DNA Barcoding Methods for Invertebrates

  • Nathaniel Evans
  • Gustav PaulayEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 858)


Invertebrates comprise approximately 34 phyla, while vertebrates represent one subphylum and insects a (very large) class. Thus, the clades excepting vertebrates and insects encompass almost all of animal diversity. Consequently, the barcoding challenge in invertebrates is that of barcoding animals in general. While standard extraction, cleaning, PCR methods, and universal primers work for many taxa, taxon-specific challenges arise because of the shear genetic and biochemical diversity present across the kingdom, and because problems arising as a result of this diversity, and solutions to them, are still poorly characterized for many metazoan clades. The objective of this chapter is to emphasize general approaches, and give practical advice for overcoming the diverse challenges that may be encountered across animal taxa, but we stop short of providing an exhaustive inventory. Rather, we encourage researchers, especially those working on poorly studied taxa, to carefully consider methodological issues presented below, when standard approaches perform poorly.

Key words

DNA barcoding Invertebrates CO1 Cytochrome c oxidase subunit I 


  1. 1.
    Hebert PDN, Cywinska A, Ball SL, Dewaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci 270:313–321Google Scholar
  2. 2.
    Lynch M, Koskella B, Schaack S (2006) Mutation pressure and the evolution of organelle genomic architecture. Science 311: 1727–1730PubMedGoogle Scholar
  3. 3.
    Davison A, Blackie RLE, Scothern GP (2009) DNA barcoding of stylommatophoran land snails: a test of existing sequences. Mol Ecol Resour 9:1092–1101PubMedGoogle Scholar
  4. 4.
    Creer S, Fonseca VG, Porazinska DL et al (2010) Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises. Mol Ecol 19(Suppl 1):4–20PubMedGoogle Scholar
  5. 5.
    Hassanin A (2006) Phylogeny of Arthropoda inferred from mitochondrial sequences: strategies for limiting the misleading effects of multiple changes in pattern and rates of substitution. Mol Phylogenet Evol 38: 100–116PubMedGoogle Scholar
  6. 6.
    Knowlton N (1993) Sibling species in the sea. Ann Rev Ecol Syst 24:189–216Google Scholar
  7. 7.
    Verheyen E, Salzburger W, Snoeks J, Meyer A (2003) Origin of the superflock of cichlid fishes from Lake Victoria, East Africa. Science 300:325–329PubMedGoogle Scholar
  8. 8.
    Gregory TR, Mable BK (2005) Polyploidy in animals. In: Gregory TR (ed) The evolution of the genome. Academic, Waltham, MA, pp 428–501Google Scholar
  9. 9.
    Landry C, Geyer LB, Arakaki Y, Uehara T, Palumbi SR (2003) Recent speciation in the Indo-West Pacific: rapid evolution of gamete recognition and sperm morphology in cryptic species of sea urchin. Proc R Soc Lond B Biol Sci 270:1839–1847Google Scholar
  10. 10.
    Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS Biol 3:e422PubMedGoogle Scholar
  11. 11.
    Coyne J, Orr H (2004) Speciation. Sinauer Associates, Sunderland, MA, p 545Google Scholar
  12. 12.
    Mayr E (1963) Animal species and their evolution. Harvard University Press, Cambridge, p 797Google Scholar
  13. 13.
    Meyer C, Geller J, Paulay G (2005) Fine scale endemism on coral reefs: archipelagic differentiation in turbinid gastropods. Evolution 59:113–125PubMedGoogle Scholar
  14. 14.
    Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  15. 15.
    Chen I-P, Tang C-Y, Chiou C-Y et al (2009) Comparative analyses of coding and noncoding DNA regions indicate that Acropora (Anthozoa: Scleractina) possesses a similar evolutionary tempo of nuclear vs. mitochondrial genomes as in plants. Mar Biotechnol 11:141–152PubMedGoogle Scholar
  16. 16.
    Huang D, Meier R, Todd PA, Chou LM (2008) Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding. J Mol Evol 66:167–174PubMedGoogle Scholar
  17. 17.
    McFadden CS, Benayahu Y, Pante E et al (2010) Limitations of mitochondrial gene barcoding in Octocorallia. Mol Ecol Resour 11:1–13Google Scholar
  18. 18.
    Ortman BD (2008) DNA barcoding the medu­sozoa and ctenophora. Ph.D. Dissertation, University of Connecticut, Storrs, CTGoogle Scholar
  19. 19.
    Shearer TL, Coffroth MA (2008) DNA BARCODING: barcoding corals: limited by interspecific divergence, not intraspecific variation. Mol Ecol Resour 8:247–255PubMedGoogle Scholar
  20. 20.
    Signorovitch AY, Dellaporta SL, Buss LW (2006) Caribbean placozoan phylogeography. Biol Bull 211:149–156PubMedGoogle Scholar
  21. 21.
    Signorovitch AY, Buss LW, Dellaporta SL (2007) Comparative genomics of large mitochondria in placozoans. PLoS Genet 3:e13PubMedGoogle Scholar
  22. 22.
    Wörheide G, Erpenbeck D, Menke C (2008) The Sponge Barcoding Project: aiding in the identification and description of poriferan taxa. In: Custódio M, Lôbo-Hajdu G, Haidu E, Muricy G (eds) Porifera research: biodiversity, innovation and sustainability. Museu Nacional de Rio de Janiero Book Series. Rio de Janeiro, Brazil, pp 123–128Google Scholar
  23. 23.
    Dawson MN, Jacobs DK (2001) Molecular evidence for cryptic species of Aurelia aurita (Cnidaria, Scyphozoa). Biol Bull 200:92PubMedGoogle Scholar
  24. 24.
    Spiess A-N-L, Mueller N, Ivell R (2004) Trehalose is a potent pcr enhancer: lowering of DNA melting temperature and thermal ­stabilization of Taq polymerase by the disaccharide trehalose. Clin Chem 50:1256–1259PubMedGoogle Scholar
  25. 25.
    Kreader CA (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62:1102–1106PubMedGoogle Scholar
  26. 26.
    Templado J, Paulay G, Gittenberger A, Meyer C (2010) Sampling the marine realm. In: Eymann J, Degreef J, Häuser C et al (eds) Manual on field recording techniques and protocols for all taxa biodiversity inventories. vol 8. ABC Taxa. Belgian National Focal Point for the GTI, Brussels, pp 273–307Google Scholar
  27. 27.
    Eymann J, Degreef J, Häuser C, Monje JC, Samyn Y, Van den Spiegel D (eds) (2010) Manual on field recording techniques and protocols for all taxa biodiversity inventories. vol 8. ABC Taxa. Belgian National Focal Point for the GTI, BrusselsGoogle Scholar
  28. 28.
    Gaither M, Szabó Z, Crepeau M et al (2011) Preservation of corals in salt-saturated DMSO buffer is superior to ethanol for PCR experiments. Coral Reefs 30:329–333Google Scholar
  29. 29.
    Ivanova NV, Dewaard JR, Hebert PDN (2006) An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol Ecol Notes 6:998–1002Google Scholar
  30. 30.
    Rosengarten RD, Sperling EA, Moreno MA, Leys SP, Dellaporta SL (2008) The mitochondrial genome of the hexactinellid sponge Aphrocallistes vastus: evidence for programmed translational frameshifting. BMC Genomics 9:33PubMedGoogle Scholar
  31. 31.
    Sinniger F, Pawlowski J (2009) The partial mitochondrial genome of Leiopathes glaberrima (Hexacorallia: Antipatharia) and the first report of the presence of an intron in COI in black corals. Galaxea 11:21–26Google Scholar
  32. 32.
    Fukami H, Chen CA, Chiou C-Y, Knowlton N (2007) Novel group I introns encoding a putative homing endonuclease in the mitochondrial cox1 gene of Scleractinian corals. J Mol Evol 64:591–600PubMedGoogle Scholar
  33. 33.
    Milbury CA, Gaffney PM (2005) Complete mitochondrial DNA sequence of the eastern oyster Crassostrea virginica. Mar Biotechnol 7:697–712PubMedGoogle Scholar
  34. 34.
    Hajibabaei M, DeWaard JR, Ivanova NV et al (2005) Critical factors for assembling a high volume of DNA barcodes. Philos Trans R Soc Lond B Biol Sci 360:1959–1967PubMedGoogle Scholar
  35. 35.
    DeWaard J, Ivanova N, Hajibabaei M, Hebert P (2008) Assembling DNA barcodes. Analytical protocols. In: Martin C (ed) Methods in molecular biology. Humana, Totowa, pp 275–293Google Scholar
  36. 36.
    Bickley J, Hopkins D (1999) Inhibitors and enhancers of PCR. In: Saunders GC, Parkes HC (eds) Analytical molecular biology: quality and validation. Royal Society of Chemistry, Cambridge, UK, pp 81–102Google Scholar
  37. 37.
    Ralser M, Querfurth R, Warnatz H-J et al (2006) An efficient and economic enhancer mix for PCR. Biochem Biophys Res Comm 347:747–751PubMedGoogle Scholar
  38. 38.
    Hecker KH, Roux KH (1996) High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20:478–485PubMedGoogle Scholar
  39. 39.
    Siddall ME, Fontanella FM, Watson SC et al (2009) Barcoding bamboozled by bacteria: convergence to metazoan mitochondrial primer targets by marine microbes. Syst Biol 58:445–451PubMedGoogle Scholar
  40. 40.
    Schubart C (2009) Mitochondrial DNA and decapod phytogenies: the importance of pseudogenes and primer optimization. In: Martin JW, Crandall KA, Felder DL (eds) Decapod crustacean phylogenetics. CRC, Boca Raton, FL, pp 47–64Google Scholar
  41. 41.
    Hoareau TB, Boissin E (2010) Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata. Mol Ecol Resour 10:960–967PubMedGoogle Scholar
  42. 42.
    Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species. Heredity 101:301–320PubMedGoogle Scholar
  43. 43.
    Chen C, Chiou CY, Dai CF, Chen CA (2008) Unique mitogenomic features in the scleractinian family pocilloporidae (Scleractinia: Astrocoeniina). Mar Biotech 10:538–553Google Scholar
  44. 44.
    Rawlings TA, Collins TM, Bieler R (2003) Changing identities: tRNA duplication and remolding within animal mitochondrial genomes. Proc Natl Acad Sci USA 100:15700–15705PubMedGoogle Scholar
  45. 45.
    Walther E, Schofl G, Mrotzek G et al (2011) Paralogous mitochondrial control region in the giant tiger shrimp, Penaeus monodon (F.) affects population genetics inference: a cautionary tale. Mol Phylgenet Evol 58:404–408Google Scholar
  46. 46.
    Machida R, Miya M, Nishida M, Nishida S (2006) Molecular phylogeny and evolution of the pelagic copepod genus Neocalanus (Crustacea: Copepoda). Marine Biol 148:1071–1079Google Scholar
  47. 47.
    Erpenbeck D, Hooper JNA, Worheide G (2006) CO1 phylogenies in diploblasts and the “Barcoding of Life” – are we sequencing a suboptimal partition? Mol Ecol Notes 6:550–553Google Scholar
  48. 48.
    Derycke S, Vanaverbeke J, Rigaux A et al (2010) Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes. PLoS One 5:e13716PubMedGoogle Scholar
  49. 49.
    Simon C, Buckley TR, Frati F, Stewart JB, Beckenbach AT (2006) Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA. Ann Rev Ecol Syst 37:545–579Google Scholar
  50. 50.
    Simpson R, Wilding C, Grahame J (2005) Intron analyses reveal multiple calmodulin copies in Littorina. J Mol Evol 60:505–512PubMedGoogle Scholar
  51. 51.
    Chenuil A, Hoareau TB, Egea E et al (2010) An efficient method to find potentially universal population genetic markers, applied to metazoans. BMC Evol Biol 10:276PubMedGoogle Scholar
  52. 52.
    Hwang UW, Kim W (1999) General properties and phylogenetic utilities of nuclear ­ribosomal DNA and mitochondrial DNA commonly used in molecular systematics. Korean J Parasitol 37:215PubMedGoogle Scholar
  53. 53.
    Eickbush TH, Eickbush DG (2007) Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175:477–485PubMedGoogle Scholar
  54. 54.
    Harris DJ, Crandall KA (2000) Intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Decapoda: Cambaridae): implications for phylogenetic and microsatellite studies. Mol Biol Evol 17:284PubMedGoogle Scholar
  55. 55.
    Derycke S, Fonseca G, Vierstraete A et al (2008) Disentangling taxonomy within the Rhabditis (Pellioditis) marina (Nematoda, Rhabditidae) species complex using molecular and morhological tools. Zool J Linn Soc 152:1–15Google Scholar
  56. 56.
    Bhadury P, Austen MC (2010) Barcoding marine nematodes: an improved set of nematode 18S rRNA primers to overcome eukaryotic co-interference. Hydrobiologia 641: 245–251Google Scholar
  57. 57.
    Sonnenberg R, Nolte A (2007) An evaluation of LSU rDNA D1-D2 sequences for their use in species identification. Front Zool 4:6PubMedGoogle Scholar
  58. 58.
    Markmann M, Tautz D (2005) Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ri-bosomal RNA signature sequences. Philos Trans R Soc Lond B Biol Sci 360:1917–1924PubMedGoogle Scholar
  59. 59.
    Cárdenas P, Rapp HT, Schander C, Tendal OS (2010) Molecular taxonomy and phylogeny of the Geodiidae (Porifera, Demospongiae, Astrophorida)–combining phylogenetic and Linnaean classification. Zoolog Scripta 39:89–106Google Scholar
  60. 60.
    McLain DK, Li J, Oliver JH (2001) Interspecific and geographical variation in the sequence of rDNA expansion segment D3 of Ixodes ticks (Acari: Ixodidae). Heredity 86:234–242PubMedGoogle Scholar
  61. 61.
    Benesh DP, Hasu T, Suomalainen L-R, Valtonen ET, Tiirola M (2006) Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes. Int J Parasitol 36:247–254PubMedGoogle Scholar
  62. 62.
    Martínez-Aquino A, Reyna-Fabián ME, Rosas-Valdez R, Razo-Mendivil U, de León GP-P, García-Varela M (2009) Detecting a complex of cryptic species within Neoechi­norhynchus golvani (Acanthocephala: Neoech­inorhynchidae) inferred from ITSs and LSU rDNA gene sequences. Int J Parasitol 95:1040–1047Google Scholar
  63. 63.
    Steinauer ML, Nickol BB, Ortí G (2007) Cryptic speciation and patterns of phenotypic variation of a highly variable acanthocephalan parasite. Mol Ecol 16:4097–4109PubMedGoogle Scholar
  64. 64.
    Sikes JM, Bely AE (2008) Radical modification of the A-P axis and the evolution of asexual reproduction in Convolutriloba acoels. Evol Dev 10:619–631PubMedGoogle Scholar
  65. 65.
    Telford MJ, Herniou EA, Russell RB, Littlewood DT (2000) Changes in mitochondrial genetic codes as phylogenetic characters: two examples from the flatworms. Proc Natl Acad Sci USA 97:11359–11364PubMedGoogle Scholar
  66. 66.
    Chang C-H, Rougerie R, Chen J-H (2009) Identifying earthworms through DNA barcodes: pitfalls and promise. Pedobiologia 52:171–180Google Scholar
  67. 67.
    Aguado MT, Nygren A, Siddall ME (2007) Cladistics analysis of nuclear and mitochondrial genes. Cladistics 23:552–564Google Scholar
  68. 68.
    Carr CM (2010) The polychaeta of canada: exploring diversity and distribution patterns using DNA barcodes. MSc Thesis, University of Guelph, Guelph, ONGoogle Scholar
  69. 69.
    James SW, Porco D, Decaëns T et al (2010) DNA barcoding reveals cryptic diversity in Lumbricus terrestris L., 1758 (Clitellata): resurrection of L. herculeus (Savigny, 1826). PLoS One 5:e15629PubMedGoogle Scholar
  70. 70.
    Zhou H, Zhang Z, Chen H et al (2010) Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China. Chin J Oceanol Limnol 28:899–910Google Scholar
  71. 71.
    Bely AE, Weisblat DA (2006) Lessons from leeches: a call for DNA barcoding in the lab. Evol Dev 8:491–501PubMedGoogle Scholar
  72. 72.
    Costa FO, Henzler CM, Lunt DH et al (2009) Probing marine Gammarus (Amphipoda) taxonomy with DNA barcodes. Syst Biod 7:365Google Scholar
  73. 73.
    Costa FO, DeWaard JR, Boutillier J, Ratnasingham S, Dooh RT, Hajibabaei M, Hebert PDN (2007) Biological identifications through DNA barcodes: the case of the Crustacea. Can J Fish Aquat Sci 64:272–295Google Scholar
  74. 74.
    Böttger-Schnack R, Machida RJ (2010) Comparison of morphological and molecular traits for species identification and taxonomic grouping of oncaeid copepods. Hydrobiologia 666:111–125Google Scholar
  75. 75.
    Bradford T, Adams M, Humphreys W, Austin A, Cooper S (2010) DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia’s arid zone. Mol Ecol Resour 10:41–50PubMedGoogle Scholar
  76. 76.
    Goolsby JA, DE Barro PJ, Makinson JR, Pemberton RW, Hartley DM, Frohlich DR (2006) Matching the origin of an invasive weed for selection of a herbivore haplotype for a biological control programme. Mol Ecol 15:287–297PubMedGoogle Scholar
  77. 77.
    Murienne J, Edgecombe GD, Giribet G (2010) Including secondary structure, fossils and molecular dating in the centipede tree of life. Mol Phylogenet Evol 57:301–313PubMedGoogle Scholar
  78. 78.
    Navajas M, Navia D (2010) DNA-based methods for eriophyoid mite studies: review, critical aspects, prospects and challenges. Exp Appl Acarol 51:257–271PubMedGoogle Scholar
  79. 79.
    Barrett RDH, Hebert PDN (2005) Identifying spiders through DNA barcodes. Can J Zool 83:481–491Google Scholar
  80. 80.
    Radulovici AE, Sainte-Marie B, Dufresne F (2009) DNA barcoding of marine crustaceans from the Estuary and Gulf of St Lawrence: a regional-scale approach. Mol Ecol Resour 9:181–187PubMedGoogle Scholar
  81. 81.
    Ros VID, Breeuwer JAJ (2007) Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding. Exp Appl Acarol 42:239–262PubMedGoogle Scholar
  82. 82.
    Hurst GDD, Jiggins FM (2005) Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. Proc R Soc Lond B Biol Sci 272:1525–1534Google Scholar
  83. 83.
    Engelstädter J, Hurst GDD (2009) The ecology and evolution of microbes that manipulate host reproduction. Ann Rev Ecol Evol Syst 40:127–149Google Scholar
  84. 84.
    Cohen BL, Bitner MA, Harper EM et al (2011) Vicariance and convergence in Magellanic and New Zealand long-looped brachiopod clades (Pan-Brachiopoda: Terebratelloidea). Zoolog J Linn Soc 162. doi:  10.1111/j.1096-3642.2010.00682.x
  85. 85.
    Lüter C, Cohen B (2002) DNA sequence evidence for speciation, paraphyly and a Mesozoic dispersal of cancellothyridid articulate brachiopods. Mar Biol 141:65–74Google Scholar
  86. 86.
    Gómez A, Wright PJ, Lunt DH et al (2007) Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon. Proc R Soc Lond B Biol Sci 274:199–207Google Scholar
  87. 87.
    Kon T, Nohara M, Nishida M et al (2006) Hidden ancient diversification in the circumtropical lancelet Asymmetron lucayanum complex. Mar Biol 149:875–883Google Scholar
  88. 88.
    Jennings RM, Bucklin A, Pierrot-Bults A (2010) Barcoding of arrow worms (Phylum Chaetognatha) from three oceans: genetic diversity and evolution within an enigmatic phylum. PLoS One 5:e9949PubMedGoogle Scholar
  89. 89.
    Sinniger F, Reimer JD, Pawlowski J (2008) Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia). Zoolog Sci 25:1253–1260PubMedGoogle Scholar
  90. 90.
    Concepcion GT, Crepeau MW, Wagner D et al (2007) An alternative to ITS, a hypervariable, single-copy nuclear intron in corals, and its use in detecting cryptic species within the octocoral genus Carijoa. Coral Reefs 27:323–336Google Scholar
  91. 91.
    Coleman AW, van Oppen MJH (2008) Secondary structure of the rRNA ITS2 region reveals key evolutionary patterns in acroporid corals. J Mol Evol 67:389–396PubMedGoogle Scholar
  92. 92.
    Chiou CY, Chen IP, Chen C et al (2008) Analysis of Acropora muricata Calmodulin (CaM) indicates that scleractinian corals possess the ancestral exon/intron organization of the eumetazoan CaM gene. J Mol Evol 66:317–324PubMedGoogle Scholar
  93. 93.
    Flot J-F, Magalon H, Cruaud C et al (2008) Patterns of genetic structure among Hawaiian corals of the genus Pocillopora yield clusters of individuals that are compatible with morphology. C R Biol 331:239–247PubMedGoogle Scholar
  94. 94.
    Miranda LS, Collins AG, Marques AC (2010) Molecules clarify a cnidarian life cycle – the “hydrozoan” Microhydrula limopsicola is an early life stage of the Staurozoan Haliclystus antarcticus. PLoS One 5:e10182PubMedGoogle Scholar
  95. 95.
    Moura CJ, Harris DJ, Cunha MR, Rogers AD (2008) DNA barcoding reveals cryptic diversity in marine hydroids (Cnidaria, Hydrozoa) from coastal and deep-sea environments. Zoolog Scripta 37:93–108Google Scholar
  96. 96.
    Dawson MN (2004) Some implications of molecular phylogenetics for understanding biodiversity in jellyfishes, with emphasis on Scyphozoa. Hydrobiologia 530–531: 249–260Google Scholar
  97. 97.
    Ortman BD, Bucklin A, Pagès F, Youngbluth M (2010) DNA barcoding the Medusozoa using mtCOI. Deep Sea Res II 57: 2148–2156Google Scholar
  98. 98.
    Henderson M, Okamura B (2004) The phylogeography of salmonid proliferative kidney disease in Europe and North America. Proc R Soc Lond B Biol Sci 271:1729Google Scholar
  99. 99.
    Whipps CM, Kent ML (2006) Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites (Myxozoa: Myxosporea). J Eukaryot Microbiol 53:364–373PubMedGoogle Scholar
  100. 100.
    Podar M, Haddock SH, Sogin ML, Harbison GR (2001) A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes. Mol Phylogenet Evol 21:218–230PubMedGoogle Scholar
  101. 101.
    Gorokhova E, Lehtiniemi M, Viitasalo-fro S, Haddock SHD (2009) Molecular evidence for the occurrence of ctenophore Mertensia ovum in the northern Baltic Sea and implications for the status of the Mnemiopsis leidyi invasion. Limnol Oceanogr 54:2025–2033Google Scholar
  102. 102.
    Obst M, Funch P, Giribet G (2005) Hidden diversity and host specificity in cycliophorans: a phylogeographic analysis along the North Atlantic and Mediterranean Sea. Mol Ecol 14:4427–4440PubMedGoogle Scholar
  103. 103.
    Lessios HA (2008) The great American schism: divergence of marine organisms after the rise of the Central American Isthmus. Ann Rev Ecol Evol Syst 39:63–91Google Scholar
  104. 104.
    Fuchs J, Iseto T, Hirose M, Sundberg P, Obst M (2010) The first internal molecular phylogeny of the animal phylum Entoprocta (Kamptozoa). Mol Phyl Evol 56:370–379Google Scholar
  105. 105.
    Todaro MA, Kånneby T, Dal Zotto M, Jondelius U (2011) Phylogeny of thaumastodermatidae (gastrotricha: macrodasyida) inferred from nuclear and mitochondrial sequence data. PLoS One 6:e17892PubMedGoogle Scholar
  106. 106.
    Sørensen MV, Sterrer W, Giribet G (2006) Cladistics four molecular loci and morphology. Cladistics 22:32–58Google Scholar
  107. 107.
    Cannon JT, Rychel AL, Eccleston H, Halanych KM, Swalla BJ (2009) Molecular phylogeny of hemichordata, with updated status of deep-sea enteropneusts. Mol Phyl Evol 52:17–24Google Scholar
  108. 108.
    Smith SE, Douglas R, Burke K, Swalla BJ (2003) Morphological and molecular identification of Saccoglossus species (Hemichordata: Harrimaniidae) in the Pacific Northwest. Can J Zool 141:133–141Google Scholar
  109. 109.
    Giribet G, Sorensen MV, Funch P et al (2004) Investigations into the phylogenetic position of Micrognathozoa using four molecular loci. Cladistics 20:1–13Google Scholar
  110. 110.
    Doucet-Beaupré H, Breton S, Chapman EG et al (2010) Mitochondrial phylogenomics of the Bivalvia (Mollusca): searching for the origin and mitogenomic correlates of doubly uniparental inheritance of mtDNA. BMC Evol Biol 10:50PubMedGoogle Scholar
  111. 111.
    Campbell DC, Johnson PD, Williams JD et al (2008) Identification of “extinct” freshwater mussel species using DNA barcoding. Mol Ecol Resour 8:711–724PubMedGoogle Scholar
  112. 112.
    Ghiselli F, Milani L, Passamonti M (2011) Strict sex-specific mtDNA segregation in the germ line of the DUI species Venerupis philippinarum (Bivalvia: Veneridae). Mol Biol Evol 28:949–961PubMedGoogle Scholar
  113. 113.
    Allcock AL, Barratt I, Eléaume M et al (2010) Cryptic speciation and the circumpolarity debate: a case study on endemic Southern Ocean octopuses using the COI barcode of life. Deep Sea Res IIGoogle Scholar
  114. 114.
    Kelly RP, Sarkar IN, Eernisse DJ, Desalle R (2007) DNA barcoding using chitons (genus Mopalia). Mol Ecol Notes 7:177–183Google Scholar
  115. 115.
    Dinapoli A, Klussmann-Kolb A (2010) The long way to diversity–phylogeny and evolution of the Heterobranchia (Mollusca: Gastropoda). Mol Phylogenet Evol 55:60–76PubMedGoogle Scholar
  116. 116.
    Barr NB, Cook A, Elder P et al (2009) Application of a DNA barcode using the 16S rRNA gene to diagnose pest Arion species in the USA. J Molluscan Stud 75:187–191Google Scholar
  117. 117.
    Ladoukakis ED, Theologidis I, Rodakis GC, Zouros E (2011) Homologous recombination between highly diverged mitochondrial sequences: examples from maternally and paternally transmitted genomes. Mol Biol Evol 28:1–40Google Scholar
  118. 118.
    Breton S, Stewart DT, Shepardson S et al (2011) Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)? Mol Biol Evol 28:1645–1659PubMedGoogle Scholar
  119. 119.
    Bourlat SJ, Nakano H, Åkerman M et al (2008) Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding. Mol Ecol Resour 8:18–22PubMedGoogle Scholar
  120. 120.
    Bhadury P, Austen MC, Bilton DT et al (2007) Exploitation of archived marine nematodes – a hot lysis DNA extraction protocol for molecular studies. Zoolog Scripta 36:93–98Google Scholar
  121. 121.
    De Ley P, De Ley IT, Morris K et al (2005) An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philos Trans R Soc Lond B Biol Sci 360: 1945–1958PubMedGoogle Scholar
  122. 122.
    Mateos E, Giribet G (2008) Exploring the molecular diversity of terrestrial nemerteans (Hoplonemertea, Monostilifera, Acteonemertidae) in a continental landmass. Zoolog Scripta 37:235–243Google Scholar
  123. 123.
    Maslakova S, Norenburg J (2008) Revision of the smiling worms, genus Prosorhochmus Keferstein, 1862, and description of a new species, Prosorhochmus belizeanus sp. nov. (Prosorhochmidae, Hoplonemertea, Nemertea) from Florida and Belize. J Nat History 42:1219–1260Google Scholar
  124. 124.
    Sundberg P, Thuroczy Vodoti E, Strand M (2010) DNA barcoding should accompany taxonomy – the case of Cerebratulus spp (Nemertea). Mol Ecol Resour 10:274–281PubMedGoogle Scholar
  125. 125.
    Daniels SR, Ruhberg H (2010) Molecular and morphological variation in a South African velvet worm Peripatopsis moseleyi (Onychophora, Peripatopsidae): evidence for cryptic speciation. J Zool 282:171–179Google Scholar
  126. 126.
    Trewick SA (2000) Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora). Mol Ecol 9:269–281PubMedGoogle Scholar
  127. 127.
    Podsiadlowski L, Braband A, Mayer G (2008) The complete mitochondrial genome of the onychophoran Epiperipatus biolleyi reveals a unique transfer RNA set and provides further support for the ecdysozoa hypothesis. Mol Biol Evol 25:42–51PubMedGoogle Scholar
  128. 128.
    Santagata S, Cohen BL (2009) Phoronid phylogenetics (Brachiopoda; Phoronata): evidence from morphological cladistics, small and large subunit rDNA sequences, and mitochondrial cox1. Zool J Linn Soc 157:34–50Google Scholar
  129. 129.
    Voigt O, Collins AG, Pearse VB et al (2004) Placozoa – no longer a phylum of one. Current Biol 14:944–945Google Scholar
  130. 130.
    Sanna D, Lai T, Francalacci P et al (2009) Population structure of the Monocelis lineata (Proseriata, Monocelididae) species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Gen Mol Biol 32:864–867Google Scholar
  131. 131.
    Moszczynska A, Locke SA, McLaughlin JD et al (2009) Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths. Mol Ecol Resour 9:75–82Google Scholar
  132. 132.
    Zarowiecki MZ, Huyse T, Littlewood DTJ (2007) Making the most of mitochondrial genomes–markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea). Int J Parasitol 37:1401–1418PubMedGoogle Scholar
  133. 133.
    Pöppe J, Sutcliffe P, Hooper JNA et al (2010) CO I barcoding reveals new clades and radiation patterns of Indo-Pacific sponges of the family Irciniidae (Demospongiae: Dictyoceratida). PLoS One 5:e9950PubMedGoogle Scholar
  134. 134.
    Wang X, Lavrov DV (2008) Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the Demospongiae. PLoS One 3:e2723PubMedGoogle Scholar
  135. 135.
    Watanabe KI, Bessho Y, Kawasaki M, Hori H (1999) Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema. J Mol Biol 286:645–650PubMedGoogle Scholar
  136. 136.
    Derry AM, Hebert PDN, Prepas EE (2003) Evolution of rotifers in saline and subsaline lakes: a molecular phylogenetic approach. Limn Oceanograph 48:675–685Google Scholar
  137. 137.
    Fontaneto D, Kaya M, Herniou EA, Barraclough TG (2009) Extreme levels of hidden diversity in microscopic animals (Rotifera) revealed by DNA taxonomy. Mol Phy Evol 53:182–189Google Scholar
  138. 138.
    Gómez A, Serra M, Carvalho GR, Lunt DH (2002) Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (Rotifera). Evolution 56:1431–1444PubMedGoogle Scholar
  139. 139.
    Du X, Chen Z, Deng Y, Wang Q (2009) Comparative analysis of genetic diversity and population structure of Sipunculus nudus as revealed by mitochondrial COI sequences. Biochem Genet 47:884–891PubMedGoogle Scholar
  140. 140.
    Kawauchi GY, Giribet G (2010) Are there true cosmopolitan sipunculan worms? A genetic variation study within Phascolosoma perlucens (Sipuncula, Phascolosomatidae). Marine Biol 157:1417–1431Google Scholar
  141. 141.
    Blaxter M, Mann J, Chapman T, Thomas F, Whitton C, Floyd R, Abebe E (2005) Defining operational taxonomic units using DNA barcode data. Philos Trans R Soc Lond B Biol Sci 360:1935–1943PubMedGoogle Scholar
  142. 142.
    Sands CJ, Convey P, Linse K, McInnes SJ (2008) Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada. BMC Ecol 8:7PubMedGoogle Scholar
  143. 143.
    Schill RO (2007) Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades. J Limnol 66:164–170Google Scholar
  144. 144.
    Cesari M, Bertolani R, Rebecchi L, Guidetti R (2009) DNA barcoding in Tardigrada: the first case study on Macrobiotus macro-calix Bertolani & Rebecchi 1993 (Eutar-digrada, Macrobiotidae). Mol Ecol Resour 9:699–706PubMedGoogle Scholar
  145. 145.
    Stefaniak L, Lambert G, Gittenberger A et al (2009) Genetic conspecificity of the worldwide populations of Didemnum vexillum Kott, 2002. Aquat Invasion 4:29–44Google Scholar
  146. 146.
    Nydam ML, Harrison RG (2007) Genealogical relationships within and among shallow-water Ciona species (Ascidiacea). Marine Biol 151:1839–1847Google Scholar
  147. 147.
    Bourlat SJ, Nielsen C, Lockyer AE et al (2003) Xenoturbella is a deuterostome that eats molluscs. Nature 424:925–928PubMedGoogle Scholar
  148. 148.
    Meyer CP (2003) Molecular systematics of cowries (Gastropoda: Cypraeidae) and diversification patterns in the tropics. Biol J Linn Soc 79:401–459Google Scholar
  149. 149.
    Kojima S, Segawa R, Hashimoto J, Ohta S (1997) Molecular phylogeny of vestimentiferans collected around Japan, revealed by the nucleotide sequences of mitochondrial DNA. Marine Biol 127:507–513Google Scholar
  150. 150.
    Prendini L (2005) Systematics of the group of African whip spiders (Chelicerata: Amblypygi): Evidence from behaviour, morphology and DNA. Organ Div Evol 5:203–236Google Scholar
  151. 151.
    Schwendinger PJ, Giribet G (2005) The systematics of the south-east Asian genus Fangensis Rambla (Opiliones: Cyphophthalmi: Stylocellidae). Invertebr Syst 19:297–323Google Scholar
  152. 152.
    Fukami H, Budd AF, Levitan DR et al (2004) Geographic differences in species boundaries among members of the Montastraea annularis complex based on molecular and morphological markers. Evolution 58:324–337PubMedGoogle Scholar
  153. 153.
    Dawson MN (2005) Incipient speciation of Catostylus mosaicus (Scyphozoa, Rhizostomeae, Catostylidae), comparative phylogeography and biogeography in south-east Australia. J Biog 32:515–533Google Scholar
  154. 154.
    Martínez DE, Iñiguez AR, Percell KM et al (2010) Phylogeny and biogeography of Hydra (Cnidaria: Hydridae) using mitochondrial and nuclear DNA sequences. Mol Phyl Evol 57:403–410Google Scholar
  155. 155.
    Palumbi SR, Martin A, Romano S et al (2002) The simple fool’s guide to PCR, Version 2.0. Department of Zoology and Kewalo Marine Laboratory, Honolulu, HIGoogle Scholar
  156. 156.
    Apakupakul K, Siddall ME, Burreson EM (1999) Higher level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences. Mol Phylogenet Evol 12:350–359PubMedGoogle Scholar
  157. 157.
    Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The Characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491–499PubMedGoogle Scholar
  158. 158.
    White T, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR Protocols: a guide to methods and applications. Academic, New York, pp 315–322Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Florida Museum of Natural HistoryUniversity of FloridaGainesvilleUSA

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