biological identifications through dna barcodes: the case...

Biological identifications through DNA barcodes:the case of the Crustacea

Filipe O. Costa, Jeremy R. deWaard, James Boutillier, Sujeevan Ratnasingham,Robert T. Dooh, Mehrdad Hajibabaei, and Paul D.N. Hebert

Abstract: The ability of a 650 base pair section of the mitochondrial cytochrome c oxidase I (COI) gene to providespecies-level identifications has been demonstrated for large taxonomic assemblages of animals such as insects,birds, and fishes, but not for the subphylum Crustacea, one of the most diverse groups of arthropods. In this study,we test the ability of COI to provide identifications in this group, examining two disparate levels in the taxonomichierarchy — orders and species. The first phase of our study involved the development of a sequence profile for23 dominant crustacean orders, based upon the analysis of 150 species, each belonging to a different family. TheCOI amino acid data placed these taxa into cohesive assemblages whose membership coincided with currentlyaccepted boundaries at the order, superorder, and subclass levels. Species-level resolution was subsequently exam-ined in an assemblage of Decapoda and in representatives of the genera Daphnia (Cladocera) and Gammarus(Amphipoda). These studies revealed that levels of nucleotide sequence divergence were from 19 to 48 times greaterbetween congeneric species than between individuals of a species. We conclude that sequence variation in the COIbarcode region will be very effective for discriminating species of Crustacea.

Résumé : Il a été démontré que l’utilisation d’une section de 650 paires de bases du gène mitochondrial de la cyto-chrome c oxydase I (COI) permet de faire des identifications au niveau spécifique de grands ensembles taxonomiquesd’animaux tels que les insectes, les oiseaux et les poissons, mais pas encore dans le sous-phylum des crustacés, l’undes groupes les plus diversifiés d’arthropodes. Nous testons dans notre étude le potentiel de l’utilisation de COI pourfaire des identifications dans ce groupe en examinant deux niveaux disparates de la hiérarchie taxonomique — lesordres et les espèces. La première phase de notre recherche consiste en l’établissement de profils de séquences pour23 des ordres principaux de crustacés, d’après l’analyse de 150 espèces, appartenant chacune à une famille différente.Les données sur les acides aminés de COI permettent de placer ces taxons en ensembles cohésifs dont la compositioncoïncide avec les frontières couramment acceptées aux niveaux de l’ordre, du super-ordre et de la sous-classe. Nousavons ensuite étudié la détermination au niveau spécifique chez un ensemble de décapodes et chez des représentantsdes genres Daphnia (Cladocera) et Gammarus (Amphipoda). Ces études révèlent que les niveaux de divergence desséquences de nucléotides sont de 19 à 48 fois plus importants entre les espèces d’un même genre qu’entre les indivi-dus d’une même espèce. Nous concluons que la variation des séquences dans la région du code-barre de COI devraitpermettre de séparer de façon très efficace les espèces de crustacés.

[Traduit par la Rédaction] Costa et al. 295

Introduction

The ideal DNA-based identification system would employa single gene for the placement of any organism in the fulltaxonomic hierarchy from kingdom to species. The ability ofa ~658 base pair (bp) section of the mitochondrial cyto-chrome c oxidase I (COI) gene to provide this resolution hasnow been demonstrated for many animal lineages, includinglepidopterans (Hebert et al. 2003; Janzen et al. 2005;Hajibabaei et al. 2006), birds (Hebert et al. 2004), spiders

(Barrett and Hebert 2005), ants (Smith et al. 2005), andfishes (Ward et al. 2005) (but see Dasmahapatra and Mallet2006 for a discussion of success rates). Although COI ap-pears to have high potential as the foundation for an identifi-cation system, it is critical to verify that it can deliversimilar taxonomic resolution in other groups. The presentstudy addresses this issue by examining the patterning ofCOI diversity in the subphylum Crustacea, the most ancientand structurally diverse group of arthropods. The diversityof modern crustaceans, in addition to their remarkable vari-

Can. J. Fish. Aquat. Sci. 64: 272–295 (2007) doi:10.1139/F07-008 © 2007 NRC Canada

272

Received 20 January 2006. Accepted 26 October 2006. Published on the NRC Research Press Web site at http://cjfas.nrc.ca on24 February 2007.J19120

F.O. Costa.1,2 Instituto do Mar (IMAR), Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.J.R. deWaard, S. Ratnasingham, R.T. Dooh, M. Hajibabaei, and P.D.N. Hebert. Biodiversity Institute of Ontario, University ofGuelph, ON N1G 2W1, Canada.J. Boutillier. Pacific Biological Station, Fisheries and Oceans Canada, 3190 Hammond Bay Road, Nanaimo, BC V9T 6N7, Canada.1Corresponding author (e-mail: [email protected]).2Present address: School of Biological Sciences, University of Wales, Bangor, Bangor, Gwynedd, LL57 2UW, United Kingdom.

ety in morphologies, habitats, and ecologies, is reflected bytheir assignment to six classes, 48 orders, 849 different fam-ilies, and about 52 000 species (Martin and Davis 2001). Bycomparison, the more than one million described species ofinsects are assigned to about 1000 families, but just 30 or-ders and a single class (Borror et al. 1989; Nielsen 1995).

While the higher level systematics (i.e., class, subclass) ofthe Crustacea are not fully stabilized (Martin and Davis2001), ordinal boundaries have been nearly constant formore than 20 years despite ongoing morphological and mo-lecular studies. As such, this taxonomic rank represents auseful criterion to judge the ability of a DNA-based identifi-cation system to deliver coarse taxonomic placement of anunknown taxon. However, as the primary task of taxonomylies in identification of species, it is also critical to test theeffectiveness of DNA barcodes at this level. The presentstudy examines the effectiveness of a 658 bp section near the5′ end of COI (hereafter referred to as the COI barcode) asan identification tool at these two disparate levels in the tax-onomic hierarchy — orders and species. Its effectiveness atthe ordinal level was tested in a two-step process: 150 spe-cies, each in a different family and including representativesfrom 23 of the 48 crustacean orders, were sequenced to es-tablish a COI ordinal profile. The effectiveness of this pro-file in ordinal-level placements was subsequently evaluatedby determining its success in the assignment of 100 newtaxa to the proper order. The present study also sought to de-termine if the COI barcode region can enable species-levelassignments within the Crustacea; this issue was examinedin representatives of nine genera of Decapoda and in singlegenera of Cladocera and Amphipoda.

Materials and methods

Taxon sampling

COI ordinal profileOne hundred and fifty species, each belonging to a differ-

ent family and including representatives from 23 of 48 crus-tacean orders, were sequenced to establish a COI ordinalprofile (sequences for 75 of these families were acquiredfrom GenBank, see below). More than 10 families were ex-amined from each of four orders (Amphipoda, Calanoida,Decapoda, Isopoda), between four and nine families fromeight other orders (Anostraca, Cladocera, Cumacea, Diplo-straca, Harpacticoida, Podocopida, Sessilia, Stomatopoda),while one to two families were examined from the other 11orders. Sequences were also obtained for 100 additional spe-cies (53 were acquired from GenBank, see below) in fami-lies included in the profile, but belonging to different generato provide a group of test taxa (details on specimens are pro-vided in Appendix A, Tables A1 and A2).

Decapod case studyFamily- and species-level resolution was analyzed in a

data set that comprised members of the order Decapoda.Most newly analyzed decapods were collected on researchcruises led by Fisheries and Oceans Canada (DFO) off thePacific coast of Canada in 2002 and 2004, but a few specieswere collected in Resolute Bay, Nunavut; in the Gulf ofGdansk, Poland; and off Lizard Island, Australia. These

specimens were identified to the species level after collec-tion and were subsequently preserved in 100% ethanol. Intotal, sequences were newly gathered for 146 specimens,representing 57 species, and these results were merged with173 sequences from GenBank, representing 71 species(complete list of decapods in Appendix A, Tables A3 andA4).

Selected crustacean generaTo obtain detailed information on the patterning of COI

diversity among closely related Crustacea, additional datawere assembled on two genera to enable a comparison ofcongeneric versus conspecific divergences. This analysis wasperformed for 13 species in the cladoceran genus Daphnia(Adamowicz et al. 2004) and for 12 species in the amphipodgenus Gammarus (F.O. Costa and P.D.N. Hebert, unpub-lished data). A complete list of species used in these analy-ses is provided in Appendix A, Tables A5 and A6.

GenBank dataWith more than 5000 entries, COI is the most intensively

studied crustacean gene, representing nearly 50% of theGenBank mitochondrial DNA data for this subphylum (Gen-Bank search, 30 June 2005). Although these entries provideCOI sequences for over 600 species, very few include acomplete sequence for the gene. Because the partial se-quences were acquired with varied primers, the opportunitiesfor sequence comparisons are limited. From sequences avail-able in GenBank (September 2004), 1585 providing cover-age for the COI barcode region were extracted. Use of thesedata was further limited by the short lengths of some se-quences. Only those with a minimum of 500 bp of the COIbarcode were used, with a single exception to accommodatea small group of 498 bp sequences for the crab Carcinus(Roman and Palumbi 2004). The data from GenBank do pro-vide valuable information for analyses that require a broadtaxonomic coverage, such as an examination of shifts inguanine–cytosine (GC) usage among crustacean lineages.For this purpose, a total of 617 sequences were extracted andanalyzed at various taxonomic levels.

DNA extraction, amplification, and sequencingIn the case of newly analyzed specimens for the ordinal

study, DNA was extracted using the proteinase K protocol(Schwenk et al. 1998) or Isoquick (Orca Research Inc.,Bothell, Washington). A 658 bp fragment from the 5′ end ofCOI was amplified using the primer pair LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198(5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmeret al. 1994). Each polymerase chain reaction (PCR) had a to-tal volume of 50 µL and contained 5 µL of 10× PCR buffer,1.5 mmol·L–1 of MgCl2, 50 µmol·L–1 of each dNTP, 1 unit(U) of Taq polymerase (1 U ≈ 16.67 nkat), 0.3 µmol·L–1 ofeach primer, and 2–4 µL of DNA template. The PCR thermalregime consisted of 60 s at 94 °C; five cycles of 30 s at94 °C, 90 s at 45 °C, and 60 s at 72 °C; 35 cycles of 30 s at94 °C, 90 s at 51 °C, and 60 s at 72 °C; followed by a finalextension of 5 min at 72 °C. The PCR product was subse-quently gel-purified with Qiaex II kit (Qiagen Inc., Valencia,California.) and sequenced in one direction on an ABI 377

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Costa et al. 273

automated sequencer (Applied Biosystems, Inc., Foster City,California).

A slightly different protocol was used in the decapod casestudy; DNA was isolated using either the GenElute kit(Sigma Inc., St. Louis, Missouri) or the Chelex dry releasemethod (Hajibabaei et al. 2005). The COI barcode was ampli-fied using HCO2198 as reverse primer combined with one ofthree forward primers: LCO1490, CrustF1 (5′-TTTTCTACA-AATCATAAAGACATTGG-3′), or CrustF2 (5′-GGTTCTTCT-CCACCAACCACAARGAYATHGG-3′). Each polymerasechain reaction had a total volume of 50 µL and contained5 µL of 10× PCR buffer, 2.2 mmol·L–1 of MgCl2, 50 µmol·L–1of each dNTP, 1.5 U of Taq polymerase, 0.2 µmol·L–1 ofeach primer, and 1 µL of DNA template. The PCR thermalregime was identical to the one described above except forthe CrustF2 primer, which was as follows: 1 cycle of 60 s at94 °C; 35 cycles of 30 s at 94 °C, 90 s at 42 °C, and 60 s at72 °C; followed by a final cycle of 5 min at 72 °C. The PCRproduct was subsequently gel-purified with UltraClean™15DNA purification kit (MoBio Laboratories, Inc., SolanaBeach, California) and sequenced in one or both directionsusing an ABI 377 or ABI 3730. For sequencing reactions weused BigDye® Terminator v3.1 cycle sequencing kit (Ap-plied Biosystems, Inc., Foster City, California).

Data analysisSequence data, electropherograms, and primer details for

each newly analyzed specimen are available in the com-pleted project file entitled “Crustacean Barcodes” on theBarcode of Life Data Systems (BOLD) website (http://www.barcodinglife.org). COI sequences were edited usingSeqApp 1.9 sequence editor (http://iubio.bio.indiana.edu/soft/molbio/seqapp/) and Sequencher (Gene codes Corpora-tion, Ann Arbor, Michigan) and aligned using CLUSTAL W(Thompson et al. 1994) implemented in MEGA3 (Kumar etal. 2004). To accommodate GenBank data, all sequenceswere subsequently pruned to 658 bp.

Neighbor-joining (NJ) analysis, implemented in MEGA3(Kumar et al. 2004), was employed for graphical representa-tion of the patterns of COI divergences among crustaceanspecies. The Kimura two-parameter (K2P) distance modelwas used to calculate nucleotide divergences (Kimura 1980),while Poisson correction (Nei and Kumar 2000) was used tocompute amino acid divergences for the ordinal profile. Boththe ordinal amino acid NJ tree and the Decapod nucleotideNJ tree were subjected to 10 000 bootstraps.

The first phase of the ordinal analysis involved the gener-ation of an NJ tree for the 150 profile taxa. This profile wassubsequently used as a classification engine by rerunning theNJ analysis following repeated addition of a single test taxonto the data set. After each analysis, the test species was as-signed membership in the same taxonomic group as its near-est neighboring taxon. For example, a test taxon wasidentified as a member of the order Anostraca if it groupedmost closely with any one of the seven anostracan familiesincluded in the profile. The success of the classification was

quantified by determining the proportion of test taxa as-signed to their proper order.

Levels of COI nucleotide divergences among conspecificand congeneric individuals were compared in Daphnia,Gammarus, and in those genera from the decapod data setthat included at least three species and two specimens ofeach species. For this purpose, the “Distance Summary” im-plemented in BOLD (Ratnasingham and Hebert 2007) wasused. This feature performs an automated computation ofpairwise divergences at the species, genus, and family levelfor all possible specimen combinations in the data set. Theaverage values obtained for conspecific and congeneric di-vergences were applied in the calculation of the taxonomicresolution ratio, which is defined as the quotient betweencongeneric divergences and conspecific divergences.

Results

Ordinal profileThe amino acid NJ tree for representatives of 150 crusta-

cean families showed high cohesion of taxonomic groups.Most allied species formed assemblages that mirrored estab-lished higher-taxonomic categories — orders, superorders,or subclasses (Fig. 1; a detailed tree can be found in Supple-mental Appendix S1).3 For example, members of the ordersSessilia and Pedunculata formed a cohesive group as super-order Thoracica, while members of the subclasses Copepodaand Phyllopoda formed distinctive assemblages. Members ofthe orders Amphipoda, Anostraca, Isopoda, Mysida, andStomatopoda were also joined in cohesive clusters. Therewere, however, a few deviations from this pattern; membersof the order Decapoda split into two groups, with Stomato-poda embedded within. As well, two species in the ordersCumacea and Amphipoda (Pseudocuma similis and The-misto gaudichaudii, respectively) were genetically divergentfrom allied taxa, grouping instead with species from ordersrepresented by a single family. A few other species, such asthe parasitic isopods, (Bopyroides hippolytes, Olencira prae-gustator, Rocinela angustata), occupied isolated positions inthe tree.

The ordinal profile was subsequently used to classify 100newly analyzed species. In 95 of these cases, the test taxonwas assigned to the correct order. In three cases, the test taxawere assigned only at a rank above the order level, namelyDrepanopus sp., Eurycercus longirostris, and Mytilocyprisambiguosa, which were assigned to subclasses Copepodaand Phyllopoda and class Ostracoda, respectively. Two mis-assignments involved taxa in the order Mysidacea, namelyParamesopodopsis rufa and Tenagomysis australis.

Decapod studyMembers of most (13 of 17) decapod families represented

by more than one species formed a cohesive cluster. How-ever, species in the families Palaemonidae, Portunidae, andOplophoridae were each split into two separate clusters,while Paguridae clustered with Lithodidae. The NJ tree for127 species of Decapoda did, however, reveal the regular

© 2007 NRC Canada

274 Can. J. Fish. Aquat. Sci. Vol. 64, 2007

3 Supplementary data for this article are available on the journal Web site (http://cjfas.nrc.ca) or may be purchased from the Depository ofUnpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ONK1A 0R6, Canada. DUD 5134. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml.

© 2007 NRC Canada

Costa et al. 275F

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ability of COI sequences to provide species-level resolution(Fig. 2; the complete tree can be found in Supplemental Ap-pendix S2).3 COI sequences formed assemblages congruentwith known species boundaries for all taxa from the PacificCoast of Canada, Resolute Bay, and the Baltic Sea. We had

comparable results with GenBank sequences (correct assign-ment = 93.0%), with only 5 congeneric taxa of unsettledspecies status not being discriminated with the COI se-quences. All five taxa belong to the mitten crab genus Erio-cheir, where species-level taxonomy has been problematic

© 2007 NRC Canada


Fig. 2. Three illustrative branches of the neighbor-joining tree based on mitochondrial cytochrome c oxidase I (COI) barcode nucleo-tide distances (K2P) for 127 decapod species representing 30 families. Bootstrap values for 10 000 replicates are shown near the re-spective branches. The complete tree is displayed in Supplemental Appendix S2.3

(Chu et al. 2003), and its members show incomplete resolu-tion in the NJ tree with E. formosa and E. rectus in one clus-ter and E. hepuensis, E. sinensis, and E. japonica in asecond cluster.

COI divergences among members of a species versuscongeners

For members of the order Decapoda, the average K2P dis-tance within species averaged 0.46%, while congeneric di-vergences averaged 17.16%, but ranged from a low 4.92% inthe crab genus Chionoecetes to a high of 31.39% in theamphipod genus Gammarus (Table 1). Within-species diver-gences were below 1% for 86% of the within-species com-parisons in decapods. The equivalent value in Gammaruswas only 75%, while in Daphnia it was 59%. Athough theDaphnia data set showed higher within-species distances,

the congeneric distances were are also high, averaging25.28%, so even in this genus there was a clear separation ofconspecific and congeneric distances (Fig. 3). The distribu-tion of K2P distances within different taxonomic categories(species, genus) for the Decapoda and as well for the generaPandalus, Daphnia, and Gammarus is shown (Fig. 3).

Variation of GC content in crustacean COI genesThe availability of 25 complete mitochondrial sequences

enabled examination of the relationship between GC contentof the COI barcode region and that in the whole mitochon-drial genome. This analysis revealed that the GC content ofthe barcode region was a very strong predictor (r2 = 0.81,p < 0.001) of genomic shifts in nucleotide usage, motivatingan examination of GC content in all crustaceans where 5′-COI information was available. The GC content of the

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Costa et al. 277

TaxonaPairwisedivergences

No. ofcomparisons

Min.distance

Meandistanceb

Max.distance TRRc Reference

Order Decapoda Within a species 144 0.00 0.46±0.05 2.57 37.6 This study(54 species, 31 genera,

138 sequences)Within a genus 409 4.92 17.16±0.18 23.66Within a family 459 11.27 19.75±0.17 49.93Within a order 8441 15.30 27.65±0.05 49.00

Genus Cancer Within a species 5 0.16 0.50±0.15 0.97 34.6 This study(3 species, 7 sequences) Within a genus 16 14.97 17.24±0.45 20.01

Genus Chionoecetes Within a species 5 0.00 0.26±0.08 0.47 21.5 This study(3 species, 7 sequences) Within a genus 16 4.92 5.67±0.09 6.28

Genus Crangon Within a species 5 0.00 0.94±0.41 2.13 20.1 This study(3 species, 7 sequences) Within a genus 16 17.31 18.91±0.41 21.74

Genus Eualus Within a species 22 0.00 0.59±0.14 2.57 32.5 This study(5 species, 16 sequences) Within a genus 98 15.17 19.24±0.23 23.64

Genus Pandalus Within a species 24 0.00 0.48±0.11 1.86 34.4 This study(6 species, 21 sequences) Within a genus 186 9.85 16.49±0.18 20.82

Genus Spirontocaris Within a species 8 0.15 0.47±0.11 1.20 32.9 This study(5 species, 11 sequences) Within a genus 47 10.80 15.40±0.29 18.74

GenBank DecapodaGenus Callinectes

(3 species, 14 sequences)

Within a species

Within a genus

47

44

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15.86

0.57±0.03

17.03±0.10

1.08

18.09

29.9 Pfeiler et al. 2005

Darden et al., unpub-lished datad

Genus Cherax


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Within a genus

22

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10.43

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15.95±0.42

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22.63

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Miller et al. 2004Genus Raymunida


Within a species

Within a genus

26

110

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7.6

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12.08±0.21

2.17

16.41

48.3 Macpherson andMachordom 2001

Other crustaceansOrder Cladocera

Genus Daphnia


Within a species

Within a genus

472

2688

0.00

13.18

1.32±0.05

25.28±0.05

4.30

30.65

19.2 Adamowicz et al.2004

Order AmphipodaGenus Gammarus


Within a species

Within a genus

284

2062

0

5.58

0.74±0.05

25.33±0.06

3.09

31.39

26.1 Costa and Hebert,unpublished datae

aNumber of species with more than one sequence and number of sequences analysed are shown in parentheses.bData reported as K2P distance (%) ± SE.cTaxonomic resolution ratio (see Data analysis in Materials and methods).dR.L. Darden, B.R. Kreiser, and A. Place, Biological Sciences, University of Southern Mississippi, 118 College Drive #5018, Hattiesburg, MS 39406,

USA.eF.O. Costa and P.D.N. Hebert, School of Biological Sciences, University of Wales, Bangor, Bangor, Gwynedd, LL57 2UW, United Kingdom.

Table 1. Pairwise mitochondrial cytochrome c oxidase I (COI) barcode nucleotide divergences for Decapoda and selected crustaceangenera, using K2P distances (%).

barcode region varied among the 617 species of crustaceansfrom 29.9% to 49.6% (Table 2). The highest GC contentwas observed in Parartemia longicaudata from saline lakesin Australia, while the lowest GC content was found in the

amphipod Hyperia galba, a parasite of jellyfish. Members ofthe orders Amphipoda and Isopoda possessed the highestand lowest average GC content, respectively (41.2% vs.38.7%). In all cases, the GC content decreased from the first

© 2007 NRC Canada


Fig. 3. Frequency distribution of conspecific (solid bars) and congeneric (shaded bars) mitochondrial cytochrome c oxidase I (COI)barcode distances (K2P) from pairwise comparisons among members of the order Decapoda and selected crustacean genera: (a) orderDecapoda, (b) genus Pandalus, (c) genus Daphnia, and (d) genus Gammarus. Values below 0.1% are not displayed.

to the third codon position, ranging from 48% to 52% in theformer, but only from 23% to 31.6% in the latter. Variationin GC content was consistently higher in the third codonbase, while the second base showed the least variation.

Discussion

This study establishes that the barcode region of COI hasconsiderable potential as the foundation for a DNA bar-coding identification system for crustaceans. A COI profilebased on single representatives of 150 crustacean familiesproved 95% effective in placing newly encountered speciesto the right order. This high success makes it clear that aprofile based on even a few thousand species will provide ahighly effective tool for taxonomic assignments at this level.Further parameterization of the ordinal profile should beginby adding sequence records for each of the 849 crustaceanfamilies. Higher sampling intensity should be directed tofamilies with accelerated rates of molecular evolution, andthe present results provide some directions in this regard.Parasitic lineages often show rate acceleration (Hassanin2006; J.R. deWaard and P.D.N. Hebert, unpublished data),and this pattern was apparent in the current study, as evi-denced by the isolated positions of parasitic isopods in theNJ tree. As a result, particular sampling intensity should bedirected to crustacean lineages with parasitic lifestyles tosubdivide the misleading long branches and to create an ef-fective system for placements deep in the taxonomic hierar-chy.

The present study suggests that a COI-based system willregularly deliver species-level resolution for crustacean lin-eages. In fact, levels of sequence divergence among conge-neric species of crustaceans averaged 17.16%, the highestvalue yet reported for any animal group. By comparison,congeneric species of lepidopterans show just 6.1% variation(Hebert et al. 2003), birds show 7.93% variation (Hebert etal. 2004), and fishes possess 9.93% divergence (Ward et al.2005). Of course, the actual level of divergence among con-geners is less critical than the ratio of the genetic divergencebetween species to that within species. Levels of intra-specific variation in crustaceans averaged 0.46%, valuesslightly higher than those reported in other groups (mostrange from 0.25% to 0.30%), but the interspecific to intra-specific ratios of sequence divergence were very large, rang-ing from 19.2 to 48.3. As a consequence, species recognition

was straightforward in most cases (~95%). Based on theseobservations, we conclude that a COI barcoding system willdeliver levels of species resolution comparable with thoseseen in tropical lepidopterans (98%, Hajibabaei et al. 2006),marine gastropods (96%, Meyer and Paulay 2005), fishes(100%, Ward et al. 2005), and birds (95%, Hebert et al.2004).

We emphasize the need for a critical assessment of bothbarcode results and current taxonomic systems in cases ofdiscordance. For example, the lack of barcode divergencebetween members of the mitten crab genus Eriocheir reflectsa case where there is a growing consensus that some recog-nized species do not merit this status (discussed in detail inChu et al. 2003 and Tang et al. 2003). In other cases, levelsof barcode variation within species have surely been inflatedby a failure of current taxonomic systems to recognize validspecies. For example, the presence of two genetically diver-gent groups in the hermit crabs Pagurus longicarpus andPagurus pollicaris is thought to reflect overlooked species(Young et al. 2002). Similarly, the freshwater shrimp Para-tya australiensis includes several highly divergent mitochon-drial lineages that are now thought to represent species thatdiversified in the Pliocene (Baker et al. 2004). Finally, bothmorphological and biogeographic data suggest the possibil-ity of overlooked species within Cherax tenuimanus andCherax preissi (Munasinghe et al. 2003).

No single approach can provide a definitive conclusion onspecies boundaries (but see Lee 2003). We emphasize thatDNA barcoding is not a substitute for conventional taxo-nomic approaches. It seeks instead to flag cases of deepgenetic divergence among individuals grouped as a singlespecies that may indicate overlooked species. For example,we noted particularly high intraspecific divergences (3.1%)in Gammarus oceanicus, but individuals from the Baltic Sea,Iceland, and Hudson Bay showed an average within-speciesK2P divergence of only 0.43%. The extreme divergencevalues all reflected samples of this species from the St. Law-rence estuary, perhaps reflecting an overlooked species en-demic to this region. In contrast with these cases, there areother cases where species recognized through past taxo-nomic work show little or no barcode divergence. Some ofthese cases may represent instances where current taxo-nomic systems inappropriately recognize variation as reflect-ing species status. However, other cases may reflect veryyoung species. We emphasize that the recognition of taxo-

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Costa et al. 279

Codon position

Taxona Min. Mean Max. 1st 2nd 3rd

Subphylum Crustacea (617) 29.86 38.98±0.14 49.62 49.35±0.14 43.05±0.06 24.21±0.33Order Amphipoda (48) 35.02 40.39±0.53 49.39 47.54±0.42 43.61±0.17 29.82±1.34Order Anostraca (35) 35.81 39.46±0.53 49.62 51.07±0.35 44.27±0.19 23.03±1.38Subclass Copepoda (61) 33.50 38.69±0.38 44.73 48.34±0.28 42.02±0.12 25.71±0.97Order Decapoda (138) 32.62 39.29±0.27 47.04 50.82±0.25 42.96±0.10 23.92±0.64Order Isopoda (36) 33.17 38.93±0.54 45.69 47.95±0.55 44.00±0.33 24.83±1.23Subclass Phyllopoda (55) 34.48 40.10±0.36 46.35 51.42±0.33 43.93±0.08 24.81±0.89

aNumber of species analyzed are shown in parentheses.

Table 2. Variation in GC content in the mitochondrial cytochrome c oxidase I (COI) barcode region amongcrustacean taxa.

nomic boundaries in such cases is always demanding, oftensubjective, and best pursued through a weight of evidenceapproach that employs molecular, morphological, and eco-logical traits to reach a decision.

As the creation of a comprehensive COI barcode databasefor crustaceans will be a substantial undertaking, we stressthat benefits will be diverse. COI can, for example, serve asa sentinel gene enabling the detection of lineages with un-usual patterns of nucleotide usage or exceptional rates ofevolution. The analysis of GC content in the present studydemonstrates that the COI barcode region can be a predictorof the nucleotide usage of the entire mitochondrial genome.Similarly, the molecular rate acceleration of parasitic lin-eages revealed previously using other gene regions (Hassa-nin 2006; J.R. deWaard and P.D.N. Hebert, unpublisheddata) was evident in the current data set. As such, it will aidmultigene systematics by allowing such studies to target taxashowing unusual attributes of nucleotide usage or evolution-ary rates. The benefits of a barcode system will also extendinto ecological work. For example, an estimated one-third ofmarine arthropods (thus mainly crustaceans) in 138 studiesfrom the North Atlantic could not be identified to a specieslevel (Schander and Willassen 2005). Schander andWillassen (2005) pointed out several relevant means bywhich improved ability to recognize marine species willbenefit marine biological and ecological sciences in a num-ber of ways (see also Böttger-Schnack et al. 2004). A partic-ular gain could arise through species-level identification oflarvae, enabling studies of larval dispersal and ecology, andbenthic adult connectivity, all critical for management ofcrustacean fisheries (Tully et al. 2003). DNA barcoding willalso allow the identification of crustacean prey items instomach contents of fishes, birds, and other predators, aidingparameterization of food web models. For example, morpho-logical approaches did not allow the species identification ofseveral crustacean prey in the stomach contents of fulmars(Fulmarus glacialis) (Phillips et al. 1999). Other potentialbenefits of a COI barcode identification system for crusta-ceans include the identification of parasitic crustaceans atany developmental stage (e.g., Øines and Heuch 2005), aswell as the detection of invasive crustacean species(Armstrong and Ball 2005). In summary, the present studyreveals that a large-scale effort to assemble DNA barcode re-cords for crustaceans will deliver a highly effective identifi-cation system with impacts on a broad range of researchthemes.

Acknowledgements

The “Fundação para a Ciência e a Tecnologia” (Portugal)provided a postdoctoral fellowship (BPD/11588/2002) toF.O. Costa. This work was supported by grants to PDNHfrom the Natural Sciences and Engineering Research Coun-cil of Canada (NSERC) and from the Gordon and BettyMoore Foundation. We thank Monika Normant for decapodsamples from the Baltic Sea.

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Appendix A

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GenBank accession No. Class Order Family Species Length

AY145426 Maxillopoda Calanoida Acartiidae Acartia omorii 624EF432739 Malacostraca Isopoda Aegidae Rocinela angustata 654AF436034 Malacostraca Decapoda Aeglidae Aegla uruguayana 657AF531742 Maxillopoda Calanoida Aetideidae Gaetanus tenuispinus 627AF436036 Malacostraca Decapoda Albuneidae Blepharipoda occidentalis 657DQ889084 Malacostraca Amphipoda Ampeliscidae Byblis gaimardi 623DQ889076 Malacostraca Anaspidacea Anaspididae Anaspides tasmaniae 657DQ889126 Malacostraca Amphipoda Anisogammaridae Ramellogammarus sp. 654DQ889078 Malacostraca Isopoda Arcturidae Arcturus baffini 644DQ889096 Maxillopoda Arguloida Argulidae Dolops sp. 624DQ889079 Malacostraca Isopoda Armadillidiidae Armadillidium sp. 657NC_001620 Branchiopoda Anostraca Artemiidae Artemia franciscana 624DQ889085 Malacostraca Isopoda Asellidae Caecidotea sp. 652DQ889120 Malacostraca Decapoda Atyidae Paratya australiensis 657AF242660 Maxillopoda Sessilia Balanidae Semibalanus balanoides 642DQ889092 Malacostraca Cumacea Bodotriidae Cyclaspis caprella 633DQ889082 Malacostraca Isopoda Bopyridae Bopyroides hippolytes 629DQ889083 Branchiopoda Cladocera Bosminidae Bosmina sp. 639AF209064 Branchiopoda Anostraca Branchinectidae Branchinecta paludosa 639AF209059 Branchiopoda Anostraca Branchipodidae Parartemia contracta 639AF044057 Malacostraca Decapoda Bresiliidae Rimicaris exoculata 605DQ889086 Branchiopoda Diplostraca Caenestheriidae Caenestheriella setosa 639DQ889087 Maxillopoda Calanoida Calanidae Calanus glacialis 624AF436025 Malacostraca Decapoda Callianassidae Callichirus major 649DQ882037 Malacostraca Decapoda Calocarididae Calocaris investigatoris 658

Table A1. List of specimens used in the profile of 150 crustacea families, sorted by family name.

© 2007 NRC Canada



DQ889125 Malacostraca Decapoda Cambaridae Procambarus clarkii 654DQ882040 Malacostraca Decapoda Cancridae Cancer magister 655AY145434 Maxillopoda Calanoida Candaciidae Candacia ethiopica 624AF315015 Maxillopoda Harpacticoida Canuellidae Coullana sp. 657DQ889075 Malacostraca Amphipoda Caprellidae Aeginina longicornis 644AY428048 Maxillopoda Sessilia Catophragmidae Catomerus polymerus 643DQ356555 Maxillopoda Calanoida Centropagidae Boeckella gracilis 636DQ889088 Branchiopoda Diplostraca Cercopagididae Cercopagis pengoi 639AF209062 Branchiopoda Anostraca Chirocephalidae Artemiopsis stefanssoni 639DQ889089 Maxillopoda Sessilia Chthamalidae Chamaesipho sp. 657DQ889090 Branchiopoda Cladocera Chydoridae Chydorus sp. 639AF513651 Maxillopoda Calanoida Clausocalanidae Pseudocalanus mimus 639AF315009 Maxillopoda Harpacticoida Cletodidae Cletocamptus deitersi 657AY327387 Maxillopoda Harpacticoida Cletopsyllidae Bathycletopsyllus sp. 603AF205247 Malacostraca Stomatopoda Coronididae Parvisquilla multituberculata 648AY174366 Maxillopoda Sessilia Coronulidae Chelonibia testudinaria 586DQ889128 Malacostraca Decapoda Crangonidae Sclerocrangon boreas 657DQ889090 Malacostraca Amphipoda Crangonyctidae Crangonyx sp. 637DQ889093 Branchiopoda Diplostraca Cyclestheriidae Cyclestheria hislopi 639AF255791 Malacostraca Isopoda Cymothoidae Olencira praegustator 583DQ889094 Ostracoda Podocopida Cyprididae Cypridopsis vidua 624NC_000844 Branchiopoda Diplostraca Daphniidae Daphnia pulex 624AJ534411 Ostracoda Podocopida Darwinulidae Vestalenula molopoensis 534DQ889095 Maxillopoda Calanoida Diaptomidae Diaptomus sp. 608AF520442 Malacostraca Cumacea Diastylidae Diastylopsis thilenuisi 657DQ882097 Malacostraca Decapoda Diogenidae Paguristes turgidus 658DQ889097 Malacostraca Decapoda Dromiidae — 644DQ889118 Malacostraca Amphipoda Epimeriidae Paramphithoe hystrix 644AB091772 Maxillopoda Calanoida Eucalanidae Eucalanus bungii 900AF531749 Maxillopoda Calanoida Euchaetidae Paraeuchaeta biloba 618DQ889098 Malacostraca Euphausiacea Euphausiidae Euphausia superba 633DQ889127 Malacostraca Amphipoda Eusiridae Rhachotropis aculeata 641DQ882090 Malacostraca Decapoda Galatheidae Munida quadrispina 658DQ889100 Malacostraca Amphipoda Gammaridae Gammarus lacustris 654DQ889101 Malacostraca Decapoda Gnathophyllidae Gnathophyllum americanus 657AF048822 Malacostraca Stomatopoda Gonodactylidae Gonodactylus graphurus 645AF317338 Malacostraca Decapoda Grapsidae Grapsus albolineatus 573AF520447 Malacostraca Cumacea Gynodiastylidae Gynodiastylis sp. 657L43049 Maxillopoda Harpacticoida Harpactidae Tigriopus californicus 606AF205252 Malacostraca Stomatopoda Hemisquillidae Hemisquilla ensigera 645AF531745 Maxillopoda Calanoida Heterorhabdidae Heterorhabdus farrani 642AF048823 Malacostraca Stomatopoda Heterosquillidae Heterosquilla tricarinata 655DQ882084 Malacostraca Decapoda Hippolytidae Lebbeus groenlandicus 505DQ889102 Branchiopoda Cladocera Holopedidae Holopedium gibberum 639AF370852 Cephalocarida Brachypoda Hutchinsoniellidae Hutchinsoniella macracantha 634AY152752 Malacostraca Amphipoda Hyalellidae Hyalella sp. 637AY639937 Malacostraca Amphipoda Hyalidae Parhyale hawaiiensis 900DQ889133 Malacostraca Amphipoda Hyperiidae Themisto gaudichaudii 657DQ889111 Malacostraca Isopoda Idoteidae Mesidotea entomon 657AF451354 Malacostraca Amphipoda Iphimediidae Gnathiphimedia sexdentata 624DQ889106 Malacostraca Amphipoda Ischyroceridae Ischyrocerus latipes 643AF352297 Malacostraca Cumacea Lampropidae Lampropis quadriplicata 626AF436030 Malacostraca Decapoda Laomediidae Jaxea nocturna 593DQ889131 Malacostraca Mysidacea Lepidomysidae Tasmanomysis oculata 633DQ889107 Branchiopoda Cladocera Leptodoridae Leptodora kindtii 638AF137516 Malacostraca Cumacea Leuconidae Eudorella pusilla 657DQ889130 Maxillopoda Cyclopoida Lichomolgidae Stellicola sp. 654

Table A1 (continued).

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AF255780 Malacostraca Isopoda Ligiidae Ligia occidentalis 583AJ534412 Ostracoda Podocopida Limnocytheridae Limnocythere inopinata 533DQ882086 Malacostraca Decapoda Lithodidae Lithodes couesi 639DQ889115 Malacostraca Lophogastrida Lophogastridae Neognathophausia ingens 630DQ889108 Malacostraca Decapoda Luciferidae Lucifer sp. 656DQ889109 Branchiopoda Diplostraca Lynceidae Lynceus sp. 639DQ889077 Malacostraca Amphipoda Lysianassidae Anonyx nugax 643DQ889117 Branchiopoda Cladocera Macrothricidae Ophryoxus gracilis 639DQ889114 Malacostraca Decapoda Majidae Naxia sp. 657AF474106 Maxillopoda Calanoida Metridinidae Metridia lucens 648DQ889112 Branchiopoda Cladocera Moinidae Moina sp. 639DQ889119 Malacostraca Mysidacea Mysidae Paramysis intermedia 618AF520450 Malacostraca Cumacea Nannastacidae Campylaspis sp. 657AF125435 Malacostraca Decapoda Nematocarcinidae Nematocarcinus ensifer 600DQ889104 Malacostraca Decapoda Nephropidae Homarus americanus 654DQ889116 Ostracoda Podocopida Notodromadidae Notodromas monacha 523AF205234 Malacostraca Stomatopoda Odontodactylidae Odontodactylus scyllarus 644EF432738 Malacostraca Amphipoda Oedicerotidae Monoculodes borealis 644DQ882168 Malacostraca Decapoda Oplophoridae Systellaspis braueri 654DQ882047 Malacostraca Decapoda Oregoniidae Chionoecetes bairdi 658NC_003058 Malacostraca Decapoda Paguridae Pagurus longicarpus 657DQ889110 Malacostraca Decapoda Palaemonidae Macrobrachium sp. 656NC_004251 Malacostraca Decapoda Palinuridae Panulirus japonicus 657DQ882111 Malacostraca Decapoda Pandalidae Pandalus danae 658DQ882143 Malacostraca Decapoda Panopeidae Rhithropanopeus harrisii 658AF474110 Maxillopoda Calanoida Paracalanidae Paracalanus parvus 648AF493624 Malacostraca Decapoda Parastacidae Cherax crassimanus 600DQ882133 Malacostraca Decapoda Pasiphaeidae Pasiphaea pacifica 658AF217843 Malacostraca Decapoda Penaeidae Penaeus monodon 657AF255775 Malacostraca Isopoda Phreatoicidae Colubotelson thomsoni 583AF317341 Malacostraca Decapoda Plagusiidae Plagusia immaculata 573DQ889099 Branchiopoda Cladocera Podonidae Evadne spinifera 639AF209063 Branchiopoda Anostraca Polyartemiidae Polyartemiella hazeni 639DQ889121 Branchiopoda Cladocera Polyphemidae Polyphemus pediculus 639AY145429 Maxillopoda Calanoida Pontellidae Calanopia thompsoni 624AY189478 Malacostraca Amphipoda Pontogammaridae Obesogammarus crassus 654DQ889122 Malacostraca Amphipoda Pontoporeiidae Pontoporeia femorata 653DQ889123 Malacostraca Isopoda Porcellionidae Porcellio spinicornis 654DQ889124 Malacostraca Decapoda Portunidae Portunus pelagicus 657AF399980 Malacostraca Decapoda Potamidae Potamon fluviatilis 539AF205253 Malacostraca Stomatopoda Protosquillidae Chorisquilla excavata 644AF137514 Malacostraca Cumacea Pseudocumatidae Pseudocuma similis 657AY145436 Maxillopoda Calanoida Pseudodiaptomidae Pseudodiaptomus marinus 624AF205245 Malacostraca Stomatopoda Pseudosquillidae Pseudosquilla ciliata 646AF346400 Malacostraca Decapoda Raninidae Ranina ranina 632AY456188 Maxillopoda Pedunculata Scalpellidae Pollicipes polymerus 658DQ889105 Malacostraca Decapoda Scyllaridae Ibacus peronii 654DQ882149 Malacostraca Decapoda Sergestidae Sergestes similis 658DQ889129 Branchiopoda Diplostraca Sididae Sida crystallina 639AF370851 Remipedia Nectiopoda Speleonectidae Speleonectes gironensis 613AF255785 Malacostraca Isopoda Sphaeromatidae Sphaeroma quadridentatum 544AF531751 Maxillopoda Calanoida Spinocalanidae Spinocalanus abyssalis 507NC_006081 Malacostraca Stomatopoda Squillidae Squilla mantis 658AF125441 Malacostraca Decapoda Stenopodidae Stenopus hispidus 600AF531752 Maxillopoda Calanoida Stephidae Stephos longipes 621AF209065 Branchiopoda Anostraca Streptocephalidae Streptocephalus dorothae 639DQ889103 Malacostraca Decapoda Sundathelphusidae Holthuisana transversa 653


© 2007 NRC Canada



AF205257 Malacostraca Stomatopoda Takuidae Taku spinosocarinatus 647AY152751 Malacostraca Amphipoda Talitridae Orchestia uhleri 637DQ889132 Maxillopoda Calanoida Temoridae Temora stylifera 636AB126701 Maxillopoda Sessilia Tetraclitidae Tetraclita japonica 658AF209066 Branchiopoda Anostraca Thamnocephalidae Thamnocephalus platyurus 639AF474112 Maxillopoda Calanoida Tortanidae Tortanus derjugini 617DQ889134 Malacostraca Decapoda Trapeziidae Trapezia rufopunctata 644DQ889135 Branchiopoda Notostraca Triopsidae Triops australiensis 639DQ882062 Malacostraca Decapoda Varunidae Eriocheir sinensis 623DQ889081 Malacostraca Decapoda Xanthidae Atergatis floridus 656DQ889113 Ostracoda Myodocopida — — 655

Table A1 (concluded).


DQ889136 Branchiopoda Cladocera Macrothricidae Acantholeberis curvirostris 639DQ889137 Branchiopoda Cladocera Chydoridae Acroperus harpae 639DQ889138 Branchiopoda Cladocera Chydoridae Alona setulosa 639DQ889139 Branchiopoda Cladocera Chydoridae Alonella exigua 639DQ882032 Malacostraca Decapoda Crangonidae Argis lar 658AY456187 Maxillopoda Arguloida Argulidae Argulus americanus 658AY531772 Malacostraca Isopoda Asellidae Asellus aquaticus 651AF308940 Branchiopoda Anostraca Thamnocephalidae Branchinella pinnata 639DQ889140 Branchiopoda Diplostraca Cercopagididae Bythotrephes sp. 639AF332791 Maxillopoda Calanoida Calanidae Calanoides acutus 639AY428047 Maxillopoda Pedunculata Scalpellidae Calantica spinosa 677DQ889141 Malacostraca Amphipoda Caprellidae Caprella unica 641AY616445 Malacostraca Decapoda Portunidae Carcinus aestuarii 498DQ889142 Maxillopoda Calanoida Centropagidae Centropages furcatus 636AY188999 Branchiopoda Anostraca Chirocephalidae Chirocephalus ruffoi 654DQ882053 Malacostraca Decapoda Majidae Chorilia longipes 655AF234819 Maxillopoda Sessilia Chthamalidae Chthamalus anisopoma 656AF462313 Maxillopoda Calanoida Clausocalanidae Clausocalanus minor 648AF520446 Malacostraca Cumacea Diastylidae Colurostylis longicaudata 658DQ882057 Malacostraca Decapoda Crangonidae Crangon alaskensis 658AF255776 Malacostraca Isopoda Phreatoicidae Crenoicus buntiae 580AF332789 Maxillopoda Calanoida Calanidae Ctenocalanus citer 639AF520449 Malacostraca Cumacea Nannastacidae Cumella sp. 658DQ889143 Branchiopoda Diplostraca Sididae Diaphanosoma sp. 639AF137510 Malacostraca Cumacea Diastylidae Diastylis sculpta 658DQ889144 Malacostraca Amphipoda Pontoporeiidae Diporeia hoyi 649DQ889145 Maxillopoda Calanoida Clausocalanidae Drepanopus sp. 621DQ889146 Branchiopoda Cladocera Macrothricidae Drepanothrix dentata 639AF451352 Malacostraca Amphipoda Iphimediidae Echiniphimedia echinata 624DQ889147 Malacostraca Amphipoda Gammaridae Echinogammarus ischnus 640AF493633 Malacostraca Decapoda Parastacidae Engaeus strictifrons 600AF520445 Malacostraca Cumacea Bodotriidae Eocuma longicorne 657AF451342 Malacostraca Amphipoda Epimeriidae Epimeria reoproi 644DQ889148 Malacostraca Amphipoda Ischyroceridae Ericthonius sp. 644DQ882073 Malacostraca Decapoda Hippolytidae Eualus biunguis 624AF209061 Branchiopoda Anostraca Chirocephalidae Eubranchipus sp. 639AF234820 Maxillopoda Sessilia Chthamalidae Euraphia eastropacensis 598DQ889149 Branchiopoda Cladocera Chydoridae Eurycercus longirostris 639AY145427 Maxillopoda Calanoida Temoridae Eurytemora pacifica 624

Table A2. List of 100 specimens used as test taxa in the amino acid profile of the crustacea.

© 2007 NRC Canada

Costa et al. 285


DQ889150 Malacostraca Amphipoda Eusiridae Eusirus cuspidatus 644DQ889151 Malacostraca Isopoda Sphaeromatidae Exosphaeroma sp. 654AF395115 Malacostraca Decapoda Penaeidae Fenneropenaeus sp. 657AF255781 Malacostraca Isopoda Idoteidae Glyptidotea lichtensteini 574AF205231 Malacostraca Stomatopoda Gonodactylidae Gonodactylellus hendersoni 648AF205224 Malacostraca Stomatopoda Gonodactylidae Gonodactylinus viridis 627AF205242 Malacostraca Stomatopoda Gonodactylidae Gonodactylopsis komodoensis 645DQ889152 Branchiopoda Cladocera Chydoridae Graptoleberis testudinaria 639AF205239 Malacostraca Stomatopoda Protosquillidae Haptosquilla glyptocercus 646AF317340 Malacostraca Decapoda Varunidae Hemigrapsus sanguineus 573AF061781 Malacostraca Cumacea Lampropidae Hemilamprops californicus 658DQ882079 Malacostraca Decapoda Oregoniidae Hyas lyratus 658DQ889153 Malacostraca Amphipoda Hyperiidae Hyperia galba 643DQ889154 Malacostraca Amphipoda Lysianassidae Ichnopus spinicornis 626DQ889155 Malacostraca Isopoda Idoteidae Idotea granulosa 636AF451347 Malacostraca Amphipoda Iphimediidae Iphimediella rigida 618AF339473 Malacostraca Decapoda Palinuridae Jasus edwardsii 639AY145428 Maxillopoda Calanoida Pontellidae Labidocera rotunda 624DQ889156 Branchiopoda Notostraca Triopsidae Lepidurus sp. 639AF520444 Malacostraca Cumacea Bodotriidae Leptocuma sp. 633DQ889157 Branchiopoda Diplostraca Limnadiidae Limnadia sp. 639DQ889158 Maxillopoda Calanoida Centropagidae Limnocalanus macrurus 631DQ889159 Malacostraca Mysidacea Mysidae Limnomysis benedeni 634AF260843 Malacostraca Isopoda Cymothoidae Lironeca vulgaris 617DQ882088 Malacostraca Decapoda Lithodidae Lopholithodes foraminatus 658DQ889160 Branchiopoda Cladocera Macrothricidae Macrothrix sp. 639AF137520 Malacostraca Cumacea Bodotriidae Mancocuma stellifera 675AJ319742 Ostracoda Podocopida Cyprididae Mesocyprideis irsacae 462DQ889161 Malacostraca Mysidacea Mysidae Mysis americana 654DQ889162 Ostracoda Podocopida Cyprididae Mytilocypris ambiguosa 656AF332793 Maxillopoda Calanoida Calanidae Nannocalanus minor 613AF513650 Maxillopoda Calanoida Calanidae Neocalanus robustior 639AF205235 Malacostraca Stomatopoda Gonodactylidae Neogonodactylus bredini 648DQ882094 Malacostraca Decapoda Oplophoridae Notostomus japonicus 647DQ889163 Malacostraca Euphausiacea Euphausiidae Nyctiphanes australis 633AY428049 Maxillopoda Sessilia Chthamalidae Octomeris angulosa 639DQ889164 Malacostraca Amphipoda Lysianassidae Onisimus glacialis 639DQ889165 Malacostraca Decapoda Cambaridae Orconectes propinquus 653DQ889166 Maxillopoda Calanoida Centropagidae Osphranticum labronectum 636AF137512 Malacostraca Cumacea Diastylidae Oxyurostylis smithi 658DQ882103 Malacostraca Decapoda Palaemonidae Palaemon elegans 658DQ882106 Malacostraca Decapoda Pandalidae Pandalopsis dispar 657DQ356574 Maxillopoda Calanoida Centropagidae Parabroteas sarsia 636AF052393 Malacostraca Mysidacea Mysidae Paramesopodopsis rufa 651AF255783 Malacostraca Isopoda Idoteidae Paridotea ungulata 564DQ889167 Branchiopoda Cladocera Podonidae Pleopis sp. 639DQ889168 Branchiopoda Cladocera Chydoridae Pleuroxus denticulatus 639DQ889169 Branchiopoda Cladocera Podonidae Podon leuckarti 639AF352302 Malacostraca Cumacea Bodotriidae Pomacuma australiae 675DQ889170 Malacostraca Amphipoda Gammaridae Pontogammarus maeoticus 657AF283874 Malacostraca Decapoda Galatheidae Raymunida dextralis 657AF531746 Maxillopoda Calanoida Eucalanidae Rhincalanus gigas 633DQ889171 Branchiopoda Diplostraca Daphniidae Scapholeberis rammneri 639DQ889172 Branchiopoda Diplostraca Daphniidae Simocephalus vetulus 639AF260846 Malacostraca Isopoda Sphaeromatidae Sphaeramene polytylotus 579DQ882157 Malacostraca Decapoda Hippolytidae Spirontocaris lamellicornis 658AF052394 Malacostraca Mysidacea Mysidae Tenagomysis australis 645


© 2007 NRC Canada



AY430813 Maxillopoda Sessilia Chthamalidae Tetrachthamalus oblitteratus 603DQ889173 Malacostraca Decapoda Scyllaridae Thenus orientalis 657AF352301 Malacostraca Cumacea Bodotriidae Vaunthompsonia cristata 658DQ889174 Malacostraca Amphipoda Gammaridae Weiprechtia pinguis 644

Table A2 (concluded).

© 2007 NRC Canada

Costa et al. 287

Gen

Ban

kac

cess

ion

No.

Pro

cess

IDS

peci

esFa

mil

yC

ount

ryA

rchi

vety

peP

rim

erpa

ira

Len

gth

DQ

8820

26F

CD

PA13

5-04

Aca

ntho

lith

odes

hisp

idus

Lit

hodi

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

29F

CD

PA02

0-04

Arg

isal

aske

nsis

Cra

ngon

idae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

28F

CD

PA02

1-04

Arg

isal

aske

nsis

Cra

ngon

idae

Can

ada

Spe

cim

enFo

lA–F

olB

654

DQ

8820

27F

CD

PA10

6-04

Arg

isal

aske

nsis

Cra

ngon

idae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

32F

CD

PA02

2-04

Arg

isla

rC

rang

onid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB65

8D

Q88

2031

FC

DPA

023-

04A

rgis

lar

Cra

ngon

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

658

DQ

8820

30F

CD

PA10

7-04

Arg

isla

rC

rang

onid

aeC

anad

aS

peci

men

FolA

–Fol

B65

0D

Q88

2033

FC

DPA

050-

04B

enth

eoge

nnem

abo

real

isB

enth

esic

ymid

aeC

anad

aT

issu

eFo

lA–F

olB

618

DQ

8820

34F

CD

PA05

1-04

Ben

theo

genn

ema

bore

alis

Ben

thes

icym

idae

Can

ada

Tis

sue

FolA

–Fol

B61

6D

Q88

2035

FC

DPA

111-

04C

aloc

arid

essp

inul

icau

daA

xiid

aeC

anad

aS

peci

men

FolA

–Fol

B63

8D

Q88

2037

FC

DPA

052-

04C

aloc

aris

inve

stig

ator

isC

aloc

arid

idae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2036

FC

DPA

053-

04C

aloc

aris

inve

stig

ator

isC

aloc

arid

idae

Can

ada

Tis

sue

FolA

–Fol

B61

4D

Q88

2040

FC

DPA

061-

04C

ance

rm

agis

ter

Can

crid

aeC

anad

aT

issu

eC

rust

F1–

FolB

655

DQ

8820

38F

CD

PA15

0-04

Can

cer

mag

iste

rC

ancr

idae

Can

ada

Tis

sue

FolA

–Fol

B62

4D

Q88

2039

FC

DPA

151-

04C

ance

rm

agis

ter

Can

crid

aeC

anad

aT

issu

eFo

lA–F

olB

638

DQ

8820

41F

CD

PA06

2-04

Can

cer

oreg

onen

sis

Can

crid

aeC

anad

aT

issu

eC

rust

F1–

FolB

616

DQ

8820

42F

CD

PA06

3-04

Can

cer

oreg

onen

sis

Can

crid

aeC

anad

aT

issu

eC

rust

F1–

FolB

511

DQ

8820

44F

CD

PA16

6-05

Can

cer

prod

uctu

sC

ancr

idae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2043

FC

DPA

064-

04C

ance

rpr

oduc

tus

Can

crid

aeC

anad

aT

issu

eFo

lA–F

olB

605

DQ

8820

45F

CD

PA06

5-04

Chi

onoe

cete

san

gula

tus

Ore

goni

idae

Can

ada

Tis

sue

FolA

–Fol

B62

0D

Q88

2046

FC

DPA

066-

04C

hion

oece

tes

angu

latu

sO

rego

niid

aeC

anad

aT

issu

eFo

lA–F

olB

520

DQ

8820

49F

CD

PA06

7-04

Chi

onoe

cete

sba

irdi

Ore

goni

idae

Can

ada

Tis

sue

FolA

–Fol

B65

2D

Q88

2048

FC

DPA

068-

04C

hion

oece

tes

bair

diO

rego

niid

aeC

anad

aT

issu

eC

rust

F1–

FolB

643

DQ

8820

47F

CD

PA13

8-04

Chi

onoe

cete

sba

irdi

Ore

goni

idae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

51F

CD

PA06

9-04

Chi

onoe

cete

sta

nner

iO

rego

niid

aeC

anad

aT

issu

eFo

lA–F

olB

523

DQ

8820

50F

CD

PA07

0-04

Chi

onoe

cete

sta

nner

iO

rego

niid

aeC

anad

aT

issu

eFo

lA–F

olB

656

DQ

8820

54F

CD

PA07

2-04

Cho

rili

alo

ngip

esM

ajid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB55

2D

Q88

2053

FC

DPA

073-

04C

hori

lia

long

ipes

Maj

idae

Can

ada

Tis

sue

FolA

–Fol

B65

5D

Q88

2052

FC

DPA

139-

04C

hori

lia

long

ipes

Maj

idae

Can

ada

Spe

cim

enFo

lA–F

olB

656

DQ

8820

55F

CD

PA02

4-04

Cra

ngon

abys

soru

mC

rang

onid

aeC

anad

aT

issu

eFo

lA–F

olB

653

DQ

8820

56F

CD

PA02

5-04

Cra

ngon

abys

soru

mC

rang

onid

aeC

anad

aT

issu

eFo

lA–F

olB

645

DQ

8820

57F

CD

PA11

6-04

Cra

ngon

alas

kens

isC

rang

onid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB65

8D

Q88

2058

FC

DPA

117-

04C

rang

onal

aske

nsis

Cra

ngon

idae

Can

ada

Spe

cim

enFo

lA–F

olB

655

DQ

8820

60F

CD

PA02

6-04

Cra

ngon

com

mun

isC

rang

onid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB57

4D

Q88

2061

FC

DPA

027-

04C

rang

onco

mm

unis

Cra

ngon

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

658

DQ

8820

59F

CD

PA15

4-04

Cra

ngon

com

mun

isC

rang

onid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB63

0D

Q88

2062

FC

DPA

074-

04E

rioc

heir

sine

nsis

Var

unid

aeP

olan

dT

issu

eC

rust

F1–

FolB

623

DQ

8820

65F

CD

PA12

3-04

Eua

lus

avin

usH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

64F

CD

PA12

4-04

Eua

lus

avin

usH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

63F

CD

PA12

5-04

Eua

lus

avin

usH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8820

69F

CD

PA03

4-04

Eua

lus

barb

atus

Hip

poly

tida

eC

anad

aS

peci

men

FolA

–Fol

B65

1D

Q88

2070

FC

DPA

035-

04E

ualu

sba

rbat

usH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

Tab

leA

3.L

ist

ofde

capo

dsp

ecim

ens

for

whi

chC

OI

barc

odes

wer

eac

quir

edin

this

stud

y,to

geth

erw

ith

thei

rco

untr

yof

coll

ecti

on,

type

ofar

chiv

eat

the

Bio

dive

rsit

yIn

stit

ute

ofO

ntar

io,

prim

erpa

irus

ed,

and

sequ

ence

leng

th.

© 2007 NRC Canada


Gen

Ban

kac

cess

ion

No.

Pro

cess

IDS

peci

esFa

mil

yC

ount

ryA

rchi

vety

peP

rim

erpa

ira

Len

gth

DQ

8820

66F

CD

PA10

9-04

Eua

lus

barb

atus

Hip

poly

tida

eC

anad

aS

peci

men

FolA

–Fol

B64

5D

Q88

2067

FC

DPA

110-

04E

ualu

sba

rbat

usH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

639

DQ

8820

68F

CD

PA14

0-04

Eua

lus

barb

atus

Hip

poly

tida

eC

anad

aS

peci

men

FolA

–Fol

B64

3D

Q88

2074

FC

DPA

036-

04E

ualu

sbi

ungu

isH

ippo

lyti

dae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2073

FC

DPA

037-

04E

ualu

sbi

ungu

isH

ippo

lyti

dae

Can

ada

Tis

sue

FolA

–Fol

B62

4D

Q88

2072

FC

DPA

038-

04E

ualu

sbi

ungu

isH

ippo

lyti

dae

Can

ada

Tis

sue

FolA

–Fol

B61

5D

Q88

2071

FC

DPA

039-

04E

ualu

sbi

ungu

isH

ippo

lyti

dae

Can

ada

Tis

sue

FolA

–Fol

B62

4D

Q88

2075

FC

DPA

087-

04E

ualu

sga

imar

diH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

659

DQ

8820

78F

CD

PA04

0-04

Eua

lus

suck

leyi

Hip

poly

tida

eC

anad

aT

issu

eFo

lA–F

olB

624

DQ

8820

76F

CD

PA12

6-04

Eua

lus

suck

leyi

Hip

poly

tida

eC

anad

aS

peci

men

FolA

–Fol

B65

6D

Q88

2077

FC

DPA

127-

04E

ualu

ssu

ckle

yiH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

657

DQ

8820

79F

CD

PA07

1-04

Hya

sly

ratu

sO

rego

niid

aeC

anad

aT

issu

eFo

lA–F

olB

658

DQ

8820

80F

CD

PA13

0-04

Hym

enod

ora

fron

tali

sO

plop

hori

dae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2081

FC

DPA

141-

04H

ymen

odor

afr

onta

lis

Opl

opho

rida

eC

anad

aT

issu

eFo

lA–F

olB

608

DQ

8820

84F

CD

PA08

1-04

Leb

beus

groe

nlan

dicu

sH

ippo

lyti

dae

Can

ada

Spe

cim

enFo

lA–F

olB

639

DQ

8820

83F

CD

PA03

2-04

Leb

beus

groe

nlan

dicu

sH

ippo

lyti

dae

Can

ada

Tis

sue

FolA

–Fol

B50

5D

Q88

2082

FC

DPA

033-

04L

ebbe

usgr

oenl

andi

cus

Hip

poly

tida

eC

anad

aT

issu

eFo

lA–F

olB

614

DQ

8820

86F

CD

PA05

4-04

Lit

hode

sco

uesi

Lit

hodi

dae

Can

ada

Tis

sue

FolA

–Fol

B63

9D

Q88

2085

FC

DPA

055-

04L

itho

des

coue

siL

itho

dida

eC

anad

aT

issu

eFo

lA–F

olB

619

DQ

8820

89F

CD

PA05

6-04

Lop

holi

thod

esfo

ram

inat

usL

itho

dida

eC

anad

aT

issu

eC

rust

F1–

FolB

638

DQ

8820

88F

CD

PA13

6-04

Lop

holi

thod

esfo

ram

inat

usL

itho

dida

eC

anad

aT

issu

eFo

lA–F

olB

658

DQ

8820

87F

CD

PA14

8-04

Lop

holi

thod

esfo

ram

inat

usL

itho

dida

eC

anad

aT

issu

eC

rust

F1–

FolB

642

DQ

8820

90F

CD

PA05

8-04

Mun

ida

quad

risp

ina

Gal

athe

idae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2092

FC

DPA

059-

04M

unid

aqu

adri

spin

aG

alat

heid

aeC

anad

aS

peci

men

FolA

–Fol

B65

8D

Q88

2091

FC

DPA

060-

04M

unid

aqu

adri

spin

aG

alat

heid

aeC

anad

aT

issu

eFo

lA–F

olB

603

DQ

8820

93F

CD

PA13

7-04

Mun

idop

sis

quad

rata

Gal

athe

idae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2094

FC

DPA

131-

04N

otos

tom

usja

poni

cus

Opl

opho

rida

eC

anad

aT

issu

eFo

lA–F

olB

647

DQ

8820

95F

CD

PA08

9-04

Orc

onec

tes

imm

unis

Cam

bari

dae

Can

ada

Spe

cim

enFo

lA–F

olB

632

DQ

8820

96F

CD

PA07

5-04

Orc

onec

tes

lim

osus

Cam

bari

dae

Can

ada

Tis

sue

Cru

stF

1–Fo

lB63

2D

Q88

2097

FC

DPA

134-

04Pa

guri

stes

turg

idus

Dio

geni

dae

Can

ada

Spe

cim

enFo

lA–F

olB

658

DQ

8821

00F

CD

PA14

5-04

Pagu

rist

estu

rgid

usD

ioge

nida

eC

anad

aS

peci

men

FolA

–Fol

B61

4D

Q88

2099

FC

DPA

146-

04Pa

guri

stes

turg

idus

Dio

geni

dae

Can

ada

Spe

cim

enFo

lA–F

olB

645

DQ

8820

98F

CD

PA14

7-04

Pagu

rist

estu

rgid

usD

ioge

nida

eC

anad

aS

peci

men

FolA

–Fol

B64

3D

Q88

2101

FC

DPA

160-

05Pa

laem

onsp

.Pa

laem

onid

aeP

olan

dS

peci

men

FolA

–Fol

B65

8D

Q88

2103

FC

DPA

157-

05Pa

laem

onel

egan

sPa

laem

onid

aeP

olan

dS

peci

men

FolA

–Fol

B65

8D

Q88

2104

FC

DPA

158-

05Pa

laem

onel

egan

sPa

laem

onid

aeP

olan

dS

peci

men

FolA

–Fol

B65

8D

Q88

2102

FC

DPA

159-

05Pa

laem

onel

egan

sPa

laem

onid

aeP

olan

dS

peci

men

FolA

–Fol

B65

8D

Q88

2105

FC

DPA

161-

05Pa

laem

onel

egan

sPa

laem

onid

aeP

olan

dS

peci

men

FolA

–Fol

B65

8D

Q88

2107

FC

DPA

018-

04Pa

ndal

opsi

sdi

spar

Pand

alid

aeC

anad

aS

peci

men

FolA

–Fol

B65

4D

Q88

2108

FC

DPA

019-

04Pa

ndal

opsi

sdi

spar

Pand

alid

aeC

anad

aS

peci

men

FolA

–Fol

B65

3D

Q88

2106

FC

DPA

105-

04Pa

ndal

opsi

sdi

spar

Pand

alid

aeC

anad

aS

peci

men

FolA

–Fol

B65

7D

Q88

2112

FC

DPA

001-

04Pa

ndal

usda

nae

Pand

alid

aeC

anad

aT

issu

eFo

lA–F

olB

626

DQ

8821

11F

CD

PA11

3-04

Pand

alus

dana

ePa

ndal

idae

Can

ada

Spe

cim

enFo

lA–F

olB

658

Tab

leA

3(c

onti

nued

).

© 2007 NRC Canada

Costa et al. 289

Gen

Ban

kac

cess

ion

No.

Pro

cess

IDS

peci

esFa

mil

yC

ount

ryA

rchi

vety

peP

rim

erpa

ira

Len

gth

DQ

8821

10F

CD

PA11

4-04

Pand

alus

dana

ePa

ndal

idae

Can

ada

Spe

cim

enFo

lA–F

olB

654

DQ

8821

09F

CD

PA11

5-04

Pand

alus

dana

ePa

ndal

idae

Can

ada

Spe

cim

enFo

lA–F

olB

654

DQ

8821

13F

CD

PA00

2-04

Pand

alus

goni

urus

Pand

alid

aeC

anad

aT

issu

eFo

lA–F

olB

614

DQ

8821

16F

CD

PA00

3-04

Pand

alus

hyps

inot

usPa

ndal

idae

Can

ada

Tis

sue

FolA

–Fol

B60

4D

Q88

2115

FC

DPA

004-

04Pa

ndal

ushy

psin

otus

Pand

alid

aeC

anad

aT

issu

eFo

lA–F

olB

656

DQ

8821

14F

CD

PA00

5-04

Pand

alus

hyps

inot

usPa

ndal

idae

Can

ada

Tis

sue

FolA

–Fol

B65

8D

Q88

2118

FC

DPA

006-

04Pa

ndal

usjo

rdan

iPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

652

DQ

8821

17F

CD

PA00

7-04

Pand

alus

jord

ani

Pand

alid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB61

3D

Q88

2120

FC

DPA

008-

04Pa

ndal

usjo

rdan

iPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

658

DQ

8821

19F

CD

PA00

9-04

Pand

alus

jord

ani

Pand

alid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB65

8D

Q88

2121

FC

DPA

010-

04Pa

ndal

usm

orta

gui

trid

ens

Pand

alid

aeC

anad

aT

issu

eC

rust

F1–

FolB

616

DQ

8821

23F

CD

PA01

1-04

Pand

alus

mor

tagu

itr

iden

sPa

ndal

idae

Can

ada

Tis

sue

Cru

stF

1–Fo

lB61

8D

Q88

2122

FC

DPA

012-

04Pa

ndal

usm

orta

gui

trid

ens

Pand

alid

aeC

anad

aT

issu

eC

rust

F1–

FolB

615

DQ

8821

26F

CD

PA01

3-04

Pand

alus

plat

ycer

osPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

538

DQ

8821

25F

CD

PA01

4-04

Pand

alus

plat

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osPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

658

DQ

8821

24F

CD

PA15

3-04

Pand

alus

plat

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osPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

643

DQ

8821

29F

CD

PA01

5-04

Pand

alus

sten

olep

isPa

ndal

idae

Can

ada

Spe

cim

enFo

lA–F

olB

602

DQ

8821

28F

CD

PA01

6-04

Pand

alus

sten

olep

isPa

ndal

idae

Can

ada

Spe

cim

enC

rust

F2–

FolB

653

DQ

8821

27F

CD

PA01

7-04

Pand

alus

sten

olep

isPa

ndal

idae

Can

ada

Spe

cim

enFo

lA–F

olB

617

DQ

8821

30F

CD

PA05

7-04

Para

lom

ism

ulti

spin

aL

itho

dida

eC

anad

aT

issu

eFo

lA–F

olB

615

DQ

8821

33F

CD

PA04

1-04

Pasi

phae

apa

cifi

caPa

siph

aeid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB65

8D

Q88

2134

FC

DPA

042-

04Pa

siph

aea

paci

fica

Pasi

phae

idae

Can

ada

Spe

cim

enC

rust

F1–

FolB

648

DQ

8821

35F

CD

PA04

3-04

Pasi

phae

apa

cifi

caPa

siph

aeid

aeC

anad

aS

peci

men

Cru

stF

1–Fo

lB65

7D

Q88

2132

FC

DPA

128-

04Pa

siph

aea

paci

fica

Pasi

phae

idae

Can

ada

Spe

cim

enFo

lA–F

olB

659

DQ

8821

31F

CD

PA12

9-04

Pasi

phae

apa

cifi

caPa

siph

aeid

aeC

anad

aS

peci

men

FolA

–Fol

B65

8D

Q88

2137

FC

DPA

044-

04Pa

siph

aea

tard

aPa

siph

aeid

aeC

anad

aT

issu

eFo

lA–F

olB

616

DQ

8821

36F

CD

PA04

5-04

Pasi

phae

ata

rda

Pasi

phae

idae

Can

ada

Tis

sue

Cru

stF

2–Fo

lB65

6D

Q88

2139

FC

DPA

047-

04Pa

siph

aea

tard

aPa

siph

aeid

aeC

anad

aS

peci

men

Cru

stF

2–Fo

lB65

8D

Q88

2138

FC

DPA

048-

04Pa

siph

aea

tard

aPa

siph

aeid

aeC

anad

aS

peci

men

Cru

stF

2–Fo

lB64

1D

Q88

2143

FC

DPA

162-

05R

hith

ropa

nope

usha

rris

iiPa

nope

idae

Pol

and

Spe

cim

enFo

lA–F

olB

658

DQ

8821

42F

CD

PA16

3-05

Rhi

thro

pano

peus

harr

isii

Pano

peid

aeP

olan

dS

peci

men

FolA

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B65

8D

Q88

2140

FC

DPA

164-

05R

hith

ropa

nope

usha

rris

iiPa

nope

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Pol

and

Spe

cim

enFo

lA–F

olB

658

DQ

8821

41F

CD

PA16

5-05

Rhi

thro

pano

peus

harr

isii

Pano

peid

aeP

olan

dS

peci

men

FolA

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B65

8D

Q88

2144

FC

DPA

079-

04Sc

lero

cran

gon

bore

asC

rang

onid

aeC

anad

aS

peci

men

FolA

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B65

9D

Q88

2145

FC

DPA

080-

04Sc

lero

cran

gon

bore

asC

rang

onid

aeC

anad

aS

peci

men

FolA

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B65

9D

Q88

2146

FC

DPA

082-

04Sc

lero

cran

gon

bore

asC

rang

onid

aeC

anad

aS

peci

men

FolA

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B56

4D

Q88

2148

FC

DPA

086-

04Sc

lero

cran

gon

bore

asC

rang

onid

aeC

anad

aS

peci

men

FolA

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B65

8D

Q88

2147

FC

DPA

088-

04Sc

lero

cran

gon

bore

asC

rang

onid

aeC

anad

aS

peci

men

FolA

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B65

9D

Q88

2150

FC

DPA

142-

04Se

rges

tes

sim

ilis

Ser

gest

idae

Can

ada

Spe

cim

enFo

lA–F

olB

646

DQ

8821

52F

CD

PA14

3-04

Serg

este

ssi

mil

isS

erge

stid

aeC

anad

aS

peci

men

FolA

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B64

8D

Q88

2151

FC

DPA

144-

04Se

rges

tes

sim

ilis

Ser

gest

idae

Can

ada

Spe

cim

enFo

lA–F

olB

647

DQ

8821

49F

CD

PA13

3-04

Serg

este

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