Molecular Phylogenetics and Evolution 76 (2014) 181–188

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Molecular markers from different genomic compartments reveal crypticdiversity within glaucophyte species

http://dx.doi.org/10.1016/j.ympev.2014.03.0191055-7903/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Department of Biology, University of New Brunswick,10 Bailey Drive, Fredericton, New Brunswick E3B 5A3, Canada. Fax: +1 506 4533583.

E-mail address: areyes@unb.ca (A. Reyes-Prieto).

Jasmine Chong a,b, Christopher Jackson a,b, Jong Im Kim c,d, Hwan Su Yoon c, Adrian Reyes-Prieto a,b,⇑a Department of Biology, University of New Brunswick, 10 Bailey Drive, Fredericton, New Brunswick E3B 5A3, Canadab Integrated Microbiology Program, Canadian Institute for Advanced Research, Canadac Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Koread Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 October 2013Revised 14 March 2014Accepted 18 March 2014Available online 28 March 2014

Keywords:GlaucophytaCyanophoraGlaucocystisCryptic species diversityArchaeplastida

Glaucophytes are the least studied of the three major Archaeplastida (Plantae sensu lato) lineages. It hasbeen largely recognized that comprehensive investigations of glaucophyte genetic and species diversitywill shed light on the early evolution of photosynthetic eukaryotes. Here we used molecular phylogenet-ics and genetic distance estimations of diverse molecular markers to explore strain and species diversitywithin the glaucophyte genera Cyanophora and Glaucocystis. Single gene and concatenated maximumlikelihood analyses of markers from three different genetic compartments consistently recovered similarintrageneric genetic groups. Distance analyses of plastid (psbA and rbcL) and mitochondrial (cob and cox1)genes, and the nuclear internal transcribed spacer (ITS) region, revealed substantial genetic divergencebetween some Cyanophora paradoxa and Glaucocystis nostochinearum strains. The genetic distances esti-mated between some glaucophyte strains currently considered the same species are similar or greaterthan divergence values calculated between different species in other unicellular algae, such as certaingreen algae and diatoms. The analyzed molecular markers are prospective candidates for future studiesof species diversity in glaucophytes. Overall, our results unveil previously unrecognized cryptic diversitywithin Cyanophora and Glaucocystis species.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

The glaucophytes (Glaucophyta Skuja, 1954, or Glauco-cystophyta; Kies and Kremer, 1986), green plants (land plantsand green algae; Viridiplantae Cavalier-Smith, 1981) and red algae(Rhodophyta Wettstein, 1922) are the three groups of photosyn-thetic eukaryotes bearing primary plastids formally united in the‘‘supergroup’’ Archaeplastida (Adl et al., 2005, 2012). The Archaep-lastida are monophyletic in diverse molecular analyses, supportingthe hypothesis for the unique origin of primary plastids as a resultof endosymbiosis between a heterotrophic eukaryote and a photo-synthetic cyanobacterium (Burki et al., 2008; Hackett et al., 2007;McFadden and van Dooren, 2004; Palmer, 2003; Price et al., 2012;Rodríguez-Ezpeleta et al., 2005). However, conflicting phylogeneticresults have been interpreted as evidence of separate origins(Howe et al., 2008; Stiller, 2003). Understanding the origin and

diversity of the Archaeplastida lineages is fundamental to compre-hending the evolution of eukaryotes as primary producers(Bhattacharya et al., 2004; Keeling, 2010; Reyes-Prieto et al., 2007).

Genomic information from Viridiplantae is relatively abundantand the amount of red algal data has increased substantially in re-cent times (Bhattacharya et al., 2013; Collén et al., 2013; Matsuzakiet al., 2004; Schönknecht et al., 2013). However, glaucophyte geno-mic data is currently very limited with only one published genome(Price et al., 2012). Additionally, species diversity within glauco-phytes is poorly understood, as are the phylogenetic relationshipsamong the different genera. This limited sampling and understudyof glaucophytes restricts our ability to discern between differenthypotheses regarding the evolution of primary photosyntheticeukaryotes, such as the presumed unique origin of Archaeplastidaand the internal relationships between the three major lineages(Burki et al., 2012; Chan et al., 2011; Reyes-Prieto andBhattacharya, 2007). Further, limited taxon sampling may biasphylogenetic inferences towards erroneous monophyleticassociations (Heath et al., 2008; Hedtke et al., 2006).

Glaucophytes are freshwater unicellular or colonial organismsbearing blue-green plastids historically referred as ‘‘cyanelles’’ or

182 J. Chong et al. / Molecular Phylogenetics and Evolution 76 (2014) 181–188

‘‘muroplasts’’ (Hall and Claus, 1963; Löffelhardt and Bohnert, 2004).These plastids are characterized by both a remnant peptidoglycancell wall between the two plastid membranes, and by carboxy-some-like structures localized in the organelle stroma (Löffelhardtand Bohnert, 2004). Both plastid structures are presumably ances-tral traits derived from their cyanobacterial relatives and conservedin glaucophytes during the evolution of the group. Several molecu-lar phylogenetic studies using single gene (Bhattacharya andSchmidt, 1997; Bhattacharya et al., 1995; Helmchen et al., 1995;Marin et al., 1998; Maruyama et al., 2008; Yang et al., 2011) andmultiple-gene (Burki et al., 2008; Hackett et al., 2007; Reeb et al.,2009; Rodríguez-Ezpeleta et al., 2005) data sets have shown thatglaucophytes form a monophyletic group. However, despite theimportance of glaucophytes in understanding early evolution ofphotosynthetic eukaryotes, morphological and molecular studiesinvestigating diversity within this algal group are scarce. Gen-ome-scale comparative analyses have included sequence data fromonly two taxa: Cyanophora paradoxa (strain CCMP 329) and Glauco-cystis nostochinearum (strain UTEX 64) (Burki et al., 2012; Priceet al., 2012; Rodríguez-Ezpeleta et al., 2005) and although dozensof glaucophyte strains are available in algal culture collections, onlyfew have been studied. The vast majority of available glaucophyteisolates have no sequence data in public databases, and so the ex-tent of genetic variation at genus and species levels is unknown.

To distinguish between alternative scenarios regarding thediversification history of Archaeplastida, we require comprehen-sive studies of diversity within the glaucophytes, and generationof genomic data from a broad glaucophyte taxon sampling. In thisstudy we address a basic question: what is the level of geneticdiversity within glaucophyte genera and species? We use bothmolecular phylogenetics and DNA barcode-like methods to inves-tigate levels of genetic divergence between 17 glaucophyte strainsfrom different culture collections, and four novel Glaucocystisisolates from North America and Korea.

2. Material and methods

2.1. Glaucophyte strains and cell cultures

A total of 17 glaucophyte strains were obtained from the AlgalCulture Collection of the University of Göttingen (SAG, Germany),the Microbial Culture Collection at the National Institute for Envi-ronmental Studies (NIES, Japan), and The Culture Collection ofAlgae of the University of Texas (UTEX, USA) (supplementalTable S1). The analyzed strains included three Cyanophora species(5 strains total), two Cyanoptyche gloeocystis strains, one Gloeocha-ete wittrockiana isolate and 9 strains originally identified as Glauco-cystis nostochinearum. Additionally, four novel Glaucocystis sp.strains were collected using a 20 lm mesh plankton net (Bokyeongco., Pusan, Korea) from a pond at Boothbay Railway Village, Booth-bay Maine, USA (strain BBH), Sportsmans Lake, Friday Harbor,Washington, USA (strain HS30), and Samhogi, Taean, Chungnam,Korea (strains KRJ1 and KRJ2). Individual cells were isolated underan inverted microscope (Leica DMI 3000B, Wetzlar, Germany)using mouth-picking techniques. Cell cultures from single isolateswere established and maintained in liquid DY-V culture media(Andersen et al., 1997) at 18 �C on a 14/10 h light/dark cycle.

2.2. DNA extraction, gene amplification and sequencing

Cells were harvested at stationary phase by centrifugation for15 min at 1800–2000g. We extracted total DNA from all strains fol-lowing the protocol for fresh plant tissue using the DNeasy PlantMini Kit (Qiagen, Hilden, Germany). Sequences of differentmolecular markers were amplified by PCR. A region of the plastid

gene psbA (D1 protein of the photosystem II) was amplified withdifferent combinations of the forward primers psbAF1, psbAFn2or psbAF91 with the reverse primer psbAR2 (Yoon et al., 2002)using a denaturation step at 94 �C for 4 min, then 38 cycles of94 �C for 1 min, 46–50 �C for 30 s and 72 �C for 2 min, concludingwith a 10 min extension at 72 �C. Partial sequence of the mitochon-drial gene cob (apocytochrome b) was amplified using alternativecombinations of the forward cobF81 and cobF199 primers andthe cobR1054 and cob1084 reverse counterparts. The protocol forcob amplification comprised an initial denaturation step at 98 �Cfor 40 s, then 31 cycles of 98 �C for 10 s, 48 �C for 30 s and 62 �Cfor 30 s, with a final extension of 10 min at 72 �C. The nuclear inter-nal transcribed spacer (ITS) region was amplified using the primercombination P1 and G4 (see Saunders and Moore, 2013) for regularPCR or nested reactions with the primer pair GlaITSF1 andGlaITSR1. Conditions for ITS amplification comprised an initialdenaturation step at 94 �C for 4 min, followed by 38 cycles of94 �C for 1 min, 45 �C for 45 s and 72 �C for 2 min with a finalextension of 10 min at 72 �C. Additionally, we attempted amplifica-tion of the 50 region of the mitochondrial gene cox1 (CO1-5P) usingthe primer pair GWSLF2 and GWSRx (Saunders and Moore, 2013),and the nuclear 18S rRNA region using alternative combinations ofthe forward primers 18SFn2 or 18SFn42 with the reverse primers18SRn1760 and 18SRn1772. PCR products were purified and eitherdirectly sequenced or cloned (CloneJET PCR Cloning Kit, Fermentas)prior to sequencing by standard dye-terminator methods.Sequences of all primers used are provided in supplementalTable S2. Finally, complete sequences of the plastid gene encodingthe ribulose-1,5-bisphosphate carboxylase: oxygenase large sub-unit (rbcL) and the mitochondrial gene cox1 of particular Cyano-phora species (supplemental Table S3), obtained from parallelDNA sequencing by synthesis (Illumina) projects in our lab, wereused for additional pairwise distance estimations (Section 2.4).GenBank accession numbers of DNA sequences generated are pro-vided in supplemental Table S1.

2.3. Phylogenetic analysis

DNA sequences were aligned with MAFFT v7 (Katoh and Standley, 2013) and manually refined using Se-Al v2.0a11 (http://tree.bio.ed.ac.uk/software/seal/). Regions with missing data on bothends of the multiple DNA alignments were excluded from furtheranalyses. All multiple alignments are available upon request. Thebest-fit nucleotide substitution model for each multiple alignmentwas selected with JModelTest 2.1.4 (Darriba et al., 2012). Maxi-mum likelihood (ML) trees were estimated using the correspond-ing best-fit substitution model (AIC criterion) and using thenearest neighbor interchange (NNI) topology improvement imple-mented in PhyML 3.0 (Guindon et al., 2009). Branch support wasassessed with 500 non-parametric bootstrap replicates. Bayesianposterior probabilities were calculated with MrBayes 3.2.1(Ronquist et al., 2012) using the GTR + I + C substitution modelrunning a Metropolis-coupled Markov Chain Monte Carlo (MC3)for 2 million generations. Two independent MC3 runs were per-formed simultaneously starting from different random trees. Chainconvergence was evaluated every 150,000 generations, until theaverage standard deviation of split frequencies dropped below0.01. Trees were sampled every 100th generation. Final posteriorprobabilities were estimated after discarding trees from the first500,000 generations. Trees were visualized and graphic versionsprepared with Archaeopteryx beta v0.997 (Han and Zmasek, 2009).

2.4. Pairwise genetic distance estimation

In order to quantify the amount of genetic divergence in glauco-phyte species and strains, we generated matrices of uncorrected

J. Chong et al. / Molecular Phylogenetics and Evolution 76 (2014) 181–188 183

(p-) distances (number of nucleotide differences/total number ofnucleotides compared) from multiple DNA alignments using MEGAv5.2 (Tamura et al., 2011). Data sets of psbA and cob comprised se-quences generated by PCR from all four available glaucophyte gen-era, but rbcL and cox1 sequences were obtained only for certainCyanophora species from our independent organelle genomesequencing projects. Distance estimations of ITS data were carriedout independently for Cyanophora and Glaucocystis alignments.

3. Results and discussion

3.1. Characteristics of sequence data sets

To investigate the amount of genetic divergence between spe-cies and strains of Cyanophora and Glaucocystis (supplementalTable S1), we generated sequence sets of the plastid genes psbAand rbcL, the mitochondrial markers cob and cox1, and the nuclearloci ITS and 18S rRNA.

PCR amplification of a partial region of the psbA gene (�850nucleotides, nt) was successful for all glaucophyte strains tested.A multiple alignment (765 nt, after visual inspection and manualediting) including homologous genes from other Archaeplastida(i.e., red algae and Viridiplantae) was generated using 22 novelpsbA glaucophyte sequences and reported data from Cyanophoraparadoxa UTEX LB 555 and Glaucocystis nostochinearum UTEX B1929 (Table S1). Other than a three-nucleotide insertion found inthe 50 region of G. nostochinearum SAG 16.98 sequence (excludedbefore further analyses), the psbA alignment contained no indelsor intronic sequences. An additional glaucophyte-only psbA align-ment (795 nt; 265 encoded amino acids), corresponding to nucle-otides 144–939 of the reported C. paradoxa UTEX LB 555 gene, wasgenerated to estimate pairwise p-distance values (see Section 3.3).

The CO1-5P region has been widely used as a DNA-barcode tostudy species diversity in algal groups such as red algae and dia-toms. However, for most of the glaucophyte strains tested, PCRamplification of CO1-5P was unsuccessful, with recurrent shorttruncated reads (probably associated with secondary structuresin A + T rich templates) and occasional amplification of contami-nating bacterial sequences from non-axenic cultures. Conse-quently, we chose cob as an alternative mitochondrial marker, asboth loci present similar average p-distance values between Cyano-phora species (see Section 3.3). We successfully amplified an�875 bp region of the cob gene from 16 out of 21 glaucophytestrains (supplemental Table S1). For G. nostochinearum SAG16.98, we were only able to sequence a 650 bp fragment of thecob amplicon. A cob multiple alignment (618 nt, after visual inspec-tion and manual editing), including sequence data from viridi-plants and red algae, was generated for phylogenetic analyses.An additional glaucophyte-only alignment (753 nt; 251 encodedamino acids), corresponding to nucleotide positions 175–927 ofthe reported C. paradoxa CCMP 329 cob gene, was prepared forpairwise p-distance estimations.

We successfully amplified partial 18S rRNA sequences for alltested glaucophyte strains, but the intraspecific p-distancesshowed limited divergence (<0.8% in all cases) and these sequenceswere not further considered for genetic distance analyses. PCRamplification of the ITS region was also successful for all testedstrains (supplemental Table S1). At least three independentlyamplified PCR products from each strain were sequenced and noevidence of strain-specific ITS variation was found. Due to the pres-ence of indels and substantial intergeneric sequence divergence atthis locus, multiple alignments of ITS sequences for all glaucophytegenera could not be constructed. We therefore prepared an ITSalignment with Cyanophora sequences only (553 nt), and a sepa-rate alignment with Glaucocystis sequences (542 nt).

3.2. Maximum likelihood phylogenetic analysis of organelle andnuclear markers

To verify the monophyly of the glaucophyte strains under studywe estimated ML trees from the multiple alignments of psbA andcob including orthologs from viridiplants and red algae. The MLtree estimated from the psbA data recovered the monophyly ofglaucophytes (85% bootstrap support; BS), regardless of whetherred algae or green plants were arbitrarily defined as outgroup(Fig. S1; only the ML tree rooted with red algae is shown). Simi-larly, ML analysis of cob data (Fig. S2) depicted glaucophytes as amonophyletic group (98% BS). In the cob ML tree, only the Cyano-phora genus clade was strongly supported (97% BS). The phyloge-netic affiliation of Gloeochaete wittrockiana (SAG 46.84) andCyanoptyche gloeocystis (SAG 4.97 and SAG 34.90) was not consis-tent between these single-gene trees.

The psbA and cob alignments containing Archaeplastida homo-logues were 5% and 17% shorter, respectively, than glaucophyte-only counterparts. We therefore estimated ML trees from these lat-ter alignments to test the effect of a larger data set on resolutionwithin the glaucophyte clade. The glaucophyte-only alignmentswere also used for p-distance analyses (see Section 3.3), allowingdirect comparison between the ML and distance results. Overall,the topologies of the glaucophyte-only psbA (Fig. 1A) and cob(Fig. 1B) ML trees were consistent with phylogenies includingother Archaeplastida homologues, with well-supported Cyanopho-ra and Glaucocystis clades (P95% BS, in both cases) and similarintrageneric clusters resolved, including the Cyanophora paradoxa(CP) group and multiple Glaucocystis (Gn) groups. The Cyanophoraand Glaucocystis ITS ML analyses (Fig. 1C and D) revealed geneticgroups consistent with those obtained from psbA (Fig. 1A) andcob (Fig. 1B) sequences.

To gain further insights into the phylogenetic robustness of theintrageneric genetic groups recovered in single-gene trees, we pre-pared a four-gene concatenated alignment comprising the plastidpsbA and mitochondrial cob data together with the two nuclearmarkers 5.8S rRNA (156 nt, extracted from the ITS region), and18S rRNA. Previously reported glaucophyte sequences were alsoincluded in the concatenated multiple loci alignment (supplemen-tal Table S1). The final edited four-gene alignment was 3272 nt.The ML tree generated from this alignment (Fig. 2) resolved themonophyly of the Cyanophora sequences (100% BS) with the cladeof C. biloba and C. tetracyanea as a sister group (97% BS) to the threeC. paradoxa strains (Fig. 2, CP; 93% BS). All Glaucocystis strains wererecovered in a monophyletic group (100% BS) separated fromGloeochaete wittrockiana and Cyanoptyche gloeocystis. Within theGlaucocystis clade, six discrete groups were resolved (Fig. 2, groupsG1–G6). The three G. nostochinearum strains SAG 28.80 (no site col-lection data available, NSC), SAG 229-3 (NSC) and UTEX B 1929(NSC) constitute a single clade (group G1; 100% BS). Similarly,the G. nostochinearum strains NIES-1369 (Japan), NIES-966 (Japan),and Glaucocystis sp. KRJ2 (Korea) (group G4; 100% BS), and the iso-lates SAG 27.80 (France), HS30 (Washington, USA) and BBH (Maine,USA) (group G5; 99% BS), form well-supported groups. Otherrecovered Glaucocystis groups were SAG 45.88 (Germany) withSAG 16.98 (Germany) (group G2; 82% BS), NIES-1961 (Japan)united with KRJ1 (Korea) (group G3; 100% BS) and SAG 229.2(Denmark) affiliated with UTEX 64 (UK) (group G6; 100% BS).Given the consistency between the single-gene and concatenateddata ML analyses, hereafter we refer to the genetic group identifi-cations illustrated in the ML phylogenetic trees (Figs. 1 and 2)when discussing the p-distance results of individual genetic mark-ers (see Section 3.3).

Overall, the ML phylogenetic trees of single-gene and concate-nated sequences of plastid, mitochondrial and nuclear markersshow evidence of genetic divergence between glaucophyte strains

Fig. 1. Maximum likelihood analyses of glaucophyte molecular markers. Maximum likelihood (ML) trees estimated from (A) the plastid loci psbA, (B) the mitochondrial genecytochrome b (cob) from diverse glaucophyte genera, and the nuclear ITS region of (C) Cyanophora and (D) Glaucocystis species/strains, respectively. The psbA and cob ML treesare rooted using the Cyanophora clade as outgroup. Numbers near nodes indicate PhyML bootstrap proportion support (only values >50% are shown). Branch lengths areproportional to the number of substitutions per site (scale bar corresponds to 0.1 substitutions per site). Putative genetic groups are indicated in the Cyanophora paradoxa (CP)and within Glaucocystis (Gn) branches.

184 J. Chong et al. / Molecular Phylogenetics and Evolution 76 (2014) 181–188

currently identified as the same species. The moderate to strongbranch support (>80% BS) observed for most of the Cyanophoraand Glaucocystis internal groups (Fig. 2), and the evident diver-gence between members of the same intraspecific clades, (i.e.within the Cyanophora paradoxa group [CP], and between the sev-eral Glaucocystis nostochinearum groups), reveals potential crypticspecies diversity in these taxa.

3.3. Distance analysis of plastid, mitochondrial and nuclear markers

Genetic distance studies of short (�650–800 bp) molecularmarkers with relatively high interspecific and low intraspecificvariation (i.e., DNA-barcodes; Hebert et al., 2003), have been usedfor biodiversity assessments and to assist formal species identifica-tion in unicellular green algae (e.g., Hall et al., 2010; Bock et al.,2011; Demchenko et al., 2012), diatoms (e.g., Hamsher et al.,2011), dinoflagellates (e.g., Lin et al., 2009; Stern et al., 2010) andraphidophytes (e.g., Demura et al., 2009). In the green algal generaBracteacoccus, Chlorosarcinopsis and Scenedesmus rbcL-3P interspe-cific distances show average values of 5.6%, 1.9% and 4.8%, respec-tively (Hall et al., 2010). In the case of CO1-5P, interspecificdistances in the diatom genus Sellaphora range from 1.2% to 3.7%(Evans et al., 2007; Hamsher et al., 2011) and in Bracteacoccus

average 10.7% (Hall et al., 2010). Distances estimated with cob se-quences between different dinoflagellate species vary from zero to>20% (Lin et al., 2009). Interspecifc distances evaluated with the ITSregion show distances <1% in the raphidophyte Chattonella(Demura et al., 2009) and between 7.3–17.4% and 1.5–24.3%,respectively, in the green algal genera Chlorella (Bock et al., 2011)and Microglena (Demchenko et al., 2012).

In comparison, interspecific distances estimated in the glauco-phyte genus Cyanophora, using the plastid markers rbcL-3P andpsbA, ranged from 7.1% to 9.5% and 4.0% to 6.5%, respectively,whereas intraspecific distances in C. paradoxa from the same plas-tid markers were 4.4% and 3–4%, respectively (Tables 1 and S3).These results indicate some overlap between the Cyanophora psbAinterspecific distance values and the C. paradoxa intraspecific dis-tances. In contrast, distances estimated from psbA between certainGlaucocystis genetic groups (Table 1) varied from 2.2% to 2.9%,whereas the psbA intraspecific distance between Glaucocystisnostochinearum strains using the same molecular marker was inmost cases <2.0% (Table 1). Values of interspecific distances inthe genus Cyanophora derived from the mitochondrial markersCO1-5P and cob ranged from 13.7% to 19.5% and 11.9% to 16.2%,respectively (Tables 2 and S3). The minimum and maximum cobintraspecific distances within the C. paradoxa group were 12.2%

Fig. 2. Maximum likelihood analysis of the concatenated psbA, cob, 5.8S and 18 rRNAs sequences. Numbers near nodes indicate PhyML bootstrap proportion support (onlyvalues >50% are shown). Thick branches indicate Bayesian posterior probabilities P0.95. Branch lengths are proportional to the number of substitutions per site (scale barcorresponds to 0.1 substitutions per site). Putative genetic groups are indicated in the Cyanophora paradoxa (CP) and within Glaucocystis (Gn) branches. Original strainsampling locations are indicated in parenthesis.

Table 1Estimated psbA p-distances in Cyanophora and Glaucocystis strains.

Group Cyanophora strain A B C D

A NIES-764B UTEX LB 2766 0.045

C CP NIES-763 0.048 0.065D SAG 45.84 0.040 0.060 0.030E UTEX LB 555 0.056 0.058 0.040 0.0409

Glaucocystis strain F G H I J K L M

F G1 SAG 28.80, SAG 229-3, UTEX B 1929

G G2 SAG 45.88 0.020H SAG 16.98 0.022 0.002

I G3 NIES-1961, KRJ1 0.023 0.011 0.011

J G4 KRJ2, NIES-966, NIES-1369 0.025 0.014 0.014 0.015

K G5 SAG 27.80 0.019 0.009 0.009 0.008 0.007L HS30 0.021 0.010 0.010 0.008 0.007 0.001M BBH 0.029 0.017 0.016 0.016 0.014 0.007 0.007

N G6 SAG 229-2 0.027 0.020 0.021 0.016 0.021 0.014 0.015 0.023

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and 15.5%, again indicating some overlap of inter- and intraspecificdistance values in the Cyanophora taxa analyzed. In the case ofGlaucocystis, the cob p-distances between strains varied across awide range from zero to 15.6% (Table 2), with the majority of thepairwise comparisons between genetic groups above 10% (Table 2).The ITS interspecific distances in Cyanophora varied from 2.9% to5.3% (Table 3), whereas intraspecific distances in the C. paradoxagroup ranged from 1.2% to 2.5% (Table 3). The maximum ITSp-distance between Glaucocystis strains was 17.8% with an averagedistance between strains of 10.1% ± 5.4 SD (Table 3).

The effective use of DNA-barcodes to assist species recognition ordelimitation necessarily relies on a solid taxonomic foundation

based on morphological, geographic and ecological data. In the ab-sence of a robust taxonomic framework for glaucophytes we cannotdelineate different species based on genetic distances alone, andtherefore relative comparisons of glaucophyte intraspecifc distancesversus interspecific values in other algae should be interpreted withcaution. Nonetheless, the relative comparisons of the diversemolecular markers indicate that the genetic distance between someglaucophyte isolates currently considered strains of the same spe-cies are equal to or surpass interspecific distances estimated in otherunicellular algal groups. Overall, the p-distances measured in thisstudy reveal previously unrecognized genetic divergence withinglaucophytes and suggest cases of cryptic species diversity.

Table 2Estimated cob p-distances in Cyanophora and Glaucocystis strains.

Group Cyanophora strain A B C D

A NIES-764B UTEX LB 2766 0.119

C CP NIES 763 0.159 0.161D SAG 45.84 0.162 0.162 0.122E CCMP 329 0.158 0.155 0.129 0.155

Glaucocystis strain F G H I J K L M N O

F G1 SAG 28.80, SAG 229-3

G G2 SAG 45.88 0.117H SAG 16.98 0.100 0.089

I G3 NIES-1961 0.142 0.123 0.120

J G4 NIES-966 0.134 0.117 0.095 0.130K NIES-1369 0.126 0.110 0.094 0.123 0.018

L G5 SAG 27.80 0.146 0.133 0.128 0.139 0.127 0.119M HS30 0.137 0.132 0.121 0.129 0.131 0.125 0.078N BBH 0.137 0.130 0.124 0.129 0.130 0.123 0.075 0.005

O G6 SAG 229-2 0.133 0.133 0.140 0.141 0.148 0.141 0.156 0.152 0.152P UTEX 64 0.135 0.135 0.140 0.143 0.147 0.139 0.154 0.150 0.149 0.001

Table 3Estimated ITS p-distances in Cyanophora and Glaucocystis strains.

Group Cyanophora strain A B C D

A NIES-764B UTEX LB 2766 0.036

C CP NIES-763 0.029 0.042D SAG 45.84 0.042 0.051 0.012E CCAP 981/1 0.033 0.053 0.020 0.025

Glaucocystis strain F G H I J K L M N O

F G1 SAG 28.80. SAG 229-3

G G2 SAG 45.88 0.162H SAG 16.98 0.150 0.022

I G3 NIES-1961, KRJ1 0.156 0.062 0.061

J G4 KRJ2 0.156 0.083 0.075 0.070K NIES-966 0.148 0.079 0.069 0.069 0.026L NIES-1369 0.147 0.080 0.070 0.071 0.028 0.002

M G5 SAG 27.80 0.162 0.073 0.061 0.059 0.070 0.063 0.064N HS30 0.158 0.073 0.062 0.056 0.072 0.064 0.066 0.008O BBH 0.169 0.082 0.070 0.065 0.078 0.072 0.074 0.010 0.009

P G6 SAG 229-2 0.178 0.146 0.136 0.129 0.145 0.144 0.144 0.141 0.135 0.140

186 J. Chong et al. / Molecular Phylogenetics and Evolution 76 (2014) 181–188

4. Conclusions

The rarity of glaucophytes has been a major limiting factor instudying basic elements of their biology, such as their geographicdistribution and species diversity. The present molecular scrutinyof glaucophyte culture collections, and new isolates from the NorthEast and West USA and Korea has unveiled substantial geneticdivergence between some strains currently considered membersof the same species. The aim of this molecular survey was to estab-lish the foundation for future comparative genomic investigationsof glaucophytes, rather than species discovery or identification.Formal description of new species within C. paradoxa and G. nosto-chinearum awaits further comprehensive taxonomic investigations,including morphological, ultrastructural and biophysiological anal-yses, investigations of reproductive mechanisms, thorough surveysof ecological and geographic distributions, and detailed molecularphylogenetic assessments using a broader glaucophyte taxonsample.

Even though we were not able to generate a complete glauco-phyte database for the widely used mitochondrial CO1-5P and

plastid rbcL-3P regions, comparisons of the genetic distances be-tween these typical DNA-barcodes with distances from our alter-native markers, cob and psbA, reveal comparable divergencevalues between Cyanophora strains and species when consideringloci from the same organelle genome. Overall, the markers psbA,cob and ITS, from different sub-cellular genomic compartments, re-solved similar genetic groups in the genus Cyanophora, and withinthe clade composed of G. nostochinearum strains and the novelGlaucocystis isolates. The phylogenetic and distance surveys sug-gest that the analyzed loci are suitable candidates for furthermolecular studies of glaucophyte species diversity. However, theeffectiveness of different markers as formal DNA-barcodes is lim-ited by the lack of detailed information regarding the inheritancemechanisms of organellar and nuclear genetic material in glauco-phytes. Importantly, there is no evidence of sexual reproductionin glaucophytes, and this lack of knowledge of the reproductivemechanisms (e.g., uniparental versus biparental organelleinheritance in the case of sexual reproduction) complicates strictcomparisons between potential DNA-barcodes. With no clearunderstanding of the plastid, or mitochondrial, inheritance

J. Chong et al. / Molecular Phylogenetics and Evolution 76 (2014) 181–188 187

patterns in a putative sexual reproduction context, rigorous inter-pretations of differences observed here between organelle and nu-clear markers, such as the contrast of the psbA and ITS results fromthe novel Glaucocystis KRJ1 and KRJ2 strains (isolated from thesame pond), are not attainable and should await further investiga-tions of basic glaucophyte biology.

The perspective gained through the present study on glauco-phyte diversity using multiple markers has allowed us to identifykey taxa for further studies. Important emerging questions regard-ing glaucophyte diversity include how the different genera are re-lated to each other, and which among these groups is the earliestbranching lineage? Our phylogenetic analyses consistently sepa-rate the scarcely studied Gloeochaete and Cyanoptyche from the‘‘well known’’ Cyanophora and Glaucocystis clades. The phyloge-netic position of these two largely overlooked taxa within the‘‘Glaucophyte Tree of Life’’ is an important target for further geno-mic and biochemical/physiological investigations. Finally, if thesingle origin of primary plastids stands as a likely hypothesis, thenwe still need to answer the question: what was the earliest diverg-ing lineage during Archaeplastida evolution? Diverse comparativeanalyses have failed to provide an unambiguous and unanimousanswer concerning the early diversification of Archaeplastida,and the possibility to untangle this scenario greatly relies on a reli-able molecular knowledge base of glaucophytes. The present workadds to that effort by providing a renovated view of glaucophytediversity and guiding directions for further genomic investigationsof this algal group.

Acknowledgments

The present work was supported by the Natural Sciences andEngineering Research Council of Canada (project 402421-2011),the Canada Foundation for Innovation (project 28276), and theNew Brunswick Innovation Foundation (project RIF2012-006)awarded to ARP, and grants from the Korean Rural DevelopmentAdministration Next-generation BioGreen21 (PJ009525) to HSY.We want to acknowledge Joshua Clowater for his involvement dur-ing the early planning of the project.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2014.03.019.

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