On the utility of DNA barcoding for species differentiation among brown macroalgae (Phaeophyceae) including a novel extraction protocol

Authors


  • Communicating editor: T. Motomura.

*To whom correspondence should be addressed.
Email: dan.mcdevit@unb.ca

SUMMARY

The generation of a species-rich DNA barcode database in combination with rapid and affordable sequencing techniques will dramatically change specimen identification in ecological, biogeographical and taxonomic applications. Though cytochrome c oxidase 1 has been shown to be a useful tool for differentiating some groups of marine algae, its wide application in the Phaeophyceae has yet to be studied. The presence of polymerase chain reaction (PCR) inhibiting compounds in members of the Fucales, Laminariales and Tilopteridales, that are often co-extracted with DNA, has hampered the rapid processing associated with barcode projects. Polyphenolics and polysaccharides are present in concentrations such that DNA extraction methods typically include extensive series of washes, organelle extractions and/or cesium columns. In this paper we examine the utility of cytochrome c oxidase 1 for barcoding the Phaeophyceae and present a method for extracting PCR friendly DNA from brown macroalgae in about 2 h, dramatically reducing the time required from previous methods, some of which take days. This method is easily adapted to a 96 well, high-throughput format and may have applications in other organisms where the presence of similar PCR inhibiting compounds hinders molecular analyses. We extracted DNA from 106 isolates representing 29 species from 20 genera in nine families from five orders of Phaeophyceae. We were able to amplify the barcode marker (cytochrome c oxidase 1) from all samples and a nuclear marker (internal transcribed spacer region) from 54 selected samples. Cytochrome c oxidase 1 was able to differentiate clearly among species, showing within species divergence of 0.00–0.46%, with the exception of one previously studied genus, and between species divergences of greater than 3%.

INTRODUCTION

As our knowledge of the diversity of life broadens, including recognition of the increasing numbers of species going extinct (Wright & Muller-Landau 2006), there is an ever-pressing need to catalogue and describe this diversity. Though millions of species have been described, the total number of extant species, as well as the rate at which species are disappearing, remains an uncertainty (Godfray 2002; Blaxter 2004). For systematists, the first step in this process of cataloging life is recognizing the differences between species. Though typically an increase in diversity is correlated with an increase in morphological variability, in many cases species do not look distinct. In the algal world, morphological plasticity and convergent evolution combined with a relative paucity of characters can make assigning a collection to a family difficult at times and identification to species nearly impossible (Saunders 2005). For the researcher interested in taxonomy, this can be a welcome challenge, allowing us to agonize over the minute details of anatomy and morphology that differentiate species. However, for the majority of researchers, the task of identification is merely a limiting, at times impossible step in their protocol. The DNA barcode database combined with improving molecular techniques could provide a solution to this problem.

The DNA barcode initiative, as proposed by Hebert et al. (2003a,b), champions the use of a mitochondrial gene, cytochrome c oxidase 1 (COI), as a standardized marker for species identification. This marker has been successfully used across diverse animal taxa (Herbert et al. 2004a,b), as well as red (Saunders 2005; Robba et al. 2006) and brown (Kucera & Saunders 2008) macroalgae. However, in other groups, such as plants, COI is likely not a good candidate as a marker for species identification (Newmaster et al. 2006). Previous studies of the COI barcode in brown macroalgae have focused on groups of closely related species or difficult species complexes (Lane et al. 2007; Kucera & Saunders 2008). These studies have shown that in these difficult to differentiate groups the barcode has had either mixed results likely due to extensive hybridization (Lane et al. 2007) or was able to differentiate among only some of the accepted species, but with low levels of variation (Kucera & Saunders 2008). However, the DNA barcode has yet to be tested across a wide range of taxa in the Phaeophyceae. Therefore in this paper we test the variability of COI within the Dictyotales, Ectocarpales, Fucales, Laminariales and Tilopteridales. In addition we assess the utility of COI to differentiate between species across several members of the Fucales, Laminariales and Tilopteridales.

Ultimately, researchers would like to sample large numbers of collections from broad geographical locations, allowing them to ask previously daunting questions about ecology, local distribution and biogeography. The DNA barcode, combined with large-scale collection and processing methodology, will facilitate sufficient generation of data to address such questions. Unfortunately, a major roadblock to accomplishing this goal for some macroalgal taxa is the lack of rapid, consistent and automation-compatible DNA-extraction methods. Extraction of DNA from members of the Fucales, Laminariales and Tilopteridales of sufficient quality for molecular analyses has been a constant challenge in our laboratory. The methods (Mayes et al. 1992; Lane et al. 2006), as well as those used elsewhere (Coyer et al. 1995; Phillips et al. 2001; Hoarau et al. 2007), require large amounts of dried tissue, the use of toxic or volatile chemicals (such as cesium columns, phenol and β- mercaptoethanol) and in some cases, days to process. Commercial kits are available (e.g. ISO Plant, Nippon Gene; DNeasy Plant Mini Kit, QIAGEN) and have been successfully used on kelp (Yotsukura et al. 2001), but they are costly and still require additional cleaning steps to remove polysaccharides subsequent to the manufacturers' recommended protocols. Our procedure, developed as a modification of a previous protocol (Lane et al. 2006), uses an initial acetone wash to remove some of the polymerase chain reaction (PCR) inhibiting compounds. This step eliminates the need for an organelle extraction and decreases the total time required for extraction. This protocol was designed to enable the eventual automation of the extraction and sequencing procedure and can easily be adapted to a 96 well format, compatible with high throughput robotic systems similar to those already in use for animal tissue at the Biodiversity Institute of Ontario (Ivanova et al. 2006). In the present paper we assess the utility of the DNA barcode to discriminate among species of brown algae and establish the efficiency of our DNA extraction procedure across a range of robust members of the Phaeophyceae.

MATERIALS AND METHODS

To test the resolution of COI, 106 specimens representing 20 genera of brown algae from nine families (Alariaceae, Chordariaceae, Costariaceae, Dictyotaceae, Laminariaceae, Lessoniaceae, Phyllariaceae, Sargassaceae and Scytosiphonaceae; listed in Table 1) were dried in silica in the field and kept at −80°C until extracted. A portion of each sample was ground using a mortar and pestle in liquid nitrogen. Approximately 80 µg of ground material was suspended in 1 mL of 100% acetone at room temperature for 10 min. Samples were centrifuged briefly at 15 000 g and the acetone was removed and discarded. The acetone wash was repeated and samples were air-dried for 10 min. Next, 600 µL of DNA extraction buffer (0.1 M Tris base pH = 8, 0.3 M CaCl2, 0.05 M disodium ethylenediaminetetraacetic acid (EDTA) and 0.2 M NaCl (Saunders 1993; Saunders & Kraft 1995)), 60 µL of 10%Tween-20 and 6 µL of Proteinase K (20 mg mL−1) was added to each sample. Samples were incubated for 1 h at room temperature, placed on ice for 10 min and centrifuged at 15 000 g for 10 min at 4°C. The aqueous layer was transferred to new micro-centrifuge tubes and DNA was cleaned using the Wizard DNA Clean-up System (Promega, Madison, WI, USA) following the manufacturer's protocol with a final elution volume of 50 µL.

Table 1.  List of samples used in this study and accession numbers for barcode (cytochrome c oxidase 1 (COI)) and internal transcribed spacer (ITS) sequences (where determined), respectively
Order/family/speciesVoucherCollection informationLatitude/longitudeBOLDGenbank COI/ITS
  1. BC, B. Clarkston; BOLD, barcode of life data systems; DM, D. McDevit; DS, D. Saunders; GWS, G. Saunders; HK, H. Kucera; JD, J. DeWaard; JU, J. Utge; KR, K. Roy; LLG, L. Le Gall; RW, R. Withall; SC, S. Clayden; SH, S. Hamsher.

Dictyotales
Dictyotaceae
Dictyota binghamiae J. Agardh
 GWS003070Bamfield, BC Canada GWS, MS48.8622/−125.1768MACRO008-06FJ409140/−
GWS004536Bamfield, BC Canada GWS, BC, DM48.8622/−125.1768MACRO236-06FJ409139/−
Ectocarpales
Chordariaceae
Leathesia difformis (L) Areschoug
 GWS003387Bamfield, BC Canada GWS, BC, DM48.7862/−125.1191MACRO140-06FJ409170/−
GWS006079Fort Wetherill, RI, USA41.47910/−71.36066MACRO769-07FJ409169/−
Melanosiphon intestinalis (D.A. Saunders) M.J. Wynne
 GWS005950Blacks Harbour, NB Canada DM, GWS45.0442/−66.8105MACRO738-07FJ409176/−
GWS005953Seal Cove, NB Canada GWS44.65059/−66.82959MACRO739-07FJ409175/−
Soranthera ulvoidea Postels et Ruprecht
 GWS003389Bamfield, BC, Canada GWS, BC, DM48.7862/−125.1191MACRO142-06FJ409217/−
GWS005110Prince Rupert, BC, Canada GWS, BC, DM54.3007/−130.2507MACRO252-06FJ409216/−
Scytosiphonaceae
Petalonia fascia (O.F.Müller) Kuntze
 GWS003560Lepreau, NB, Canada GWS45.0722/−66.4690MACRO153-06FJ409184/−
GWS003561Lepreau, NB, Canada GWS45.0722/−66.4690MACRO154-06FJ409183/−
Fucales
Fucaceae
Fucus distichous Linnaeus
 HK023Kucera and Saunders (2008) MACRO863-07EU646658/EU525599
HK151Kucera and Saunders (2008) MACRO878-07EU646655/EU525600
HK240Kucera and Saunders (2008) MACRO884-07EU646651/EU525601
Fucus serratus Linnaeus
 HK620Kucera and Saunders (2008) MACRO927-07EU646715/EU525638
HK634Kucera and Saunders (2008) MACRO930-07EU646712/EU525639
HK640Kucera and Saunders (2008) MACRO932-07EU646735/−
LLG157Kucera and Saunders (2008) MACRO936-07EU646711/EU525640
Fucus spiralis Linnaeus
 CSM009AKucera and Saunders (2008) MACRO055-06EU646738/EU525641
HK003Kucera and Saunders (2008) MACRO858-07EU646725/EU525642
HK004Kucera and Saunders (2008) MACRO859-07EU646724/EU525643
Fucus vesiculosus Linnaeus
 HK033Kucera and Saunders (2008) MACRO866-07EU646756/EU525660
HK050Kucera and Saunders (2008) MACRO868-07EU646754/EU525661
HK268Kucera and Saunders (2008) MACRO888-07EU646739/EU525659
Sargassaceae
Cystoseira geminata C. Agardh
 GWS004223Vancouver Island, BC, Canada, GWS, BC, DM48.3521/−123.7281MACRO448-07FJ409138/−
GWS004226Vancouver Island, BC, Canada, GWS, BC, DM48.3521/−123.7281MACRO449-07FJ409137/−
GWS006391Vancouver Island, BC, Canada, DM, BC, KR, SH48.3521/−123.7281MACRO972-08FJ409136/−
Sargassum muticum (Yendo) Fensholt
 GWS002758Bamfield, BC, Canada, GWS, SC48.8524/−125.1224MACRO058-06FJ409215/FJ042707
GWS003402Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO560-07FJ409214/FJ042708
GWS006791Thasis, BC, Canada, DM, BC, KR, HK49.593283/−126.6165MACRO975-08FJ409213/FJ042709
Laminariales
Alariaceae
Alaria esculenta (Linnaeus) Greville
 DM05-004Beaver Harbor, NB, Canada, DM45.0717/−66.7372MACRO059-06FJ409128/FJ042728
GWS002318Cape Breton, NS, Canada, GWS45.8962/−59.9604MACRO097-06FJ409127/−
GWS005605Cape Neddick, ME, USA, GWS, BC, DM43.1658/−70.5924MACRO703-07FJ409126/FJ042760
GWS005639Fort William, ME, USA, GWS, BC, DM43.6256/−70.2138MACRO478-07FJ409125/FJ042763
GWS005961Grand Manan, NB, Canada, DM, BC44.8012/−66.788MACRO742-07FJ409124/FJ042766
GWS005962Grand Manan, NB, Canada, DM, BC44.8012/−66.788MACRO743-07FJ409123/FJ042767
GWS008090Les Escoumins, QC, Canada, GWS, DM, HK48.35062/−69.39722MACRO802-07FJ409122/−
GWS008091Les Escoumins, QC, Canada, GWS, DM, HK48.35062/−69.39722MACRO803-07FJ409121/FJ042774
Alaria marginata Postels et Ruprecht
 GWS002778Bamfield, BC, Canada, GWS, SC48.8524/−125.1224MACRO060-06FJ409133/−
GWS004710Palmerston, BC, Canada, BC, DM50.5933/−128.2583MACRO389-06FJ409132/−
GWS004948Ridley Island, BC, Canada, GWS, BC, DM54.2212/−130.3293MACRO606-07FJ409131/−
GWS005103Prince Rupart, BC, Canada, GWS, BC, DM54.3007/−130.2507MACRO610-07FJ409130/−
GWS006396Vancouver Island, BC, Canada, DM, BC, KR, SH48.3521/−123.7281MACRO973-08FJ409129/FJ042770
Lessoniopsis littoralis (Tilden) Reinke
 GWS003393Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO558-07FJ409172/−
GWS004716Palmerston, BC, Canada, BC, DM50.5933/−128.2583MACRO392-06FJ409171/−
Pleurophycus gardneri Setchell et Saunders ex Tilden
 GWS003992Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO583-07FJ409187/−
GWS004439Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4539MACRO341-06FJ409186/−
GWS004938Prince Rupart, BC, Canada, GWS, BC54.3390/−130.9365MACRO458-07FJ409185/−
Pterygophora californica Ruprecht
 GWS004197Otter Point, BC, Canada, GWS, BC, DM48.3625/−123.8048MACRO220-06FJ409193/−
GWS004274Victoria, BC, Canada, GWS, BC, DM48.4227/−123.4195MACRO226-06FJ409192FJ042739
GWS004298Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO229-06FJ409191/−
GWS004447Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO342-06FJ409190/FJ042744
GWS004468Bamfield, BC, Canada, GWS, BC, DM48.8187/−125.2084MACRO343-06FJ409189/−
GWS006350Vancouver Island, BC, Canada, DM, BC, KR, SH48.3625/−123.8048MACRO971-08FJ409188/−
Costariaceae
Costaria costata (C. Agardh) Saunders
 GWS003474Bamfield, BC, Canada, GWS, BC, DM48.8524/−125.1224MACRO566-07FJ409135/−
GWS006313Sidney, BC, Canada, DM, BC, KR, SH48.6481/−123.3936MACRO970-08FJ409134/FJ042769
Agarum clathratum Dumortier
 GWS002688Rockland, ME, USA, GWS44.0899/−69.0462MACRO550-07FJ409117/−
GWS005254Churchill, MB, Canada, GWS, BC, DM58.7703/−93.8474MACRO613-07FJ409116/FJ042754
GWS005636Cape Neddick, ME, USA, GWS, DM43.1658/−70.5924MACRO476-07FJ409115/FJ042761
GWS005637Cape Neddick, ME, USA, GWS, DM43.1658/−70.5924MACRO477-07FJ409114/FJ042762
GWS005924Letete, NB, Canada, DM45.0382/−66.8912MACRO730-07FJ409113/FJ042765
GWS006138Les Escoumins, QC, Canada, GWS, DM48.31830/−69.41359MACRO969-08FJ409112/FJ042768
Agarum fimbriatum Harvey
 GWS004270Victoria, BC, Canada, GWS, BC, DM48.4227/−123.4195MACRO589-07FJ409120/FJ042737
GWS004337Bamfield, BC, Canada, GWS, BC, DM48.8524/−125.1224MACRO592-07FJ409119/FJ042740
GWS004736Port Hardy, BC, Canada, BC, DM50.8468/−127.6442MACRO599-07FJ409118/−
Laminariaceae
Laminaria digitata (Hudson) J.V. Lamouroux
 GWS005325Churchill, MB, Canada, GWS, BC, DM58.78057/−94.27670MACRO272-06FJ409151/FJ042755
GWS005669Meadow Cove, NB, Canada, DM, LLG, DS, GWS45.0381/−66.8913MACRO490-07FJ409150/FJ042764
GWS007680St. Brides, NL, Canada, LLG, HK, DM, JU46.92068/−54.17384MACRO317-06FJ409149/FJ042772
GWS007822White Point, NS, Canada, LLG, DM46.88226/−60.35077MACRO325-06FJ409148/FJ042773
Laminaria ephemera Setchell
 GWS003985Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO211-06FJ409153/FJ042732
GWS003986Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO582-07FJ409152/FJ042733
Laminaria hyperborea (Gunnerus) Foslie
 GWS009234Fanad Head, Ireland, C.A. Maggs55.2764/−7.6344MACRO1007-08FJ409156/−
GWS009235Fanad Head, Ireland, C.A. Maggs55.2764/−7.6344MACRO1008-08FJ409155/−
GWS009236Fanad Head, Ireland, C.A. Maggs55.2764/−7.6344MACRO1009-08FJ409154/−
Laminaria setchellii Silva
 GWS003404Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO147-06FJ409160/-
GWS003991Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO212-06FJ409159/FJ042734
GWS004442Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO234-06FJ409158/−
GWS004937Prince Rupert, BC, Canada, GWS, BC54.3390/−130.9365MACRO603-07FJ409157/−
Laminaria solidungula J. Agardh
 GWS005335Churchill, MB, Canada, GWS, BC, DM58.78057/−94.27670MACRO616-07FJ409166/FJ042756
GWS005378Churchill, MB, Canada, GWS, BC, DM58.81154/−94.21970MACRO283-06FJ409165/FJ042757
GWS005385Churchill, MB, Canada, DM58.81154/−94.21970MACRO617-07FJ409164/FJ042758
GWS005386Churchill, MB, Canada, DM58.81154/−94.21970MACRO466-07FJ409163/FJ042759
GWS005422Churchill, MB, Canada, DM, JD58.81154/−94.21970MACRO976-08FJ409162/−
GWS007069Bonne Bay, NL, Canada, LLG, JU49.52826/−57.82495MACRO499-07FJ409161/−
Laminaria yezoensis Miyabe
 05-758 ARidley Island, BC, Canada, SB54.2454/−130.3386MACRO101-06FJ409168/FJ042727
GWS004941Ridley Island, BC, Canada, GWS, BC, DM54.2212/−130.3293MACRO244-06FJ409167/FJ042749
Macrocystis integrifolia Bory
 GWS002852Bamfield, BC, Canada, GWS48.8583/−125.1588MACRO066-06FJ409174/−
GWS003990Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO446-07FJ409173/−
Nereocystis luetkeana (Mertens) Postels et Ruprecht
 GWS004440Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO387-06FJ409182/FJ042742
GWS004470Bamfield, BC, Canada, GWS, BC, DM48.8187/−125.2084MACRO594-07FJ409181/−
GWS004546Bamfield, BC, Canada, GWS, BC, DM48.8622/−125.1768MACRO388-06FJ409180/FJ042745
GWS004711Palmerston, BC, Canada, BC, DM50.5933/−128.2583MACRO390-06FJ409179/FJ042747
GWS004947Ridley Island, BC, Canada, GWS, BC, DM54.2212/−130.3293MACRO460-07FJ409178/−
GWS006545Thasis, BC, Canada, BC, DM, KR, SH49.724722/−126.6425MACRO974-08FJ409177/FJ042771
Saccharina bongardiana (Postels et Ruprecht) Selivanova, Zhigadlova et G.I. Hansen
 GWS004436Botanical Beach, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO232-06FJ409198/FJ042741
GWS004942Ridley Island, BC, Canada, GWS, BC, DM54.2212/−130.3293MACRO245-06FJ409197/FJ042750
GWS004944Ridley Island, BC, Canada, GWS, BC, DM54.2212/−130.3293MACRO246-06FJ409196/FJ042751
GWS005105Butze Rapids, BC, Canada, GWS, BC, DM54.3007/−130.2507MACRO249-06FJ409195/FJ042752
GWS005108Butze Rapids, BC, Canada, GWS, BC, DM54.3007/−130.2507MACRO250-06FJ409194/FJ042753
Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl et G.W. Saunders
 GWS004199Otter Point, BC, Canada, GWS, BC, DM48.3625/−123.8048MACRO587-07FJ409204/FJ042735
GWS004269Saxe Point, BC, Canada, GWS, BC, DM48.4227/−123.4195MACRO223-06FJ409203/FJ042736
GWS004271Saxe Point, BC, Canada, GWS, BC, DM48.4227/−123.4195MACRO224-06FJ409202/FJ042738
GWS004286Maple Bay, BC, Canada, GWS, BC, DM48.8147/−123.6099MACRO227-06FJ409201/−
GWS004652Vivian Island, BC, Canada, BC, DM48.8395/−124.7010MACRO240-06FJ409200/FJ042746
GWS005400Churchill, MB, Canada, GWS, BC58.81154/−94.21970MACRO966-08FJ409199/−
Saccharina sessile (C. Agardh) C.E. Lane, C. Mayes, Druehl et G.W. Saunders
 GWS003208Bamfield, BC, Canada, GWS, RW48.7715/−125.1578MACRO080-06FJ409208/FJ042729
GWS003407Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO148-06FJ409207FJ042731
GWS004443Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO235-06FJ409206/FJ042743
GWS004714Palmerston, BC, Canada, BC, DM50.5933/−128.2583MACRO241-06FJ409205/FJ042748
Lessoniaceae
Egregia menziesii (Turner) Areschoug
 GWS003401Bamfield, BC, Canada, GWS, BC, DM48.7862/−125.1191MACRO559-07FJ409144/FJ042730
GWS003989Bamfield, BC, Canada, GWS, BC, DM48.8391/−125.2075MACRO445-07FJ409143/−
GWS004437Port Renfrew, BC, Canada, GWS, BC, DM48.5304/−124.4535MACRO593-07FJ409142/−
GWS004715Palmerston, BC, Canada, BC, DM50.5933/−128.2583MACRO391-06FJ409141/−
Eisenia arborea Areschoug
 GWS003187Bamfield, BC, Canada, RW48.8524/−125.1224MACRO083-06FJ409147/−
GWS003188Bamfield, BC, Canada, RW48.8524/−125.1224MACRO084-06FJ409146/−
GWS003932Bamfield, BC, Canada, GWS, BC, DM48.8524/−125.1224MACRO444-07FJ409145/−
Tilopteridales
Phyllariaceae
Saccorhiza dermatodea (Bac. Pyl.) Areschoug
 GWS007139Bonne Bay, NL, Canada, LLG, HK, JU49.60715/−57.94994MACRO640-07FJ409212/FJ042704
GWS007287Bonne Bay, NL, Canada, LLG, HK49.68198/−57.96275MACRO645-07FJ409211/FJ042705
GWS007802White Pt., NS, Canada, LLG, DM46.88226/−60.35077MACRO371-06FJ409210/FJ042706
GWS007821White Pt., NS, Canada, LLG, DM46.88226/−60.35077MACRO324-06FJ409209/−

Quality of the DNA was tested by PCR amplification of organellar (cytochrome c oxidase 1, COI 700 bp) and nuclear (ribosomal internal transcribed spacer region (ITS1-5.8SrDNA-ITS2), ITS 800 bp–1 kb) regions using previously published primer combinations GAZF2/GAZR2 (Lane et al. 2007) and P1/KG4 (Tai et al. 2001; Lane et al. 2006), respectively. For all PCR reactions 1 µL of DNA extract diluted 1:40 with sterile water was used in a 25 µL reaction in an Icycler (Bio-Rad Laboratories, Hercules, CA, USA), using the Takara Ex-Taq DNA polymerase kit (PanVera, Madision, WI, USA). For COI the thermal profile was as follows: an initial denaturation at 94°C for 4 min followed by 38 cycles of 94°C for 1 min, 50°C for 30 s and 72°C for 1 min, with a final extension at 72°C for 7 min and storage at 4°C. For ITS the profile was: an initial denaturation at 94°C for 4 min followed by 38 cycles of 94°C for 1 min, 45°C for 45 s and 72°C for 2 min, with a final extension at 72°C for 10 min and storage at 4°C. Primers and extra dNTPs were removed from amplified products using Exo-Sap-IT (USB, Cleveland OH, USA), a combination of Exonuclease I and Shrimp Alkaline Phosphatase, following the manufacturer's protocol. Products were then sequenced using the PE Applied Biosystems Big Dye (Foster City, CA, USA) kit following the manufacturer's protocol (ABI, Foster City, CA, USA) and analyzed using an Applied Biosystems 3130XL automated sequencer. Forward and reverse sequence reads using their respective primers were aligned and edited with Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA) and multiple sequence alignments were generated in MacClade 4.08 (Maddison & Maddison 2005). Sequence assessments were conducted in PAUP 4.0b10 (Swofford 2002). Distances were corrected using a general time reversible model and a neighbor-joining (NJ) clustering algorithm was used to provide a visual display of COI variation within and between species. Trees were constructed in PAUP and arbitrarily rooted to facilitate visualization. The ITS region from 54 selected samples was sequenced and distances were analyzed using the same method as above. Sequences generated by Kucera and Saunders (2008) from the genus Fucus were downloaded from Genbank (Table 1) and added to each alignment prior to analysis.

RESULTS

DNA extraction of 24 samples from ground material can be accomplished in approximately 2 h. PCR products of appropriate size (Fig. 1) were recovered for all 106 samples for COI and the subset of 54 samples for ITS. Subsequent sequencing of amplified fragments confirmed that PCR products were from the target organisms.

Figure 1.

Electrophoresis (0.8% agarose gel stained with SYBR Safe (Invitrogen, Burlington, ON Canada) with 1 kb DNA ladder) of polymerase chain reaction (PCR) amplification from representative samples for (a) cytochrome c oxidase 1 (COI) and (b) nuclear internal transcribed spacer (ITS) region. 1. Saccharina latissima (GWS005400), 2. Laminaria solidungula (GWS005422), 3. Costaria costata (GWS006313), 4.Agarum clathratum (GWS006138), 5. Pterygophora californica (GWS006350), 6. Cystoseira geminata (GWS006391), 7. Alaria marginata (GWS006396), 8. Nereocystis luetkeana (GWS006545), 9. Sargassum muticum (GWS006791), (C) negative control.

The amplified region for the COI is 700 bp (42 of which are complementary to the PCR primers). Across the 106 individuals from this study, within species variation was generally between 0.00 and 0.46% divergence, except in the case of Alaria marginata where within species variation was as high as 1.8% (see Lane et al. 2006). Between species variation within genera ranged from 3.04 to 10.80% divergence (Fig. 2). More specifically, within the genus Laminaria between species variation ranged from 3.04% (between L. digitata and L. hyperborea) to 5.17% (between L. yezoensis and L. hyperborea). Within the genus Saccharina variation ranged from 3.04% between S. sessile and S. bongardiana) to 6.23% (between S. sessile and S. latissima). The ITS results were wholly consistent with the COI genetic species groups, but with sequence variation between 0% and 1% divergence within species and 4.6% and 6.3% between congeneric species (Fig. 3), with the exception of Alaria esculenta and Alaria marginata, where between species variation was 0.8–1.0%. However, these values are exclusive of insertions and deletions and are thus lower than the actual divergence. These gaps make alignment of ITS sequences difficult and comparisons outside of the genus level difficult.

Figure 2.

Neighbor-joining (NJ) phylogram displaying clustering in COI sequences (this is not to be interpreted as a rigorous phylogeny) and a matrix of actual nucleotide distances within and between species of Alaria, Laminaria and Saccharina. Voucher numbers correspond to records in Table 1.

Figure 3.

Neighbor-joining (NJ) phylogram displaying clustering in internal transcribed spacer (ITS) sequences (this is not to be interpreted as a rigorous phylogeny). Voucher numbers correspond to records in Table 1.

DISCUSSION

Previous studies within the Phaeophyceae have shown that high levels of COI variation in Alaria are likely the result of incipient speciation followed by hybridization (Lane et al. 2007), and that COI can differentiate between species of Fucus except for the closely related F. spiralis and F. vesiculosus (Kucera & Saunders 2008). In these species the resolution of COI was similar to other markers commonly used for species level differentiation (such as ITS). Though both of these studies supported the utility of COI as a barcode, this is the first study to show that COI works broadly in the Phaeophyceae for species level differentiation. In a study of COI variation in 16 species of red macroalgae Saunders (2005) reported variation to be 0.0–0.3% within species and 4.5–13.6% between species. As we have shown, in brown macroalgae within species variation is generally greater than in reds (0.00–0.46%) and between species variation is generally lower than in reds (3.04–10.8%). However, in our samples the within versus between species divergence was clearly distinct, resulting in non-overlapping clusters in NJ analyses (Fig. 2), a necessary attribute if COI is to be a useful tool for the assignment of biological material to species. As evidenced by the clustering of sequences within multi-species genera such as Laminaria, Saccharina and Agarum, interspecific variation was generally more than 10-fold greater than intraspecific variation, allowing for unknown collections to be easily assigned to species groups. The COI clustering here provided equivalent resolution to the ITS (Fig. 3), a gene commonly used for species level differentiation in macroalgae (Tai et al. 2001; Lane et al. 2006, 2007). However, due to numerous insertions and deletions, the ITS region is difficult to align above the genus level, making specimen identification with alignment based methods difficult, a problem not found with COI.

In addition, we have presented a fast and easy method for isolating PCR-ready DNA from members of the Dictyotales, Ectocarpales, Fucales, Laminariales and Tilopteridales. A room temperature extraction followed by incubation on ice, combined with an acetone pre-wash removed sufficient quantities of the PCR-inhibiting compounds to allow for successful amplification of ITS and COI. This extraction protocol works just as well on other members of the Phaeophyceae (McDevit and Saunders unpubl. obs., 2007); however, we have had success in amplifying DNA from less robust thalli (e.g. members of the Ectocarpales sensu lato) by simply incubating unground material overnight in our extraction buffer followed by cleaning with the Wizard columns without the need for acetone washes. This further simplification of the extraction protocol has shortened the required contact time with the samples to approximately 30 min (after overnight lysis). In addition, the simplicity of these methods may provide insights into the modification of similar extraction protocols for other organisms that suffer from PCR inhibiting compounds.

High-throughput protocols are suitable for many applications such as DNA barcoding and whole genome research, allowing for rapid screening of large numbers of collections. The method of Lane et al. (2006) included an organelle purification prior to DNA extraction that required the suspension of ground material in Sorbitol and subsequent filtering and centrifugation. These steps, along with a phenol/chloroform clean-up step, would be difficult, if not impossible to adapt to a 96 well robotic system such as described by Ivanova et al. (2006). In contrast, the method described here was designed to be directly compatible with such robotic systems already in use at high throughput DNA barcoding facilities such as the Biodiversity Institute of Ontario (BIO) in Guelph, Ontario, Canada (Ivanova et al. 2006). The BIO extraction protocol starts with an overnight lysis (similar to our 1 h lysis) followed by DNA clean up on a 96 well vacuum filter plate (similar to our Wizard columns). A robotic workstation facilitates the liquid handling stages of this protocol enabling rapid extraction from a large number of samples (Ivanova et al. 2006). Our method could be directly used in this established high throughput barcoding process.

Cytochrome c oxidase 1 works broadly in the Phaeophyceae for species level differentiation. The COI DNA barcode, combined with rapid DNA extraction methodology can greatly increase our ability to identify brown algal collections. In our lab, the barcode has proven a useful tool for identifying unknown epi/endophytes or collections that are difficult to identify morphologically, due to factors such as lack of reproduction, condition of pressed material or stage of life history. One of the main advantages of using a molecular marker to facilitate assignment of specimens to species is that it is immediately clear when a collection has been mislabeled or misidentified.

ACKNOWLEDGMENTS

We would like to thank Natalia Ivanova for advice on method design as well as colleagues listed in Table 1 for their assistance with collections. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics Institute), and other sponsors listed at http://www.BOLNET.ca. Additional funding was provided by the Canada Research Chair Program, the Natural Science and Engineering Research Council of Canada, the Canada Foundation for Innovation and the New Brunswick Innovation Foundation grants to GWS.

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