Refinements for the Amplification and Sequencing of Red Algal DNA Barcode and Redtol Phylogenetic Markers: a Summary of Current Primers, Profiles and Strategies

Review

Algae 2013, 28(1): 31-43 http://dx.doi.org/10.4490/algae.2013.28.1.031 Open Access

Refinements for the amplification and sequencing of red algal DNA barcode and RedToL phylogenetic markers: a summary of current primers, profiles and strategies

Gary W. Saunders1,* and Tanya E. Moore1 1Centre for Environmental and Molecular Algal Research, Department of Biology, University of New Brunswick, Frederic- ton, NB E3B 5A3, Canada

This review provides a comprehensive summary of the PCR primers and profiles currently in use in our laboratory for red algal DNA barcoding and phylogenetic research. While work focuses on florideophyte taxa, many of the markers have been applied successfully to the Bangiales, as well as other lineages previously assigned to the Bangiophyceae sensu lato. All of the primers currently in use with their respective amplification profiles and strategies are provided, which can include full fragment, overlapping fragments and what might best be called “informed overlapping fragments”, i.e., a fragment for a marker is amplified and sequenced for a taxon and those sequence data are then used to identify the best primers to amplify the remaining fragment(s) for that marker. We extend this strategy for the more variable markers with sequence from the external PCR primers used to “inform” the selection of internal sequencing primers. This summary will hopefully serve as a useful resource to systematists in the red algal community.

Key Words: DNA barcode; Florideophyceae; molecular markers; phylogeny; polymerase chain reaction; primers; RedToL

INTRODUCTION

There can be little question that molecular techniques redtol/) (Vis et al. 2010). Both of these projects target a have radically altered the face of phycological research broader and more comprehensive taxonomic sampling especially in the fields of biodiversity and systematics than previous initiatives, challenging researchers to de- (Maggs et al. 2007). Whereas in the early years researchers velop more universal amplification strategies for markers focused strongly on two commonly used markers, viz., (e.g., Saunders and McDevit 2012). Not even widely used the plastid rbcL (Freshwater et al. 1994) and the nuclear markers such as the nuclear SSU and large subunit rDNA small subunit rDNA (Bird et al. 1992, Ragan et al. 1994, (Harper and Saunders 2001b) or the plastid rbcL (Fresh- Saunders and Kraft 1994), a number of alternative mark- water et al. 1994) have weathered these global initiatives ers have been developed for use with red algae from the without the need for refinement. population to the highest taxonomic levels (see Maggs et In this review, we focus strictly on the primers, profiles al. 2007, Table 6.1, for a list). Of particular importance in and strategies for the PCR amplification of markers cur- this regard have been the recent large-scale DNA barcod- rently in use in our lab (for a review of current collection ing (iBOL: http://ibol.org) (Saunders 2005) and Red Algal and DNA extraction protocols see Saunders and McDevit Tree of Life initiatives (RedToL: http://dblab.rutgers.edu/ 2012). Being partners in both the DNA barcode and Red-

This is an Open Access article distributed under the terms of the Received February 5, 2013, Accepted February 26, 2013 Creative Commons Attribution Non-Commercial License (http://cre- Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted * non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] provided the original work is properly cited. Tel: +1-506-452-6216, Fax: +1-506-453-3583

Fig. 1. Overview of PCR / sequencing strategy for the nuclear SSU. The primers G09, G15, and G16 are for chromophytic algae, but are included for reference. Primer P1 puts in context the standard forward primer that we use for amplification of the internal transcribed spacer (ITS) region.

Fig. 2. Overview of PCR / sequencing strategy for the nuclear internal transcribed spacer (ITS).

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ToL initiatives, we have been heavily involved in marker and G13 (G16) usually not necessary to generate full bidi- development and refinement to meet their respective ob- rectional reads (Fig. 1). jectives. As a result, numerous changes have been intro- Amplification profile: 94°C for 4 min; 38 cycles of 94°C duced to “tried and true” markers in an effort to improve for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min universality. 30 s; followed by 72°C final extension for 7 min. In the following section each marker has a brief summary with a corresponding figure. We have tried to adhere Internal transcribed spacer ribosomal DNA (ITS, to the same format for all figures–from top to bottom (ex- ~650-1,100 bp) amples refer to Fig. 1): marker name (e.g., small subunit ribosomal DNA), acronym (e.g., SSU), and approximate First investigated for red algae by Steane et al. (1991), size (e.g., ~1,800 bp); a schematic of the fragment(s) typi- the ITS marker is useful for species discrimination and cally amplified for each marker, their corresponding acro- detecting hybridization, in some cases population level nym / name and the primers used to amplify them when analyses, and for resolving relationships among closely more than one fragment is an option (e.g., E', a fragment related taxa. Our default methodological reference for ITS corresponding to the 5' end of the SSU is amplified with amplification is Tai et al. (2001) or, more recently, Saun- G01 and G14); a schematic showing the relative location ders and McDevit (2012) with our overall scheme first of all primers along the marker (gene regions presented summarized in Harper and Saunders (2001b, Fig. 2c, and as wider boxes than adjacent spacers); and finally a list- references therein). We amplify the ITS typically as a sin- ing of the actual PCR / sequencing primers. The typical gle fragment with the standard primers P1 and G4 (Fig. 2), primers used for PCR amplification are underlined for although it is easy to amplify the ITS1 (P1 and R1) or ITS2 each marker, while M13 linkers and sequencing primers (P5 and G4) individually. We have encountered problems are indicated in bold italics type. in using R1 with some taxa (Corallinales) (see Hind and Saunders 2013) and have developed a slightly degener- ate (more universal) version of this primer (R1s) (Fig. 2). NUCLEAR MARKERS Moving forward this modified primer will likely be used in routine applications. The primers P1 and G4 are relatively Small subunit ribosomal DNA (SSU, ~1,800 bp) universal and work on a wide variety of eukaryotes, which can be useful when trying to generate ITS data from di- The SSU was widely used in early studies of red algal vergent lineages, but detrimental for use in species prone phylogenetics (e.g., Bird et al. 1992, Ragan et al. 1994, to biological infestation. For example, recently we had Saunders and Kraft 1994) and is still useful for exploring trouble acquiring clean ITS data for Membranoptera spp. deeper relationships (e.g., West et al. 2008) with Harper (Wynne and Saunders 2012) necessitating development and Saunders (2001b, Fig. 2a) providing a summary of of the more specific MEMR4 to replace G4 and similarly PCR primers and profiles to that time. In the past it was HarvR4 for Neosiphonia (Savoie and Saunders unpub- typical to amplify this gene as four (L, M, N, O) fragments, lished). It is not unusual to have to develop primers to but with improvements in PCR reagents over the years meet particular needs, a practice that occurs routinely in we now routinely amplify this gene as two fragments (E′, our laboratory (e.g., Druehl et al. 2005). F) or as a single product (SSU full) (Fig. 1). The primers Amplification profile: 94°C for 2 min; 38 cycles of 94°C listed here remain unchanged from Harper and Saunders for 30 s, 50°C annealing for 45 s, 72°C extension for 2 min; (2001b) and the specialty primers still serve the same pur- followed by 72°C final extension for 5 min. poses, viz., H1 or H2 replace G01 and HR replaces G10 for members of the Hildenbrandiales, in which fungal con- Large subunit ribosomal DNA (LSU, ~2,700 bp) tamination (symbionts) is an issue, while G05 is a replace- ment for G10 in taxa for which the latter fails to give read- Freshwater and Bailey (1998) first used the LSU as a able sequence data (Harper and Saunders 2001b). We no phylogenetic tool for red algae while Harper and Saun- longer use all four internals to generate sequence data for ders (2001a) refined the marker by extending the portion the E’ fragment and simply use one of the forward (G02 or of the gene that was amplified (summarized in Harper G03) and one of the reverse (G10 or G11) primers (Fig. 1). and Saunders 2001b, Fig. 2b). There have been a number Similarly, for the F fragment we typically acquire internal of refinements since that time that have yet to be sum- sequence data only for G06 and G08, data for G12 (G15) marized in a review of this nature. Harper and Saunders

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Fig. 3. Overview of PCR / sequencing strategy for the nuclear LSU, as well as the D2 / D3 barcode region. Primer G4 puts in context the standard reverse primer that we use for amplification of the internal transcribed spacer (ITS) region.

Fig. 4. Overview of PCR / sequencing strategy for the nuclear EF2.

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(2001a) typically amplified the LSU as three fragments well as for sequencing with their internal primers, and it (X, Y, Z) (Fig. 3), but in recent years we commonly com- is not uncommon for species within a genus to require bine the Y and Z fragments into a single amplicon (Y & different primers. This has resulted in numerous, ongoing Z, primers T04 and T15) (Fig. 3) and at times even am- modifications to the original system. We currently ampli- plify the entire fragment as a single amplicon (LSU full, fy the entire fragment trialing combinations of external primers T01N and T15) (Fig. 3). Occasionally a fragment forward and reverse PCR primers to find the combination is from a contaminant rather than target and we circum- that works best for a sample (Fig. 4). Typically the original vent this problem by using other primer combinations L1 or the modified EF2L1Pey work in combination with (e.g., if Y brings up a contaminant rather than target we R2 or the modified EfR1 (trial progression for an isolate may try T25-T08 or T04-T19N or T04-T10N plus T33N- moves from L1-R2 then EF2L1Pey-EfR1 then Ef2Kif2- T08, etc.). More substantially, there have been a few key EfR1). Recently we have attempted to acquire EF2 from improvements to the primers in use since the publica- difficult taxa by amplifying the various forward primers tion of this system. The following fundamental changes against the internal reverse primer Mang48 to acquire an to the scheme of Harper and Saunders (2001a) have been EF2-5' fragment (Fig. 4). We then sequence with the exter- implemented over the years in a variety of manuscripts: nal PCR primers and use those data to identify the best T01N replacing T01; T10N replacing T10; T16N replacing internal forward primer from the Mang1-13 series to use T16; T19N replacing T19; T20 replacing T13 and T13N; against our external reverse PCR primers to amplify the T24U replacing T24; and T33N replacing T14 and T33 (Fig. EF2-3' fragment (Fig. 4). In all cases, we sequence with the 3). In all cases these primers, at times by slight changes PCR primers and use those data to select the best internal in position and / or addition of degeneracy, have proven sequencing primers from the forward series Mang1-13 more universal in amplification and sequencing of LSU and Mang51-58 and from the reverse series Mang21-28 data from a variety of red algae than their predecessors. and Mang31-46 (Fig. 4). In addition, Le Gall and Saunders (2007) uncovered that Amplification profile for full fragment: 94°C for 4 min; an LSU generated for the Bangiales (Harper and Saunders 38 cycles of 94°C for 1 min, 50°C annealing for 1 min, 72°C 2001a) was in fact from a contaminant. They devised a extension for 2 min; followed by 72°C final extension for strategy for these taxa amplifying the LSU as two overlap- 7 min. ping fragments with TO1N-Banrev and Banfor-T15 (Ban1 Amplification profile for two subfragments: 94°C for 4 and Ban2, respectively) (Fig. 3). Not included here are a min; 38 cycles of 94°C for 1 min, 58°C annealing for 1 min, series of specific primers to enable sequencing through 72°C extension for 2 min; followed by 72°C final extension a relatively large insert in the Z fragment of some Nema- for 7 min. liales (Huisman et al. 2004) and to amplify specifically the LSU of representative Palmariaceae and Rhodophy- semataceae from mixed host plus epi-endophyte DNA PLASTID MARKERS extractions (Clayden and Saunders 2010). We also include the D2/D3 region being promoted as a secondary DNA Photosystem I P700 chlorophyll a apoprotein A1 barcode marker and indicate the location and primers for (psaA, ~1,600 bp) this fragment (Saunders and McDevit 2012). Amplification profile: 94°C for 5 min; 38 cycles of 94°C Yoon et al. (2002) devised an amplification strategy for for 30 s, 50°C annealing for 30 s, 72°C extension for 2 min; psaA and we use their external PCR primers to amplify followed by 72°C final extension for 7 min. this marker as a single fragment (Fig. 5). These primers have not worked for all taxonomic groups that we have Elongation factor 2 (EF2, ~1,700 bp) trialed and further primer development is necessary. Ow- ing to the highly variable nature of this marker, we have Le Gall and Saunders (2007) championed EF2 as a phy- again developed a series of internal forward, GpsaA1-8F logenetic marker for red algae in a survey of Florideophy- and GpsaA9-12F, and reverse, GpsaA1-16R, primers that ceae. In that study this region was amplified as a single we select after consideration of sequence data from the product ~1,700 bp in length or as two shorter overlapping external PCR primers (Fig. 5). fragments (Le Gall and Saunders 2007, Fig. 1). In pursu- Amplification profile: 94°C for 2 min; 5 cycles of 94°C ing this marker for additional taxa we have had mixed for 30 s, 45°C annealing for 30 s, 72°C extension for 1 min; success for both amplification with their external PCR, as then 35 cycles of 94°C for 30 s, 46.5°C annealing for 30 s,

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Fig. 5. Overview of PCR / sequencing strategy for the plastid psaA.

Fig. 6. Overview of PCR / sequencing strategy for the plastid psaB.

72°C extension for 1 min; followed by 72°C final extension quence with the PCR primers and then identify the best for 7 min. internal forward and reverse primers from our newly developed series abF1-8 and abR1-6, respectively (Fig. 6). Photosystem I P700 chlorophyll a apoprotein A2 Amplification profile: 94°C for 2 min; 5 cycles of 94°C (psaB, ~1,250 bp) for 30 s, 45°C annealing for 30 s, 72°C extension for 1 min; then 35 cycles of 94°C for 30 s, 46.5°C annealing for 30 s, For this marker the external PCR primers are from Yoon 72°C extension for 1 min; followed by 72°C final extension et al. (2004) and again we amplify it as a single fragment for 7 min. (Fig. 6). As with the previous markers, we generate se-

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Fig. 7. Overview of PCR / sequencing strategy for the plastid psbA.

Fig. 8. Overview of PCR / sequencing strategy for the plastid UPA barcode marker.

Photosystem II thylakoid membrane protein D1 Plastid LSU (23S) domain V (UPA, ~370 bp) (psbA, ~950 bp) Sherwood and Presting (2007) championed UPA as a This is an easy marker to amplify with the two external universal barcode marker for the algae noting that a single PCR primers developed by Yoon et al. (2002) and works primer pair can reliably recover sequences from a broad broadly among florideophyte algae (Fig. 7). While their taxonomic range including green, red, and brown marine (loc. cit.) internal forward sequencing primer psbA500F macroalgae, diatoms, and even cyanobacteria. Whereas also works widely, we have had poor sequence for some we have had strong success with their forward primer taxa using the internal reverse primer psbA600R. We thus p23SrV_f1, the reverse primer p23SrV_r1 can result in sequence with external primers first and then pick the messy sequence data for some florideophytes. Clarkston best internal reverse option among psbA600R and the and Saunders (2010) reasoned that a repeat near the 3' newly designed psbArev1 and psbArev2. end of this primer (AGAG) was the cause of this problem Amplification profile: 94°C for 2 min; 5 cycles of 94°C and dropped the final nucleotide in designing the modi- for 30 s, 45°C annealing for 30 s, 72°C extension for 1 min; fied primer p23SnewR (Fig. 8). We have found this an eas- then 35 cycles of 94°C for 30 s, 46.5°C annealing for 30 s, ily amplified marker from a wide variety of florideophytes 72°C extension for 1 min; followed by 72°C final extension with this modification. Being a short marker, no internal for 7 min. primers are necessary to generate full bidirectional reads (Fig. 8).

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Fig. 9. Overview of PCR / sequencing strategy for the plastid rbcL. For the DNA barcode region rbcL-3P the dashed line indicates that we amplify the entire fragment, but use sequence from the external 3′ PCR primer (rbcLrevNEW) to identify the best internal forward primer (TLF1-5, ACROF1) to generate a full bidirectional read for only the 3′ end of this gene (solid line).

Amplification profile: 94°C for 2 min; 35 cycles of 94°C We are using rbcL-3P (~785 bp) as a secondary DNA for 20 s, 55°C annealing for 30 s, 72°C extension for 30 s; barcode marker (Fig. 9), however, owing to the lack of an followed by 72°C final extension for 10 min. internal “universal” primer, we amplify the entire fragment, sequence with the external reverse PCR primer and Ribulose-1,5-bisphosphate carboxylase large then use those data to identify the best internal forward subunit (rbcL, ~1,350 bp) primer (ACROF1, TLF1-5) to give a bidirectional read for only the 3' end of the marker. This marker is widely used in florideophyte phyloge- Amplification profile: 95°C for 2 min; 35 cycles of 93°C netic and barcoding studies and was first applied broadly for 1 min, 47°C annealing for 1 min, 72°C extension for 2 to these algae by Freshwater et al. (1994). We still rou- min; followed by 72°C final extension for 2 min. tinely use the forward PCR primer F57 (Freshwater and Rueness 1994), but also trial the M13-linked Ruf and RUfn for problematic taxa (Fig. 9). These last two primers are MITOCHONDRIAL MARKERS linked with one of the available M13 variants (note that the M13 variant here is different from that included be- Cytochrome c oxidase subunit 1 DNA barcode low for COI-5P, but either should work; just make sure the region (COI-5P, ~ 664 bp) M13 variants used for sequencing are a match for those employed in your primer). Thus sequencing employs Saunders and McDevit (2012) have recently reviewed only the linker portion of the primer when these are used the DNA barcode marker COI-5P in detail, but cautioned in PCR (Fig. 9). We have also developed an M13 linked that primer advances were constantly being developed. reverse primer (RUr), but typically find that our modifi- So it is that an update is already due, largely improving on cation of Vis and Sheath’s (1999) rbcL R' (rbcLrevNEW) their earlier primers by adding variants of M13 linkers to works for most red algae (Fig. 9). A key innovation here the standard primers (Fig. 10). We now have two forward is to generate sequence data from the PCR primers and and two reverse primers that in varied combinations gen- then identify the best internal primers from the forward erate PCR product for most red algae. Owing to the short and reverse series TLF1-5 (plus ACROF1) and TLR1-7, re- length of this fragment, bidirectional reads are accom- spectively (Fig. 9). This approach results in clean and full plished without the need for internal primers (Fig. 10). bidirectional reads for this marker allowing for the gen- Amplification profile: 94°C for 2 min; 5 cycles of 94°C eration of quality sequences. for 30 s, 45°C annealing for 30 s, 72°C extension for 1 min;

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Fig. 10. Overview of PCR / sequencing strategy for the mitochondrial COI-5P barcode marker. The order in which primer combinations are trialed for taxa is indicated from the first (best) to fourth option.

Fig. 11. Overview of PCR / sequencing strategy for the mitochondrial COI extended fragment.

then 35 cycles of 94°C for 30 s, 46.5°C annealing for 30 s, signal for use among closely related taxa and we wanted 72°C extension for 1 min; followed by 72°C final extension to expand on this usefulness by generating a larger COI for 7 min. fragment (Fig. 11). Either the external forward M13-linked primers outlined previously (Fig. 10) or the unlinked vari- Cytochrome c oxidase subunit 1 extended frag- ants reported with this marker are used in combination ment (COI, ~1,232 bp) with the external reverse primers from the CoTR1-6 series for amplification (Fig. 11). We then sequence with the Saunders (2008) argued that COI-5P had phylogenetic PCR primers and determine the best internal primers for

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Fig. 12. Overview of PCR / sequencing strategy for the mitochondrial COB.

Fig. 13. Overview of PCR / sequencing strategy for the mitochondrial cox2-3 intergenic spacer.

sequencing from the forward series CoTF1-5 and among and reverse (COBR1-5) sequencing primers (Fig. 12). the reverse primers GazR1, GWSRi and GWSRx. Amplification profile: 95°C for 1 min; 35 cycles of 93°C Amplification profile: 94°C for 4 min; 35 cycles of 94°C for 30 s, 42°C annealing for 30 s, 72°C extension for 1 min; for 1 min, 45°C annealing for 30 s, 72°C extension for 1 followed by 72°C final extension for 7 min. min; followed by 72°C final extension for 7 min. Cytochrome oxidase subunit 2-3 intergenic Cytochrome b (COB, ~940 bp) spacer (cox 2-3, ~350-400 bp)

This relative newcomer to red algal studies is amplified This marker was first used in red algae by Zuccarello et with the novel external primers CB44F and CB1006R de- al. (1999) and is a useful tool at the population level. We signed by Ga Hun Boo (laboratory of Prof. Sung Min Boo, a have only started to use this system and have not had to RedToL partner at Chungnam National University, South develop novel primers to date–so far the original primers Korea). Sequence is then generated with these same of Zuccarello et al. have worked on a broad spectrum of primers to identify the best internal forward (COBF1-4) taxa (Fig. 13).

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Amplification profile: 94°C for 2 min; 5 cycles of 93°C (Fig. 5), psaB (Fig. 6), psbA (Fig. 7), rbcL (Fig. 9), COI (Fig. for 1 min, 45°C annealing for 1 min, 72°C extension for 1 11) and COB (Fig. 12)]. In all of these cases we amplify min; then 30 cycles of 93°C for 30 s, 55°C annealing for 30 and sequence with the PCR primers and then use those s, 72°C extension for 30 s; followed by 72°C final extension resulting data to “inform” our choice of the best internal for 7 min. sequencing primers to use from the options. This approach has greatly improved our overall success at get- ting quality, full bidirectional reads for these markers, all DISCUSSION of which tend to lack internal conservative regions for “universal” primer design. In short, users should not be With the current rise in popularity and utility of power- constrained by the fragments and primers as presented ful next generation technologies it is understandable that here and should explore alternative fragments and novel some of our colleagues may question the usefulness of primers where necessary to circumvent problems of no summarizing single marker amplification protocols. In- amplification or contamination (e.g., Lane and Saunders deed there are currently three red algal genomes available 2005, Wynne and Saunders 2012). in GenBank (Cyanidioschyzon, Galdieria, and Chondrus) However, one word of caution is necessary whenever with two more nearing completion (Porphyridium and markers are amplified as a series of fragments–always Porphyra), while 16 additional species are in progress as be wary of inadvertently generating chimeric sequences part of the RedToL project (H. -S. Yoon personal commu- (e.g., for the LSU the X and Z fragments may be from your nication). However, in our opinion there is still consider- target species, but the Y fragment has amplified from a able value in being able to assign an unknown biological contaminant on or in the sample). We have encountered specimen to a genetic species with a simple PCR amplifi- chimeric sequences in our own data and those of col- cation and a single sequence read (both strands are not leagues between various red and brown algae and have needed for routine identifications). We also see the value, even uncovered mixed alga / invert sequences. This is at least for the near future, of targeted multi-gene phylo- going to happen, the critical point is to catch it when it genetics, which will facilitate the inclusions of hundreds does. The problem is obvious in the more variable mark- (e.g., RedToL is targeting ~650 species) rather than dozens ers where the fragments have a significant overlap (e.g., of taxa in phylogenetic analyses. We thus hope that this EF2 and rbcL) (Figs 4 & 9, respectively), but can be more summary will be of use to our colleagues. difficult to detect in markers for which fragment overlap A guiding principle for our approach to marker devel- is short and usually corresponds to conservative regions opment and use is that primers are affordable and rela- of the markers (e.g., SSU and LSU, respectively) (Figs 1 & tively easy to design and / or select once some sequence 3). We routinely generate trees for the various fragments data are in hand. We thus routinely circumvent contami- to look for phylogenetic consistency among fragments nation and amplification problems by acquiring what- for an isolate prior to combining them to generate a com- ever portion of a marker we can for a specimen. Those plete sequence. In addition, as GenBank becomes more data then “inform” our next steps with regards to amplifi- populated with red algal sequences, simply blasting the cation and or sequencing using primers currently in our fragments when you suspect a problem can highlight a lab or newly designed to meet a special need. An example chimeric sequence. On that last note, even markers am- is found in Clayden and Saunders (2010) in which LSU plified as a single fragment can be from contaminating sequences for palmarialean taxa were compared to the organisms rather than the target species–a blast through host halymeniacean data to facilitate the development the various databases such as GenBank (BOLD for COI- of the novel primers rsr1 and rsf1 for use with T01N and 5P; http://www.boldsystems.org) is typically a prudent T15, respectively (Fig. 3). These primers were then used to endeavor. amplify this marker as two overlapping fragments for the epi-endophyte Rhodonematella subimmersa (Setchell & N. L. Gardner) S. L. Clayden & G. W. Saunders from a bulk ACKNOWLEDGEMENTS DNA extraction with its host. We have extended this philosophy to markers for Members of the Saunders’ lab past and present are which relatively conservative external PCR primers have thanked for their numerous contributions to the ongoing been developed, but for which “universal” internal se- development of markers–hundreds of hours of trial and quencing primers remain elusive [i.e., EF2 (Fig. 4), psaA error have contributed greatly to the tools described in

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this review that make all of our molecular research pos- analysis. Phycologia 33:187-194. sible. We also thank Ga Hun Boo for providing the COB Harper, J. T. & Saunders, G. W. 2001a. Molecular systemat- external amplification primers to the RedToL partners, ics of the Florideophyceae (Rhodophyta) using nuclear Hwan Su Yoon for updates on RedToL genome progress large and small subunit rDNA sequence data. J. Phycol. and M. Bruce and K. Dixon for reviewing a draft of this 37:1073-1082. manuscript. Comments from three anonymous review- Harper, J. T. & Saunders, G. W. 2001b. The application of se- ers improved the final document and were sincerely ap- quences of the ribosomal cistron to the systematics and preciated. The research was supported through funding classification of the florideophyte red algae (Florideo- to GWS from the Canadian Barcode of Life Network from phyceae, Rhodophyta). Cah. Biol. Mar. 42:25-38. Genome Canada through the Ontario Genomics Insti- Hind, K. R. & Saunders, G. W. 2013. A molecular phylogenetic tute, Natural Sciences and Engineering Research Council study of the tribe Corallineae (Corallinales, Rhodophyta) of Canada and other sponsors listed at http://www.BOL- with an assessment of genus-level taxonomic features NET.ca. Additional support to GWS was provided by the and descriptions of novel genera. J. Phycol. 49:103-114. Canada Research Chair Program, the Canada Foundation Huisman, J. M., Harper, J. T. & Saunders, G. W. 2004. Phylo- for Innovation and the New Brunswick Innovation Foun- genetic study of the Nemaliales (Rhodophyta) based on dation, as well as NSF through the RedToL project (DEB large-subunit ribosomal DNA sequences supports seg- 0937975). regation of the Scinaiaceae fam. nov. and resurrection of Dichotomaria Lamarck. Phycol. Res. 52:224-234. Lane, C. E. & Saunders, G. W. 2005. Molecular investigation REFERENCES reveals epi/endophytic extrageneric kelp (Laminariales, Phaeophyceae) gametophytes colonizing Lessoniopsis Bird, C. J., Rice, E. L., Murphy, C. A. & Ragan, M. A. 1992. Phy- littoralis thalli. Bot. Mar. 48:426-436. logenetic relationships in the Gracilariales (Rhodophy- Le Gall, L. & Saunders, G. W. 2007. A nuclear phylogeny of the ta) as determined by 18S rDNA sequences. Phycologia Florideophyceae (Rhodophyta) inferred from combined 31:510-522. EF2, small subunit and large subunit ribosomal DNA: Clarkston, B. E. & Saunders, G. W. 2010. A comparison of two establishing the new red algal subclass Corallinophyci- DNA barcode markers for species discrimination in the dae. Mol. Phylogenet. Evol. 43:1118-1130. red algal family Kallymeniaceae (Gigartinales, Florideo- Maggs, C. A., Verbruggen, H. & De Clerck, O. 2007. Molecular phyceae), with a description of Euthora timburtonii sp. systematics of red algae: building future structures on nov. Botany 88:119-131. firm foundations. In Brodie, J. & Lewis, J. (Eds.) Unravel- Clayden, S. L. & Saunders, G. W. 2010. Recognition of Rubro- ling the Algae: The Past, Present, and Future of Algal Sys- intrusa membranacea gen. et comb. nov., Rhodonema- tematics. CRC Press, Boca Raton, FL, pp. 103-121. tella subimmersa gen. et comb. nov. (with a reinterpre- Ragan, M. A., Bird, C. J., Rice, E. L., Gutell, R. R., Murphy, C. tation of the life history) and the Meiodiscaceae fam. A. & Singh, R. K. 1994. A molecular phylogeny of the nov. within the Palmariales (Rhodophyta). Phycologia marine red algae (Rhodophyta) based on the nuclear 49:283-300. small-subunit rRNA gene. Proc. Natl. Acad. Sci. U. S. A. Druehl, L. D., Collins, J. D., Lane, C. E. & Saunders, G. W. 2005. 91:7276-7280. An evaluation of methods used to assess intergeneric Saunders, G. W. 2005. Applying DNA barcoding to red mac- hybridization in kelp using Pacific Laminariales (Phaeo- roalgae: a preliminary appraisal holds promise for future phyceae). J. Phycol. 41:250-262. applications. Philos Trans. R. Soc. B Biol. Sci. 360:1879- Freshwater, D. W. & Bailey, J. C. 1998. A multigene phylogeny 1888. of the Gelidiales including nuclear large-subunit rRNA Saunders, G. W. 2008. A DNA barcode examination of the red sequence data. J. Appl. Phycol. 10:229-236. algal family Dumontiaceae in Canadian waters reveals Freshwater, D. W., Fredericq, S., Butler, B. S., Hommersand, substantial cryptic species diversity. 1. The foliose Dil- M. H. & Chase, M. W. 1994. A gene phylogeny of the red sea-Neodilsea complex and Weeksia. Botany 86:773-789. algae (Rhodophyta) based on plastid rbcL. Proc. Natl. Saunders, G. W. & Kraft, G. T. 1994. Small-subunit rRNA gene Acad. Sci. U. S. A. 91:7281-7285. sequences from representatives of selected families of Freshwater, D. W. & Rueness, J. 1994. Phylogenetic relation- the Gigartinales and Rhodymeniales (Rhodophyta). ships of some European Gelidium (Gelidiales, Rho- 1. Evidence for the Plocamiales ord. nov. Can. J. Bot. dophyta) species, based on rbcL nucleotide sequence 72:1250-1263.

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