g. gut or scat contents).
74
During the last two decades, significant effort has been directed towards analyses of variation across
75
animal groups in short DNA sequences from standardized regions of the mitochondrial genome. The
76
unique combinations of fixed nucleotide differences from these regions to identify species has given rise
77
to the term DNA 'barcoding', analogous with the ubiquitous Uniform Product Code bars as identifiers on
78
manufactured products. Barcoding approaches rely upon a gap between intraspecific variation and
79
interspecific divergence (Fig. 1) and have been applied across a diverse array of studies, ranging from
80
species identification (e.g. de Oliveira Ribeiro et al 2012), the detection of cryptic conspecific species
81
(e.g. Garcia-Morales and Elias-Gutierrez 2013), hybridization between lineages (e.g. Chiesa et al. 2013)
82
and ecosystem community structure (e.g. Shokralla et al. 2012; Jackson et al. 2014). The mitochondrial
83
cytochrome c oxidase subunit I (COI) gene is generally the accepted standard for barcoding efforts,
84
yielding nearly 100% accuracy in some empirical studies of birds (Hebert et al. 2004a), insects (Hebert et
85
al. 2004b; Barrett and Hebert 2005), and fishes (Zhang and Hanner 2011). Ward et al. (2009) reported an
86
average intraspecific distance (Kimura’s two-parameter K2P) of 0.35% for 546 fish species and a mean
5
interspecific K2P distance of 8.11%.
88
Similarly, Zhang and Hanner (2011) reported
89
a 60-fold difference between intraspecific and
90
conspecific variation in 158 species around
91
Japan. In some cases, however, incomplete
92
lineage sorting can occur (Fig. 1B) when
93
insufficient generations have passed to allow
94
genetic characters to drift to fixation,
95
demonstrating reproductive isolation between
96
sister taxa (Nice and Shapiro 1999), or there
97
is recurring hybridization between two
98
lineages (e.g. Morgan et al. 2012). The
99
soundness of the barcoding approach has
A
intraspecific
variation
n individuals
87
interspecific
divergence
barcoding
gap
B
intraspecific
variation
interspecific
divergence
overlap
genetic distance
100
already been validated in other studies of diet
Figure 1. Distributions of intraspecific variation
and interspecific divergence. A; Discrete
distributions from complete coalescent lineage
sorting resulting in reciprocal monophyly. B;
Overlapping distributions from incomplete linage
sorting resulting in paraphyly or polyphyly.
101
(Blankenship and Yayanos 2005; Keskln and
Adapted from Meyer and Paulay (2005).
102
Atar 2013; Paquin et al. 2014) and
103
ichthyoplankton assemblages (Hyde et al. 2005; Leray et al. 2013).
104
In this study, we built upon previous work (NPRB Project #924) to develop molecular methods for
105
species identification in current research areas within the Alaska Fisheries Science Center (AFSC). These
106
areas included species identification of ichthyoplankton (fish eggs and larvae) in the Ecosystem and
107
Fisheries Oceanography Coordinated Investigations (EcoFOCI) program for seven flatfish species whose
108
egg size distributions overlap and cannot be distinguished using morphological characters alone.
109
Flatfishes are important as predators/prey in northeast Pacific Ocean and Bering Sea ecosystems and
110
accurate identification of eggs and larvae are crucial for determining spawning distributions and
111
seasonality. We also barcoded a variety of sculpin species likely to be encountered as prey remains in diet
112
studies conducted by the Resource Ecology and Ecosystem Modeling (REEM) program. Not all prey
113
remains can be visually identified, especially in conspecific taxa, and molecular identification of highly
114
digested prey better informs food web energetics models developed by REEM. Finally, we extended
115
species identification via barcoding to eight species of caridean shrimp of ecological importance, several
116
of economic significance, in the northeast Pacific Ocean, Bering Sea and Chukchi Sea. Identification of
117
these shrimps currently relies on minor, and sometimes subjective, differences in morphological/meristic
118
characters. Systematic relationships among species groups are also disputed, making identification even
119
more challenging. Our expectation was to provide unequivocal species identification through barcoding
120
that could be used for ecological studies (e.g. identification of juvenile instar stages), forensics (e.g. food
6
121
fraud) and perhaps further resolve systematics in this group. We combined mining public databases,
122
GenBank to obtain mtDNA sequences for species of interest, and conducted supplemental DNA
123
sequencing of voucher and field-collected samples when insufficient or no sequence data existed.
124
125
Overall Objective:
126
To expand a mtDNA database of select North Pacific, Bering Sea and Chukchi Sea marine fish and
127
invertebrate species and develop protocols for DNA-based identification.
128
Specific objectives:
129
1. Obtain mtDNA sequences and develop PCR-based restriction fragment length polymorphism
130
(RFLP) protocols to identify the eggs and/or larvae for six flatfish species of commercial and
131
ecological importance.
132
Seven flatfish species routinely encountered in field surveys, roughly divided into three groups
133
depending upon egg size, were chosen for development of PCR-RFLP protocols (Table 1).
134
135
136
137
Table 1. Pleuronectid flatfish species for molecular identification
Common name
Species name
Egg size group
Egg diameter range
(mm)
Pacific halibut
Hippoglossus stenolepsis
Large
2.9 – 3.8 1
Greenland halibut
Reinhardtius hippoglossoides
Large
3.5 – 4.5 2,3
yellowfin sole
Limanda aspera
Small
0.76 – 0.85 4
Sakhalin sole
Limanda sakhalinensis
Small
*1.2 – 1.7
longhead dab
Limanda proboscidea
Small
*1.2 – 1.7
flathead sole
Hippoglossoides elassodon
Intermediate
2.1 – 2.7 5
Bering flounder
Hippoglossoides robustus
Intermediate
2.0 -2.7 4
1
Thompson and Van Cleve (1936), 2 Jensen (1935), 3 Duffy Anderson et al. (in review), 4 PertsevaOstroumova (1961), 5 Miller (1969), * estimated range for unidentified Limanda spp. eggs (AFSC,
unpubl. data).
138
139
We were only successful in developing and validating a PCR-RFLP protocol to discriminate two of
140
seven flatfish species, Pacific halibut and Greenland halibut (see pg. 11-12). We had previously
141
developed a double restriction digest protocol, using both COI and cytochrome b (cyt b) regions
142
(Canino et al. 2011), that was capable of resolving the three Limanda species, but not the two
143
Hippoglossoides species. Sequence data from the mitochondrial NADH dehydrogenase 1(ND1), 12S,
144
16S and cyt b genes did not reveal diagnostic restriction sites for RFLP protocol development for
145
these five flatfish species.
7
146
2. Conduct mtDNA sequencing and compile a barcode database for approximately 30 species of
147
sculpins from the Gulf of Alaska, Aleutian Islands and eastern Bering Sea. This database will
148
be used in future efforts to identify sculpins as prey remains encountered in diet studies
149
conducted at the AFSC.
150
A total of 252 DNA sequences were obtained, representing 32 species of sculpins, three of which
151
have not been published in online data bases (see Table 7, p. 16-17). Comparisons of our voucher
152
sequences with those from public databases revealed a high degree of sequence similarity among
153
putative congeneric species and, unfortunately, evidence of species misidentification in those
154
databases. Misidentifications arise from several sources, including a high degree of morphological
155
similarity and unresolved systematic relationships among some sculpin species. Our efforts represent
156
a significant contribution towards more accurate identification of sculpins in diet studies conducted
157
within the large marine ecosystems of Alaska.
158
159
160
3. Conduct mtDNA sequencing on eight species of shrimp that are historically difficult to identify
using conventional taxonomic methods.
161
We tested a mtDNA barcoding approach to identify seven candidate species from the marine shrimp
162
genera Crangon and Neocrangon that occur in Alaskan waters. Major goals were to analyze sequence
163
divergence among conspecifics in different geographic regions to test for possible cryptic species and
164
to help resolve taxonomic issues in problematic species. In general, interspecific divergences were
165
comparatively large (up to 15%), comparable to means of 17.2% and 15.5% reported for decapod
166
crustaceans by Costa et al. (2007) and Silva et al. (2011), respectively. Nominal species were mostly
167
resolved into disecrete monophyletic units (p.17). However, one sample of putative Crangon
168
septemspinosa from the Chukchi Sea grouped separately from C. septemspinosa from the northwest
169
Atlantic with strong bootstrap support in a phylogenetic analysis, suggesting a potentially new,
170
cryptic species in this region. As with sculpins, we found misidentifications in public databases
171
likely resulting from strong morphological similarities in shrimps and lack of consensus on
172
systematic relationships. Barcoding results from this study show promise for application to species
173
identification (e.g. diet studies, forensics, etc.) and for resolving biogeographic distributions of these
174
ecologically important crustaceans.
175
176
METHODS
177
DATABASE MINING AND SAMPLE COLLECTION
178
Samples of Bering flounder and flathead sole were obtained from juvenile and adult specimens previously
179
collected by the AFSC and voucher tissue samples from the specimen reference collection at the
8
180
University of Washington Fish Collection (UWFC)
181
http://www.washington.edu/burkemuseum/collections/ichthyology/
182
We initially proposed to obtain sequences from a minimum of 10 individuals per each of 30 species of
183
sculpins. Previous experience (Canino et al. 2011) had indicated that this minimum number was
184
sufficient to determine whether a restriction site was fixed or polymorphic at low frequencies, an
185
important consideration for RFLP protocol development. Requests for these 30 species were submitted to
186
summer field surveys conducted by NMFS in 2013 off the west coast of Oregon and Washington, Gulf of
187
Alaska, Aleutian Islands and eastern Bering Sea, but we did not obtain minimal sample sizes in some
188
cases. As with the flatfishes, voucher specimens from the UWFC were obtained when available. COI
189
sequences from voucher and field-collected specimens were compared to those queried from GenBank
190
and FISH-BOL to ensure correct species identification. Shrimps were collected in the Gulf of Alaska,
191
Aleutian Islands and in the Chukchi Sea using a bottom trawl (Fig. 2). Whole specimens were
192
immediately preserved in 95% ethanol.
Figure 2. Collection locations for caridean shrimps.
193
194
DNA EXTRACTION, POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION AND SEQUENCING
195
DNA extractions of muscle or finclip tissues preserved in ethanol were performed using QIAGEN
196
DNeasy kits (QIAGEN, Valencia, CA) following the manufacturer’s protocol. DNA extractions and PCR
197
set-up were conducted in a PCR-free laboratory. For flatfish and sculpins a 739 base pair (bp) segment of
198
COI was PCR-amplified using a universal primer cocktail, C_FishF1t1-C_FishR1t1 (Ivanova et al. 2007),
199
containing both forward and reverse primers (Qiagen Multiplex Plus kit and Q-solution was used instead
9
200
of individual reagents in the PCR protocol). The following thermal cycler conditions were modified from
201
Ivanova et al. (2007): an initial denaturation step of 5 min at 95 °C was followed by 32 cycles of 30 s at
202
95 °C, 90 s at 57 °C, and 90 s at 72 °C. A final 10 min extension at 68 °C was added to the end of the
203
thermal cycler profile.
204
For the two flatfish species with small to intermediate egg sizes (sister taxa Hippoglossoides elassodon
205
and H. robustus), three mitochondrial genes, NADH dehydrogenase subunit 1 (ND1), 12S rRNA, 16S
206
rRNA, were sequenced for the potential development of PCR-RFLP protocols. In addition, we examined
207
a larger number of COI and cyt b sequences than we reported previously (Canino et al. 2011). Between
208
three and 23 individuals per species were sequenced for each gene region (Table 1). We were unable to
209
obtain 10 sequences each for the 16S, 12S and ND1 regions for this species pair, despite using ND1
210
primers with greater specificity in flatfish (Roje 2010) and primers for 12S (Palumbi et al. 1991) and 16S
211
(Palumbi 1996) mtDNA that amplify well in other marine fishes. We suspect lack of primer specificity,
212
rather than DNA quality, as the primary reason for PCR failures since the majority of samples amplified
213
robustly for COI and cytb gene regions.
214
215
Table 2. Mitochondrial DNA gene regions (amplicon length in base pairs) sequenced and sample sizes
for two Hippoglossoides species from the eastern North Pacific Ocean.
mtDNA gene region
Common name
Species
ND1 (831)
12S (389)
16S (576)
cytb (753)
COI (573)
flathead sole
H. elassodon
5
8
8
11
23
Bering flounder
H. robustus
8
3
4
9
23
216
217
In addition, we examined a larger number of COI and cyt b sequences than we reported previously
218
(Canino et al. 2011) to determine whether sequence data could be used to distinguish between the two
219
Hippoglossoides species. COI amplification followed the same protocol as used for sculpins and a ~1100
220
bp fragment of cyt b was PCR-amplified using primers developed by Hyde & Vetter (2007).
221
Total genomic DNA from shrimp specimens was extracted from a small piece of abdominal muscle tissue
222
using a Qiagen DNeasy Blood & Tissue Kit. Three primer pairs were used for PCR amplification of a ~
223
700 bp region of the COI gene, depending upon the taxa (Table 3). PCR amplifications for each primer
224
pair were carried out in 12.5 μL reactions following Radulovici et al. (2009) using a thermal cycler
225
profile modified from Radulovici et al. (2009): 1 min at 94 °C, 35 cycles of 40 sec at 94 °C, 40 s at 48 °-
226
54 °C depending on taxa (Table 3), and 1 min at 72 °C, and a final step of 5 min at 72 °C.
227
PCR products from fish and shrimp samples were sequenced using an ABI 3730 automated sequencer and
228
ABI Big Dye chemistry version 3.1 (Applied Biosystems, Inc). For fish samples M13 forward (5’-
10
229
TGTAAAACGACGGCCAGT-3’) and reverse (5’-CAGGAAACAGCTATGAC-3’) primers were used to
230
generate COI sequences following Ivanova et al. (2007). For shrimp samples and all other gene regions
231
(ND1, 12S, 16S, and D-loop) the same primers used in the PCR amplifications were used for sequencing.
232
Sequences were aligned in the computer program SEQUENCHER version 4.9 (Gene Codes Corp., Ann
233
Arbor, MI). COI sequences from sculpins were compared to the public database of reference sequences
234
in GenBank using the BLAST (Basic Local Alignment Search Tool) query algorithm.
235
236
237
Table 3. Primers and annealing temperatures (°C) used to PCR-amplify a portion of the mitochondrial
COI region in shrimps.
Common name
Species
sevenspine bay shrimp
Crangon septemspinosa
54
HCO2198
Folmer et al. (1994)
Alaska bay shrimp
C. alaskensis
50
CrustF1
Costa et al. (2007)
Crangon franciscorum angustimana
C. f. angustimana
as above
sand shrimp
C. crangon
as above
ridged crangon
C. dalli
as above
gray shrimp
Neocrangon communis
abyssal crangon
(°C) Primer
N. abyssorum
50
50
Reference
CrustDF1
Radulovici et al. ( 2009)
CrustDR1
Radulovici et al. ( 2009)
as above
238
239
PCR- RFLP PROTOCOL DEVELOPMENT
240
Restriction site mapping for Pacific halibut and Greenland halibut sequences from reference specimens
241
were generated in BioEdit 7.0.5.3 (Hall, 1999) to identify informative sites for PCR-RFLP protocols. The
242
presence of at least one restriction site in the mitochondrial DNA of each species was one criterion for the
243
selection of candidate restriction enzymes. This precaution greatly reduces species misidentification due
244
to mutational loss of a restriction site or failed digest reaction. Restriction site mapping for these two
245
sequences indicated that the restriction enzyme Tsp45I (recognition site ’GTSAC) could discriminate
246
them by PCR-RFLP (Table 4).
247
Restriction digests were performed in 25 uL volume reactions with 5 uL of PCR product, 1X buffer, and
248
10 units (U) of restriction enzyme Tsp45I (New England Biolabs Inc.). Digests were incubated at 65◦C for
249
1 hour and sample fragment patterns were scored visually using 3% agarose gel electrophoresis and
250
ethidium bromide stain. In order to disrupt binding of the restriction enzyme to the DNA substrate after
11
251
digestion and to ensure a more consistent migration rate of the DNA during electrophoresis (Weber and
252
Osborn, 1969), sodium dodecyl sulfate (SDS) was added for a final sample concentration of 0.1% SDS.
253
Shrimp sequences from forward and reverse directions were assembled using SEQUENCHER and
254
aligned using ClustalW as implemented in BioEdit. Sequence divergences were calculated using the
255
Kimura 2-parameter (K2P) distance method (Kimura 1980). A neighbor-joining (NJ) tree was constructed
256
using the bootstrap (BS) procedure with 10000 replications as implemented in MEGA 5.05. A haplotype
257
accumulation curve with 95% confidence intervals was assembled using the program R-package SPIDER
258
v1.1 to assess haplotype diversity in our samples.
259
260
261
Results
PCR-RFLP PROTOCOL DEVELOPMENT - FLATFISHES
262
A 739 bp region of mitochondrial COI
263
sequence was obtained for 27 Pacific
264
halibut and 23 Greenland halibut (Table 4).
265
A total of 50 halibut were tested, either by
266
restriction enzyme digests or nucleotide
267
sequence data in restriction enzyme
268
mapping analysis. Fragments visualized by
269
electrophoresis successfully resolved
270
expected haplotype patterns (Fig. 3).
271
Greenland halibut had an additional low-
272
frequency haplotype caused by loss of a
273
restriction site, but the RFLP pattern was
274
distinct from Pacific halibut.
1 2
3
4
5
6
7
8
9
10
Figure 3. PCR-RFLP banding patterns generated with
the restriction enzyme Tsp45I for Pacific halibut
(lanes 2-5) and Greenland Halibut (lanes 6 -9).
Molecular size standards are in lanes 1 and 10.
Arrow indicates the 750 base pair (bp) bands.
275
12
276
277
278
Table 4. COI restriction fragment length profiles for Pacific halibut and Greenland halibut following
digests with the enzyme Tsp45I. The number of restriction sites and resultant fragment lengths are given
for observed haplotypes.
Common name
Species name
n samples
n cut sites
Fragment sizes (bp)
Pacific halibut
Hippoglossus stenolepis
27
3
355, 157, 147, 80
Greenland halibut
Reinhardtius hippoglossoides
21
2
505, 147, 87
Reinhardtius hippoglossoides
2
1
592, 147
279
280
An 831 bp fragment of ND1 was PCR-amplified for one to` five individuals of flathead sole and Bering
281
flounder. Sequence data analysis showed this congeneric pair shared multiple haplotypes (Table 5),
282
suggesting this gene region is uninformative for resolving species identity.
283
284
Table 5. Sampling locations, sample sizes (n) and mitochondrial gene ND1 haplotype frequencies for
Bering flounder and flathead sole.
Haplotype frequency
Species
Location
n
A
B
C
D
E
F
G
flathead sole
Central Bering Sea
1
1
-
-
-
-
-
-
Bering Sea Slope
3
2
1
-
-
-
-
-
Salish Sea
1
-
-
1
-
-
-
-
Chukchi Sea
5
2
-
-
-
1
1
1
Central Bering Sea
3
1
1
-
1
-
-
-
Bering flounder
285
286
Sequence data from mitochondrial 12S, 16S and cyt b genes were also not diagnostic at the species level
287
for this pair. We were unable to PCR- amplify the control region using Hyde and Vetter’s (2007) primers.
288
Analyses of a 573 bp fragment of COI sequence data revealed unique haplotypes of H. robustus, however
289
there is a 99% sequence similarity between the most divergent of these haplotypes and the most common
290
shared haplotype, E10, found in > 50% of H. robustus and H. elassodon samples (Table 6).
291
292
BARCODING OF SCULPINS
293
A total of 252 sculpin sequences from 32 species were generated from voucher specimens obtained from
294
field samples or from the University of Washington Fish collection (Table 7). Voucher sequences from 14
295
species were correctly identified with ≥ 99% sequence similarity to entries in GenBank. However,
296
sequences from 12 of 32 species had higher than expected sequence similarity scores (99% or 100%) with
297
congeneric species and eight of our voucher specimen sequences were not found in Genbank.
13
298
299
Table 6. Sampling locations, sizes (n) and mitochondrial COI haplotype frequencies for 46 Hippoglossoides sp.samples. BS = Bering Sea, GOA =
Gulf of Alaska, AI = Aleutian Islands.
Haplotype
Species
Location
flathead
sole (H.
Central
elassodon)
BS
Bering
flounder
(H.
robustus)
n R9 E10 R11 E12 ER13 E19 R21 E22 E23 E24 E25 E26 E27 ER28 R29 ER30 R31 E32
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
BS Slope
8
-
5
-
1
-
1
-
-
1
-
-
-
-
-
-
-
-
-
GOA
6
-
4
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
AI
3
-
2
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
Salish
Sea
6
-
4
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
N.
Chukchi
Sea
7
1
3
-
-
-
-
-
-
-
-
-
-
-
-
1
1
1
-
S.
Chukchi
Sea
6
-
4
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
N. BS
2
1
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
Central
BS
8
-
5
1
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
14
300
301
302
Table 7. Sculpin voucher specimen sequence similarity scores (% maximum identity) to entries in
GenBank. Only scores ≥ 99% are shown. * denotes no entry in GenBank. Sequence similarity to
congeneric species given in parentheses.
Family
Common name
Species name
n
% identity
Cottidae
Red Irish Lord
Hemilepidotus hemilepidotus
12
99 (99, H. zapus)
Cottidae
Yellow Irish Lord
Hemilepidotus jordani
12
99, 100
Cottidae
butterfly sculpin
Hemilepidotus papilio
11
99, 100
Cottidae
Longfin Irish Lord
Hemilepidotus zapus
9
99 (99, H. jordani)
Cottidae
spectacled sculpin
Triglops scepticus
1A
*
Cottidae
roughspine sculpin
Triglops macellus
1
99
Cottidae
scissortail sculpin
Triglops forficatusB
10
99 (99, T. pingelii)
Cottidae
ribbed sculpin
Triglops pingelii
3
99, 100
Cottidae
hookhorn sculpin
Artediellus pacificus
1
*
Cottidae
threaded sculpin
Gymnocanthus pistilliger
11
99 (99, G. intermedius)
Cottidae
armorhead sculpin
Gymnocanthus galeatus
12
99, 100
Cottidae
antlered sculpin
Enophrys diceraus
5
100
Cottidae
buffalo sculpin
Enophrys bison
3
99
Cottidae
warty sculpin
Myoxocephalus verrucosus
7
* (99, 100, M. scorpius)
Cottidae
plain sculpin
Myoxocephalus jaok
13
99, 100 (99, M brandtii; 99, M. stelleri)
Cottidae
great sculpin
Myoxocephalus
polyacanthocephalus
12
99, 100 (99, M. stelleri )
Cottidae
northern sculpin
Icelinus borealis
9
99, 100
Cottidae
threadfin sculpin
Icelinus filamentosus
4
99
Cottidae
sponge sculpin
Thyriscus anoplus
1
*
Cottidae
uncinate sculpin
Icelus uncinalis
11
* (99, I. spatula; 100, I. spiniger)
Cottidae
thorny sculpin
Icelus spiniger
8
99, 100 (99, I. spatula)
Cottidae
spatulate sculpin
Icelus spatula
7
99 (99, I. spiniger)
Cottidae
roughskin sculpin
Rastrinus scutiger
7
*
Cottidae
blacknose sculpin
Icelus canaliculatus
3
*
Cottidae
wide-eye sculpin
Icelus euryops
3
*
15
303
304
Table 7 (continued).
Family
Common name
Species name
n
% identity
Hemitripteridae
bigmouth sculpin
Hemitripterus bolini
14
99, 100
Psychrolutidae
darkfin sculpin
Malacocottus zonurus
19
99 (99, M. kincaidi)
Psychrolutidae
spinyhead sculpin
Dasycottus setiger
18
99, 100
Psychrolutidae
blackfin sculpin
Malacocottus kincaidi
6
99 (99, 100, M. zonurus)
Psychrolutidae
tadpole sculpin
Psychrolutes paradoxus
Psychrolutidae
smoothcheek sculpin
Eurymen gyrinus
1
99
Rhamphocottidae
grunt sculpin
Rhamphocottus richardsonii
7
99, 100
A
11
A
99, 100
B
one sequence contains an ambiguity code; species name formerly T. forficate.
305
306
BARCODING OF CARIDEAN SHRIMP Mean intraspecific divergences among shrimps were less than 1%
307
except for Neocrangon communis, which was 4%. This was due, in part, to Chukchi Sea specimens
308
having as much as 6.9% sequence divergence from the British Columbia specimens (Table 8). Sequence
309
divergence between N. communis and N. abyssorum exceeded 15%. Crangon dalli had the highest
310
interspecific divergence estimates (all over 10%) and did not show any intraspecific variation (Table 1).
311
The lowest interspecific divergence was seen between C. crangon and C.angustimana (mean 5.7%).
312
Specimens identified as C. septemspinosa from the Chukchi Sea diverged from topotypic specimens
313
(i.e.specimens collected from within the same geographic area as the type specimen) in the Northwest
314
Atlantic by 9.1% [represented as Crangon sp. (CS) in Table 7 and highlighted in yellow in Fig. 4] and
315
possibly represent a new cryptic species. Based upon our results, it appears that Crangon septemspinosa
316
may not occur in Alaskan waters. Sequence data from an individual identified as C. septemspinosa by one
317
of us (Drumm) from the Gulf of Alaska grouped with two specimens of Crangon alaskensis from British
318
Columbia (highlighted in blue in Fig. 4) and was likely misidentified. Crangon septemspinosa is very
319
similar to C. alaskensis but lacks a keel on the fifth abdominal segment, which when present can be very
320
faint and difficult to see. Another COI sequence (GenBank Accession #AF125416.1) that came from a
321
specimen identified as C. septemspinosa grouped with C. crangon on the NJ tree [shown as Crangon sp.
322
(CA)], but with low bootstrap support. This specimen was purchased at a marine supply company in
323
California, but its origin is unknown (Shank et al. 1999).
324
We emphasize that K2P distance estimates reported here are based upon relatively small groups of
325
samples. A haplotype accumulation curve (Fig. 5) revealed that the genetic diversity was not fully
326
sampled, as indicated by the steep slope and lack of an asymptote, and that more accurate estimates of
327
genetic intraspecific variation within and interspecific divergence among these species would require
328
larger sample sizes of approximately 125-150 individuals each.
16
Figure 4. Neighbor joining tree for COI sequences from Crangon and Neocrangon. Numbers at
nodes represent percent bootstrap support. NWA, northwest Atlantic Ocean; GOA, Gulf of Alaska;
BC, British Columbia; CS, Chukchi Sea; AI, Aleutian Islands; CA, California.
15
0
5
10
h haplotypes
20
25
30
329
0
20
40
60
80
100
n individuals
Figure 5. Expected haplotype accumulation curve (in blue). Red
dashed lines represent 95% confidence limits.
17
330
331
Table 8. Intra- (bold) and interspecific Kimura 2-parameter (K2P) genetic divergences for Crangon and Neocrangon species. Ranges are given for
comparisons involving multiple specimens. Chukchi Sea (CS), California (CA).
C.
septemspinosa
C. sp
(CA)
C.
angustimana
C. sp
(CS)
C. crangon
C. dalli
N.
communis
C. septemspinosa
0.0 – 0.9
C. alaskensis
7.6 – 8.8
0.0 – 0.3
8.3 – 10.01
6.0 – 7.1
0.0 – 0.5
8.0 – 9.1
6.8 – 6.9
6.7 – 7.1
0.0
C. sp (CS)
11.3 – 12.5
7.9 – 8.1
8.7 – 9.1
9.4
-
C. crangon
8.1 – 9.5
6.4 – 6.8
5.8 – 6.6
5.6 – 5.9
7.6 – 8.0
0.0 – 0.5
C. dalli
14.2 – 15.6
13.2 – 14.0
12.9 –
13.6
13.4 – 13.6
14.4
11.9 – 12.7
0.0
N. communis
19.2 – 23.6
17.3 – 19.8
15.8 –
19.2
17.7 – 20.1
20.2 – 22.0
18.2 – 20.6
18.3 – 18.9
0.3 – 6.9
N. abyssorum
23.4 – 24.6
23.9 – 24.6
22.7 –
24.3
21.6 – 22.0
23.9
19.7 – 21.0
22.5 – 22.6
17.9 – 20.2
C. sp (CA)
C. angustimana
332
C.
alaskensis
1
range of divergences between C. septemspinosa from the NW Atlantic and specimens identified as C. septemspinosa in the Chukchi Sea.
18
N.
abyssorum
0.3
333
Discussion
334
The exponential growth of DNA barcoding studies during the last decade has generally demonstrated the
335
discriminatory power of the method for use in species identification and discovery across a wide array of
336
metazoan groups (see Taylor and Harris 2012 for review). Barcoding of wild populations can identify
337
units of concern or action in species conservation (Krishnamurthy and Francis 2012) and provide at least
338
a measure of species biodiversity (e.g. Andersen et al 2012). Barcoding approaches have been applied in
339
a variety of studies ranging from identifying eggs and larval forms to species identity of food products
340
(Rasmussen Hellberg et al. 2010) to testing for invasive species (Collins et al. 2013). However, DNA
341
barcoding of mitochondrial COI faces considerable criticisms and challenges, not only because it is
342
largely uninformative for some groups (e.g. fungi) but also because barcoding results sometimes clash
343
with established concepts of biological species (Taylor and Harris 2012). Recent advances in next-
344
generation sequencing (NGS) technology (Hohenlohe et al. 2011) provides a cost-effective means to scan
345
large portions of the nuclear genome rather than be confided to the single mitochondrial locus. Perhaps
346
the greatest challenge posed by NGS is how to cope with the enormous volumes of data it generates, and
347
whether the contemporary global barcoding effort can incorporate new methods (Taylor and Harris 2012).
348
Overall, our results validated the general utility of DNA barcoding to species identification through PCR-
349
RFLP development, but we were not able to fully accomplish our goal of discriminating all of the seven
350
flatfish species listed in the original proposal. A PCR-RFLP protocol was developed to identify
351
Greenland halibut and Pacific halibut, the two ‘large egg’ species in the group. Two RFLP haplotypes
352
identified in Greenland halibut thus far are unique and cannot be mistaken for Pacific halibut. However, a
353
screening of larger samples is warranted in order to document all RFLP haplotypes. We have already
354
developed PCR-RFLP laboratory protocols to discriminate among the three species of Limanda which
355
have ‘small eggs’ using a double-digest PCR-RFLP protocol on COI and cyt b genes (Paquin et al. 2014),
356
although we were unable to develop a single enzyme assay based upon additional sequencing conducted
357
in this study. Most single nucleotide polymorphisms (SNPs) we discovered did not create restriction sites,
358
rendering them useless for this approach. Another molecular approach directly targeting these SNPs,
359
such as multiplexed suspension arrays (Gleason and Burton 2012), could be developed for high-
360
throughput egg identification in these species.
361
DNA sequencing of the mitochondrial ND1 region in two Hippoglossoides species with ‘intermediate
362
egg’ size (Table 1) was not informative for species identification, a result similar to what we reported for
363
the COI and cyt b regions in an earlier study (Canino et al. 2011). A major obstacle to development of
364
PCR-RFLP protocols in the Limanda and Hippoglossoides species was the lack of diagnostic restriction
365
sites. One shortcoming of the PCR-RFLP approach is that it can only screen variation at restriction sites,
19
366
which are in turn only a small fraction of fixed site differences among species. Previous examination of a
367
605 bp segment of COI of H. elassodon and H. robustus revealed low nucleotide diversity and low
368
divergence between these two species (Canino et al. 2011). Congeneric K2P distances among the three
369
Limanda species ranged from 5.6% -14.5%, within ranges reported for other marine species (Ward et al.
370
2009; Zhang and Hanner 2011), while the estimate between H. elassodon and H. robustus was only 0.5%.
371
A similar lack of interspecific divergence at cyt b led Kartavtsev et al. (2007, 2008) to call for
372
synonymization of the two species. The incomplete sorting of mitochondrial lineages observed could be
373
due to several factors, including a relatively short evolutionary span since speciation or perhaps some
374
degree of introgressive hybridization, which has been reported in other paralichthyid (Xu et al. 2009) and
375
pleuronectid (Garrett 2005) flatfishes.
376
Our efforts to create a voucher collection of sculpin COI sequences for use in diet studies highlights some
377
of the potential errors DNA barcoding can introduce and perpetuate in public databases. DNA barcoding
378
can misidentify species for multiple reasons, the most common one a lack of a barcoding ‘gap’ between
379
intraspecific variation and interspecific divergence (Fig. 1). A large degree of overlap in these two
380
sources of genetic variability has been cited in cases where barcoding success has been relatively poor
381
(Meier et al. 2006) and the use of mean interspecific distances instead of the smallest ones to set threshold
382
values for species delineation can exacerbate the problem (Meier et al. 2008). In some cases, a lack of
383
consensus on the actual number of species involved and their systematic relationships contributes to
384
initial misidentifications. Species misidentification of sequences deposited in public databases is also a
385
concern, especially when there are no accompanying voucher specimens. Many sequences obtained from
386
GenBank have no vouchers and may have been misidentified prior to submission. The fact that we
387
uncovered several instances of it suggests that the problem is not trivial, researchers should scrutinize
388
such data thoroughly and, when possible, only use sequences from voucher specimens.
389
PCR-RFLP protocols, like those developed in this study, have been the most widely used method for
390
species identification for the last 15 years (Taylor and Harris 2012) but are likely to be eclipsed by
391
emerging technologies. Next-generation DNA sequencing methods have revolutionized genome scans by
392
allowing the discovery and genotype calling of thousands of SNPs (single nucleotide polymorphisms) in
393
multiple individuals (or species) at relatively low cost (Miller et al. 2007; Pompanon et al. 2012). This
394
level of resolution would greatly eliminate ambiguity of identity in congeneric species that mitochondrial
395
COI sequences sometimes fail to resolve. Once diagnostic SNPs have been developed for species of
396
interest, low-cost, high-throughput identification of fish, or fish mixtures (e.g. processed food products),
397
can be achieved using DNA microarrays (Kochzius et al. 2008, Teletchea et al. 2008; Gleason et al.
398
2012) or other screening platforms. The incipient genomics revolution in fisheries research has the
20
399
potential for unprecedented species-level identification for resolving more intractable questions in fish
400
ecology, life-history and forensics.
401
Conclusions
402
PCR-RFLP protocols that provide quick, accurate and relatively inexpensive methods for species (or
403
genus) identification have been useful tools in fisheries science. Here we expanded upon developing
404
PCR-RFLP methods for identifying the eggs and early larvae of pleuronectid flatfishes and were
405
successful in creating a new assay to distinguish between Pacific halibut and Greenland halibut.
406
However, we fell short (as we did in a previous study) in resolving the identity of two Hippoglossoides
407
species despite extensive sequencing of multiple mtDNA gene regions. Those results raise questions
408
regarding species validity, time since speciation and the possibility of introgression between lineages.
409
Further work using nuclear DNA methods may provide additional answers. Our initial efforts to barcode
410
marine sculpins and caridean shrimps were largely successful. Some groups were less represented in the
411
data set due to low (or no) availability, but overall coverage was good. Comparisons between voucher
412
specimens we collected and the public data bases revealed multiple errors and misidentifications, a
413
situation that is likely to be perpetuated with the current surge in DNA barcoding worldwide and lack of
414
taxonomic expertise and consensus regarding systematic relationships in some groups. Our sequence data
415
have voucher specimens associated with them, allowing future researchers access to the same materials
416
should they pursue further molecular work.
417
Management Implications
418
DNA barcoding approaches such as these will be of particular value in filling knowledge gaps at the
419
AFSC, where much multi-disciplinary expertise is focused on incorporating ecosystem-oriented thinking
420
into resource management. Barcoding enhances our ability to estimate species abundance at all life
421
history stages of marine fishes and to quantify food web linkages in the northeast Pacific Ocean and
422
Bering Sea. These abilities increase our capacity to detect and understand how external forces such as
423
fishing and climate change may cause shifts in ecosystem composition and function. On a practical level,
424
species identification through DNA barcoding offers a potential suite of applications to management
425
regulatory decisions, and especially in their enforcement. Barcoding has been successfully used to detect
426
seafood mislabeling, fraud and illegal fishing practices.
427
21
428
Publications
429
Drumm, D, Canino, MF, Buckley T, Paquin M. DNA barcoding analysis of marine caridean shrimps from
430
Alaska. Presented at AMSS meeting, January 20-24, 2014. Anchorage, Alaska.
431
Paquin, M.M., Buckley, T.W., Hibpshman, R.E., and Canino. M.F. 2014. DNA-based identification
432
methods of prey fish from stomach contents of 12 species of eastern North Pacific groundfish.
433
Deep Sea Research Part I: Oceanographic Research Papers 85, 110-117.
434
Canino MF, Paquin MM, Matarese AC. In prep. PCR-RFLP identification of Bathymaster sp. larvae from
435
the Gulf of Alaska.
436
437
Outreach
438
EXHIBITS/DEMONSTRATION PROJECT DEVELOPED
439
PI Canino designed an outreach activity, largely targeted towards children 5-12 years old, for use in a
440
‘hands-on exhibition’ setting. The exhibit is constructed as a fold-out box. A display panel inside explains
441
that NOAA scientists study gut contents of fish in order to understand food webs. A simple food web is
442
depicted with a stuffed great white shark (“Shredder”) as the apex predator and two fish species (red fish
443
and blue fish) as well as two crab species as prey items. The concept of using DNA (in this case
444
mitochondrial DNA) for identification of species is introduced and depicted in another display panel.
445
Children are then invited to participate in examining Shredder’s gut contents. They unzip and empty the
446
shark “gut”, enumerating the number of intact fish and crabs. There are four fish “skeletons” and crab
447
carapaces that can’t be identified visually and the children are engaged in a matching exercise of DNA
448
sequences to known sequences of fish and crabs suspended within plexiglass tubes attached to the wall of
449
the box.
450
PI Canino brought the activity to the SeaLife center in Seward and the Anchorage Museum in June, 2014,
451
demonstrating it for staff and the public. He then turned it over to the NPRB outreach director, Abigail
452
Enghirst, for loaning to the outreach community.
453
Acknowledgements
454
The authors are grateful to scientists in the Resource and Conservation Engineering Division (RACE) at
455
the Alaska Fisheries Science Center for diligent sample collection during annual field surveys. We also
456
thank Dr. Theodore Pietsch and Katherine Maslenikov at the University of Washington Fish Collection at
457
the Burke Museum of Natural History and Culture for access to voucher specimens and James Orr and
458
Duane Stevenson for examination of voucher specimens.
22
459
Literature cited
460
Andersen, K., Bird, K.L., Rasmussen, M., et al. 2012 Meta-barcoding of 'dirt' DNA from soil reflects
461
462
vertebrate biodiversity. Molecular Ecology 21:1966-1979.
Avise, J. C., and D. Walker, 1999 Species realities and numbers in sexual vertebrates: Perspectives from
463
an asexually transmitted genome. Proceedings of the National Academy of Sciences of the United
464
States of America 96: 992-995.
465
466
467
468
469
470
471
Barrett, R.D.H., and Hebert, P.D.N. 2005 Identifying spiders through DNA barcodes. Canadian Journal of
Zoology 83: 481-491.
Blankenship, L.E., and Yayanos, A.A. 2005. Universal primers and PCR of gut contents to study marine
invertebrate diets. Molecular Ecology 14: 891-899.
Brown T.A., and Clayton, D.A. 2002. Release of replication termination controls mitochondrial DNA
copy number after depletion with 2’,3’-dideoxycytidine. Nucleic Acids Research 30:2004-2010.
Canino M, Buckley T, Paquin M, Hibpshman R. 2011. Developing rapid, accurate, DNA-based
472
identification of larvae and dietary components of commercially important species. North Pacific
473
Research Board Final Report Project 924. 28 p
474
475
476
Chiesa, S., Filonzi, L., Vaghi. M., Papa R, and Marzano, F.N. 2013. Molecular barcoding of an atypical
cyprinid population assessed by cytochrome b gene sequencing. Zoological Science 30:408-413.
Collins, R.A., Armstrong, K.F., Holyoake, A.J., and Keeling, S. 2013. Something in the water:
477
biosecurity monitoring of ornamental fish imports using environmental DNA. Biological
478
Invasions 15:1209-1215.
479
480
481
482
483
Costa, F.O., DeWaard, J.R., Boutillier, J., et al.. 2007. Biological identifications through DNA barcodes:
the case of the Crustacea. Canadian Journal of Fisheries and Aquatic Sciences 64, 272-295.
de Oliveira Ribeiro, A., Caires, R.A., Mariguela, T.C., et al. 2012. DNA barcodes identify marine fishes
of Sao Paulo State, Brazil. Molecular Ecology Resources 12:1012-1020.
Drumm, D.T., Maslenikov, K.P., VanSyoc, R.J., Orr, J.W., Lauth, R.R., Stevenson, D.E., and Pietsch,
484
T.W. (in review) An annotated checklistof the marine macroinvertebrates of Alaska. NOAA
485
Professional Papers, 942 pp.
486
487
Hyde, J. R., and Vetter, R.D. 2007. The origin, evolution, and diversification of rockfishes of the genus
Sebastes (Cuvier). Molecular Phylogenetics and Evolution 44: 790-811.
23
488
489
490
491
492
493
494
495
Garcia-Morales, A.E., Elias-Gutierrez, M. 2013. DNA barcoding of freshwater Rotifera in Mexico:
Evidence of cryptic speciation in common rotifers. Molecular Ecology Resources 13:1097-1107.
Garrett, D. L., and Buth, D. 2005. A new intergeneric hybrid flatfish (Pleuronectiformes: Pleuronectidae)
from Puget Sound and adjacent waters. Copeia 2005:673-677.
Gleason, L.U., and Burton, R.S. 2012. High-throughput molecular identification of fish eggs using
multiplex suspension bead arrays. Molecular Ecology Resources 12:57-66.
Hall,T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT. Nucleic Acids Symposium Series 41:95-98.
496
Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., and Hallwachs, W. 2004a. Ten species in one:
497
DNA barcoding reveals cryptic species in the neotropical butterfly Astraptes fulgerator.
498
Proceedings of the National Academy of Sciences of the United States of America 101: 14812-
499
14817.
500
501
502
Hebert, P.D.N., Soteckle, M.Y., Zemlak, T.S., and Francis, C.M. 2004b. Identification of birds through
DNA barcodes. PLoS Biology 2:1657-1663.
Hohenlohe. P.A., Amish, S.J., Catchen, J.M., Allendorf, F.W., and Luikart, G. 2011. Next-generation
503
RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and
504
westslope cutthroat trout. Molecular Ecology Resources 11, 117-122.
505
Hyde, J.R., Lynn, E., Humphreys, R., Musyl, M., West, A.P., et al. 2005 Shipboard identification of fish
506
eggs and larvae by multiplex PCR, and description of fertilized eggs of blue marlin, shortbill
507
spearfish, and wahoo. Marine Ecology Progress Series 286: 269-277.
508
509
510
Ivanova, N. V., Zemlak, T.S., Hanner, R.H., and Hebert, P.D.N. 2007. Universal primer cocktails for fish
DNA barcoding. Molecular Ecology Notes 7: 544-548.
Jackson, J.K., Battle, J.M., White, B.P., et al. 2014. Cryptic biodiversity in streams: a comparison of
511
macroinvertebrate communities based on morphological and DNA barcode identifications.
512
Freshwater Science 33:312-324.
513
Kartavtsev, Y.P., Park, T.-J., and Vinnikov, K.A. et al. 2007. Cytochrome b (Cyt-b) gene sequence
514
analysis in six flatfish species (Teleostei, Pleuronectidae), with phylogenetic and taxonomic
515
insights. Marine Biology 152:757-773.
516
517
Kartavtsev, Y.P., Park, T.-J., and Lee, J.-S. et al. 2008. Phyhlogenetic inferences introduced on
cytochrome b gene sequences data for six flatfish species (Teleostei, Pleuronectidae) and species
24
518
synonymy between representatives of genera Pseudopleuronectes and Hippoglossoides from far
519
eastern seas. Russian Journal of Genetics 44:451-458.
520
521
522
523
524
525
526
527
528
KeskIn, E., and Atar, H.H. 2013. DNA barcoding commercially important fish species of Turkey.
Molecular Ecology Resources 13:788-797.
Kimura, M. 1980. A simple method of estimating evolutionary rate of base substitutions through
comparative studies of nucleotide sequences. Journal of Molecular Evolution 16:111–120.
Kochzius. M, Noelte, M., Weber, H., et al. 2008. DNA microarrays for identifying fishes. Marine
Biotechnology 10, 207-217.
Krishnamurthy, K.P., Francis, R.A. 2012. A critical review on the utility of DNA barcoding in
biodiversity conservation. Biodiversity and Conservation 21:1901-1919.
Leray, M., Yang, J.Y., Meyer, C.P., et al. 2013. A new versatile primer set targeting a short fragment of
529
the mitochondrial COI region for metabarcoding metazoan diversity: application for
530
characterizing coral reef fish gut contents. Frontiers in Zoology 10:34.
531
532
533
Meier, R., Shiyang, K., Vaidya, G., and Ng, P.K.L. 2006. DNA barcoding and taxonomy in Diptera: a tale
of high intraspecific variability and low identification success. Systematic Biology 55:715-728.
Meier, R., Zhang, G., and Ali, F. 2008. The use of mean instead of smallest interspecific distances
534
exaggerates the size of the “barcoding gap” and leads to misidentification. Systematic Biology
535
57:809-813.
536
Miller, M.R., Dunham, J.P., Amores, A., Cresko, W.A., and Johnson, E.A. 2007. Rapid and cost-effective
537
polymorphism identification and genotyping using restriction site associated DNA (RAD)
538
markers. Genome Research 17: 240-248.
539
Morgan, J.A.T., Harry, A.V., Welch, D.J., et al. 2012. Detection of interspecies hybridisation in
540
Chondrichthyes: hybrids and hybrid offspring between Australian (Carcharhinus tilstoni) and
541
common (C. limbatus) blacktip shark found in an Australian fishery. Conservation Genetics
542
13:455-463.
543
Nice, C.C., and Shapiro, A.M. 2001. Population genetic evidence of restricted gene flow between host
544
races in the butterfly genus Mitoura (Lepidoptera: Lycaenidae). Annals of the Entomological
545
Society of America 94:257-267.
25
546
Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L., and Grabowski, G. 1991. The simple
547
fool’s guide to PCR,. V.2.0. Spec Publ University of Hawaii, Department of Zoology & Kewalo
548
Marine Laboratory, Honolulu.
549
Palumbi, S.R. 1996. Nucleic acids II: the polymerase chain reaction. In: D.M. Hillis, C. Moritz, and B.K
550
Mable BK (Editors) Molecular Systematics, pp. 205–247. Sinauer & Associates Inc., Sunderland,
551
Massachusetts.
552
553
554
Pompanon, F., Deagle, B.E., Symondson, W.O., et al. 2012. Who is eating what: diet assessment using
next generation sequencing. Molecular Ecology 21:1931-1950.
Radulovici, A.E., Sainte-Marie, B., and Dufresne, F. 2009. DNA barcoding of marine crustaceans from
555
the Estuary and Gulf of St. Lawrence: a regional-scale approach. Molecular Ecology Resources 9
556
(Suppl. 1):181-187.
557
Rasmussen Hellberg, R.S., Morrissey, M.T., Hanner, R.H. 2010. A multiplex PCR method for the
558
identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in
559
North America. Journal of Food Science 75:C595-C606.
560
Roje, D.M. 2010. Incorporating molecular phylogenetics with larval morphology while mitigating the
561
effects of substitution saturation on phylogeny estimation: A new hypothesis of relationships for
562
the flatfish family Pleuronectidae (Percomorpha: Pleuronectiformes). Molecular Phylogenetics
563
and Evolution 56: 586-600.
564
Shank, T.M., Black, M.B., Halanych, K.M., Lutz, R.A., and Vrijenhoek, R.C. 1999. Miocene radiation
565
of deep-sea hydrothermal vent shrimp (Caridea: Bresiliidae): Evidence from mitochondrial
566
cytochrome oxidase subunit I. Molecular Phylogenetics and Evolution 13:244-254.
567
568
569
Shokralla, S., Spall, J.L., Gibson, J.F., and Hajibabaei, M. 2012. Next-generation sequencing technologies
for environmental DNA research. Molecular Ecology 21:1794-1805.
Matzen da Silva J, Creer S, dos Santos A, Costa AC, Cunha MR, et al. (2011) Systematic and
570
Evolutionary Insights Derived from mtDNA COI Barcode diversity in the Decapoda (Crustacea:
571
Malacostraca). PLoS ONE 6(5): e19449. doi:10.1371/journal.pone.0019449
572
Spies, I.B., Gaichas, S., Stevenson, D.E., Orr, J.W, and Canino, M.F. 2006. DNA-based identification of
573
Alaska skates (Amblyraja, Bathyraja and Raja: Rajidae) using cytochrome c oxidase subunit I
574
(COI) variation. J. Fish Biol. 69 (Suppl. B): 283-292.
575
576
Taylor, H.R., and Harris, W.E. 2012. An emergent science on the brink of irrelevance: a review of the
past 8 years of DNA barcoding. Molecular Ecology Resources 12:377-388.
26
577
578
579
Teletchea, F. 2009 Molecular identification methods of fish species: reassessment and possible
applications. Reviews in Fish Biology and Fisheries 19: 265-293.
Vassilenko, S.V., and Petryashov, V.V. (eds.). 2009. Illustrated Keys to Free-Living Invertebrates of
580
Eurasian Arctic Seas and Adjacent Deep Waters, Vol. 1. Rotifera, Pycnogonida, Cirripedia,
581
Leptostraca, Mysidacea, Hyperiidea, Caprellidea, Euphausiacea, Dendrobranchiata, Pleocyemata,
582
Anomura, and Brachyura. Alaska Sea Grant, University of Alaska Fairbanks, 1-186.
583
584
Ward, R.D., Hanner, R., and Hebert, P.D.N. 2009 The campaign to DNA barcode all fishes, FISH-BOL.
Journal of Fish Biology 74: 329-356.
585
Weber, K., and Osborn, M. 1969. Reliability of molecular weight determinations by dodecyl sulfate-
586
polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244: 4406-4412.
587
Xu, D., You, F., Wu, Z., Li, J., and Ni, J., et al. 2009. Genetic characterization of asymmetric reciprocal
588
hybridization between the flatfishes Paralichthys olivaceus and Paralichthys dentatus. Genetica
589
137: 151-158.
590
591
Zhang, J-B., and Hanner, R. 2011. DNA barcoding is a useful tool for the identification of marine fishes
from Japan. Biochemical Systematics and Ecology 39:31-42.
592
27