DNA Barcodes pp 207-222 | Cite as

Methods for DNA Barcoding Photosynthetic Protists Emphasizing the Macroalgae and Diatoms

Part of the Methods in Molecular Biology book series (MIMB, volume 858)

Abstract

This chapter outlines the current practices used in our laboratory for routine DNA barcode analyses of the three major marine macroalgal groups, viz., brown (Phaeophyceae), red (Rhodophyta), and green (Chlorophyta) algae, as well as for the microscopic diatoms (Bacillariophyta). We start with an outline of current streamlined field protocols, which facilitate the collection of substantial (hundreds to thousands) specimens during short (days to weeks) field excursions. We present the current high-throughput DNA extraction protocols, which can, nonetheless, be easily modified for manual molecular laboratory use. We are advocating a two-marker approach for the DNA barcoding of protists with each major lineage having a designated primary and secondary barcode marker of which one is always the LSU D2/D3 (divergent domains D2/D3 of the nuclear ribosomal large subunit DNA). We provide a listing of the primers that we currently use in our laboratory for amplification of DNA barcode markers from the groups that we study: LSU D2/D3, which we advocate as a eukaryote-wide barcode marker to facilitate broad ecological and environmental surveys (secondary barcode marker in this capacity); COI-5P (the standard DNA barcode region of the mitochondrial cytochrome c oxidase 1 gene) as the primary barcode marker for brown and red algae; rbcL-3P (the 3′ region of the plastid large subunit of ribulose-l-5-bisphosphate carboxylase/oxygenase) as the primary barcode marker for diatoms; and tufA (plastid elongation factor Tu gene) as the primary barcode marker for chlorophytan green algae. We outline our polymerase chain reaction and DNA sequencing methodologies, which have been streamlined for efficiency and to reduce unnecessary cleaning steps. The combined information should provide a helpful guide to those seeking to complete barcode research on these and related “protistan” groups (the term protist is not used in a phylogenetic context; it is simply a catch-all term for the bulk of eukaryotic diversity, i.e., all lineages excluding animals, true fungi, and plants).

Key words

Bacillariophyta Chlorophyta COI-5P DNA barcode LSU Phaeophyceae Protist rbcL-3P Rhodophyta tufA DNA barcoding 

References

  1. 1.
    Saunders GW (2005) Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Phil Trans R Soc B 360:1879–1888PubMedCrossRefGoogle Scholar
  2. 2.
    Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–322CrossRefGoogle Scholar
  3. 3.
    Hebert PDN, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B 270:S96–S99CrossRefGoogle Scholar
  4. 4.
    Saunders GW (1993) Gel purification of red algal genomic DNA: an inexpensive and rapid method for the isolation of polymerase chain reaction-friendly DNA. J Phycol 29:251–254CrossRefGoogle Scholar
  5. 5.
    Saunders GW, Kraft GT (1995) The phylogenetic affinities of Notheia anomala (Fucales, Phaeophyceae) as determined from partial small-subunit rRNA gene sequences. Phycologia 34:383–389CrossRefGoogle Scholar
  6. 6.
    Ivanova NV, Fazekas AJ, Hebert PDN (2008) Semi-automated, membrane-based protocol for DNA isolation from plants. Plant Mol Biol Rep 26:186–198CrossRefGoogle Scholar
  7. 7.
    Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7:544–548CrossRefGoogle Scholar
  8. 8.
    Hamsher SE, Evans KM, Mann DG, Poulícková A, Saunders GW (2011) Barcoding diatoms: exploring alternatives to COI-5P. Protist 162: 405–422Google Scholar
  9. 9.
    Saunders GW, Kucera H (2010) An evaluation of rbcL, tufA, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae. Crypt Algol 31:487–528Google Scholar
  10. 10.
    Maddison WP, Maddison DR (2003) MacClade, version 4.06. Sinauer, SunderlandGoogle Scholar
  11. 11.
    Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.1. http://www.geneious.com
  12. 12.
    Guillou L, Nézan E, Cueff V, Erard-Le Denn E, Cambon-Bonavita MA, Gentien P, Barbier G (2002) Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium, Dinophysis, and Karenia) from French Coasts. Protist 153:223–238PubMedCrossRefGoogle Scholar
  13. 13.
    McDonald SM, Sarno D, Zingone A (2007) Identifying Pseudo-nitzschia in natural samples using genus-specific PCR primers and clone libraries. Harmful Algae 6:849–860CrossRefGoogle Scholar
  14. 14.
    Saunders GW (2008) A DNA barcode examination of the red algal family Dumontiaceae in Canadian waters reveals substantial cryptic species diversity. 1. The foliose Dilsea-Neodilsea complex and Weeksia. Botany 86:773–789CrossRefGoogle Scholar
  15. 15.
    Kucera H, Saunders GW (2008) Assigning morphological variants of Fucus (Fucales, Phaeophyceae) in Canadian waters to recognized species using DNA barcoding. Botany 86:1065–1079CrossRefGoogle Scholar
  16. 16.
    McDevit DC, Saunders GW (2009) On the utility of DNA barcoding for species ­differentiation among brown macroalgae (Phaeophyceae) including a novel extraction protocol. Phycol Res 57:131–141CrossRefGoogle Scholar
  17. 17.
    Trobajo R, Mann DG, Clavero E, Evans KM, Vanormelingen P, McGregor RC (2011) The use of partial cox1, rbcL and LSU rDNA sequences for phylogenetics and species identification within the Nitzschia palea species complex (Bacillariophyceae). Eur J Phycol 45: 413–425CrossRefGoogle Scholar
  18. 18.
    Harper JT, Saunders GW (2001) The application of sequences of the ribosomal cistron to the systematics and classification of the florideophyte red algae (Florideophyceae, Rhodophyta). Cah Biol Mar 42:25–38Google Scholar
  19. 19.
    Harper JT, Saunders GW (2001) Molecular systematics of the Florideophyceae (Rhodophyta) using nuclear large- and small-subunit rDNA sequence data. J Phycol 37:1073–1082CrossRefGoogle Scholar
  20. 20.
    Levialdi Ghiron JH, Amato A, Montresor M, Kooistra WH (2008) Plastid inheritance in the planktonic raphid pinnate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). Protist 159:91–98PubMedCrossRefGoogle Scholar
  21. 21.
    Fama P, Wysor B, Kooistra W, Zuccarello GC (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. J Phycol 38:1040–1050CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Biology, Centre for Environmental & Molecular Algal ResearchUniversity of New BrunswickFrederictonCanada

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