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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Saunders GW (2005) Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Phil Trans R Soc B 360:1879–1888
Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–322
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–S99
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 29:251–254
Saunders GW, Kraft GT (1995) The phylogenetic affinities of Notheia anomala (Fucales, Phaeophyceae) as determined from partial small-subunit rRNA gene sequences. Phycologia 34:383–389
Ivanova NV, Fazekas AJ, Hebert PDN (2008) Semi-automated, membrane-based protocol for DNA isolation from plants. Plant Mol Biol Rep 26:186–198
Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7:544–548
Hamsher SE, Evans KM, Mann DG, Poulícková A, Saunders GW (2011) Barcoding diatoms: exploring alternatives to COI-5P. Protist 162: 405–422
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 31:487–528
Maddison WP, Maddison DR (2003) MacClade, version 4.06. Sinauer, Sunderland
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
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 153:223–238
McDonald SM, Sarno D, Zingone A (2007) Identifying Pseudo-nitzschia in natural samples using genus-specific PCR primers and clone libraries. Harmful Algae 6:849–860
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 86:773–789
Kucera H, Saunders GW (2008) Assigning morphological variants of Fucus (Fucales, Phaeophyceae) in Canadian waters to recognized species using DNA barcoding. Botany 86:1065–1079
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 57:131–141
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: 413–425
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 42:25–38
Harper JT, Saunders GW (2001) Molecular systematics of the Florideophyceae (Rhodophyta) using nuclear large- and small-subunit rDNA sequence data. J Phycol 37:1073–1082
Levialdi Ghiron JH, Amato A, Montresor M, Kooistra WH (2008) Plastid inheritance in the planktonic raphid pinnate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). Protist 159:91–98
Fama P, Wysor B, Kooistra W, Zuccarello GC (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. J Phycol 38:1040–1050
Acknowledgments
Everyone who has worked in the Saunders laboratory since our digression into the realm of DNA barcoding is thanked for his/her contributions toward the overall objective of improved protocols, better primers, and preferred markers—it has truly been a group effort. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada through the Ontario Genomics Institute, NSERC, and other sponsors listed at www.BOLNET.ca. Additional support was provided by the Canada Research Chair Program, the Canada Foundation for Innovation, and the New Brunswick Innovation Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Saunders, G.W., McDevit, D.C. (2012). Methods for DNA Barcoding Photosynthetic Protists Emphasizing the Macroalgae and Diatoms. In: Kress, W., Erickson, D. (eds) DNA Barcodes. Methods in Molecular Biology, vol 858. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-591-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-591-6_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-590-9
Online ISBN: 978-1-61779-591-6
eBook Packages: Springer Protocols