hidden biodiversity of the extremophilic cyanidiales red...

Molecular Ecology (2004)

13

, 1827–1838 doi: 10.1111/j.1365-294X.2004.02180.x

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

Hidden biodiversity of the extremophilic Cyanidiales red algae

CLAUDIA CINIGLIA,

*‡

HWAN SU YOON,

†‡

ANTONINO POLLIO,

*

GABRIELE PINTO

*

and DEBASHISH BHATTACHARYA

†

*

Dipartimento di Biologia vegetale, Università ‘Federico II’, via Foria 223, 80139 Napoli, Italy,

†

Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, Iowa 52242, USA

Abstract

The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and hightemperature conditions around hot springs. These unicellular taxa have a relatively simplemorphology and are currently classified into three genera,

Cyanidium

,

Cyanidioschyzon

and

Galdieria

. Little is known, however, about the biodiversity of Cyanidiales, theirpopulation structure and their phylogenetic relationships. Here we used a taxonomicallybroadly sampled three-gene data set of plastid sequences to infer a robust phylogeneticframework for the Cyanidiales. The phylogenetic analyses support the existence of at leastfour distinct Cyanidiales lineages: the

Galdieria

spp. lineage (excluding

Galdieria maxima

),the

Cyanidium caldarium

lineage, a novel monophyletic lineage of mesophilic

Cyanidium

spp. and the

Cyanidioschyzon merolae

plus

Galdieria maxima

lineage. Our analyses do notsupport the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algaewere ancestrally thermo-acidotolerant. We also used environmental polymerase chainreaction (PCR) for the

rbc

L gene to sample Cyanidiales biodiversity at five ecologically distinctsites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level ofsequence divergence among Cyanidiales species and the partitioning of taxa based onenvironmental conditions. Our research revealed an unexpected level of genetic diversityamong Cyanidiales that revises current thinking about the phylogeny and biodiversity ofthis group. We predict that future environmental PCR studies will significantly augmentknown biodiversity that we have discovered and demonstrate the Cyanidiales to be aspecies-rich branch of red algal evolution.

Keywords

: biodiversity, Cyanidiales, extremophile, phylogeny, plastid genes, red algae

Received 25 November 2003; revision received 13 February 2004; accepted 13 February 2004

Introduction

Extremophiles are organisms that thrive at an extremelyhigh or low pH (e.g. < 3), temperature (e.g. > 50

°

C), salinity,desiccation and pressure (Rothschild & Mancinelli 2001),relying on specialized enzymes for survival (Hough &Danson 1999). These enzymes have great potential forbiotechnological and pharmaceutical applications [e.g.

Taq

polymerase (Brock 1997)]. Despite their proclivity for hostileenvironments, extremophiles are a highly diverse groupwith an abundance of novel taxa having been discovered

recently at hot springs, in an acidic river, and at a deepocean site (DeLong & Pace 2001; Lopez-Garcia

et al

. 2001;Moon-van der Staay

et al

. 2001; Amaral Zettler

et al

. 2002).Most thermo-acidophiles are prokaryotes (i.e. Archea orBacteria) with one notable exception, the Cyanidiales.

Cyanidiales is a group of asexual, unicellular red algae,which thrive in acidic (pH 0.5–3.0) and high temperature(50–55

°

C) conditions around hot springs and/or acidicsulphur fumes (Pinto

et al

. 2003). Cyanidiales have arelatively simple morphology (see Fig. 1A) consistingof spherical thick-walled cells containing one plastid(i.e. chloroplast), 1–3 mitochondria, a nucleus, a vacuoleand storage products (Merola

et al

. 1981; Sentsova 1991; Ott& Seckbach 1994; Albertano

et al

. 2000; Pinto

et al

. 2003).Three genera (

Cyanidium

,

Cyanidioschyzon

and

Galdieria

)

Correspondence: Debashish Bhattacharya. Fax: (319) 335 1069;E-mail: [email protected]‡These authors contributed equally to this work.

1828

C . C I N I G L I A

E T A L .

© 2004 Blackwell Publishing Ltd,

Molecular Ecology

,

13

, 1827–1838

and six species are presently recognized in this order basedon morphological characters such as cell shape, number andshape of plastids, characters of the cell wall, presence–absenceof vacuoles, the pattern of cell, division and the numberof autospores in sporangia. However, species delimitationremains unclear due to a paucity of diagnostic charactersets for each taxon (Albertano

et al

. 2000; Merola

et al

. 1981;Sentsova 1991; Ott & Seckbach 1994; Pinto

et al

. 2003).Molecular phylogenetic studies suggest that the Cyanid-

iales represent one of the most ancient groups of algae,having diverged about 1.3 billion years ago at the baseof the Rhodophyta (Müller

et al

. 2001; Yoon

et al

. 2002b).Given their long evolutionary history, it is intriguing thatonly a handful of recognized morphological species havesurvived in this lineage. Three possible explanations forthis observation are (1) the Cyanidiales have always beenspecies poor, perhaps similar to other ancient algal groupssuch as the Glaucophyta (Helmchen

et al

. 1995); (2) manyCyanidiales lineages have diverged over evolutionarytime, but only a few have survived recurrent extinctions ora recent extinction event (there is, however, no fossil recordfor this group to test this idea); or (3) we grossly underes-timate the genetic diversity of Cyanidiales due to a relianceon a limited character set that may reflect strong selectionagainst morphological variation rather than genetic homo-geneity [e.g. as in

Bangia

spp. (Butterfield 2000)]. Recentanalyses (Gross

et al

. 2001; Pinto

et al

. 2003) show limitedsupport for the third hypothesis, although no detailedstudy has yet been published on Cyanidiales biodiversity.

Environmental polymerase chain reaction (PCR) (i.e.cultivation-independent surveys) has been used effectivelyto assess species biodiversity in the field (DeLong & Pace2001; Lopez-Garcia

et al

. 2001; Moon-van der Staay

et al

.2001; Amaral Zettler

et al

. 2002). In this study, we recon-structed the phylogeny of the Cyanidiales in trees thatincluded all known genera and species using a concate-nated set of three plastid genes (

psa

A,

psb

A, and

rbc

L). Inaddition, we collected environmental samples and analysedstrains isolated from different locations worldwide, as wellas employing more extensive sampling from differentsites and habitats (i.e. both extremophilic and mesophilic)in Italy to assess the diversity of the Cyanidiales. Phyloge-netic analyses of

rbc

L sequences from the environmentalsamples revealed an unanticipated level of genetic diversityin the Cyanidiales and helped us to define the majorevolutionary lineages in this group. There was also astriking pattern of intrapopulation structure that suggestsenvironmental conditions play a major role in partitioningCyanidiales genotypes.

Materials and methods

Sampling of cultured isolates and DNA-sequencing

The sources and GenBank Accession nos of the sequencesthat were determined in this study are summarized inTable 1. Twenty-seven existing cultures that have beengathered from around the world and deposited in the

Fig. 1 The Cyanidiales red algae. (A) TEM micrograph of a crypto-endolithic strain of Galdieria sulphuraria. The abbreviations denote thefollowing: m = mitochondrion, n = nucleus, p = plastid, v = vacuole. Scale bars = 1 µm. (B) The environmental Sites A–E used to collectCyanidiales at Pisciarelli in the Phlegrean Fields, Italy (see text for details).

C Y A N I D I A L E S B I O D I V E R S I T Y

1829


Molecular Ecology

, 13, 1827–1838

Table 1

Sample information and GenBank Accession nos for taxa included in the phylogenetic analyses. The Accession nos of sequencesdetermined in this study are shown in bold text

Taxa Source

psa

A

psb

A

rbc

L

Bangiales

Bangia atropurpurea

SAG 33.94 AY119698 AY119734 AY119770

Bangia fuscopurpurea

SAG 59.81 AY119699 AY119735 AY119771

Porphyra purpurea

GenBank NC_000925 NC_000925 NC_000925

Compsopogonales

Compsopogon coeruleus

SAG 36.94 AY119701 AY119737 AF087116

Cyanidiales


DBV 201 JAVA AY119693 AY119729 AY119765DBV 001 NAPS AY119694 AY119730 AY119766DBV 202 NAMN — —

AY541296

Cyanidium caldarium

RK1 NC_001840 NC_001840 NC_001840DBV 019 SIPE

AY541281 AY541289 AY541297

DBV 182 JAVA

AY541282 AY541290 AY541298

DBV 020 APAS — —

AY541299

C

. sp. — Monte Rotaro Monte Rotaro AY391362 AY391365 AY391368Monte Rotaro 19 — —

AY541300

Monte Rotaro 20 — —

AY541301

C

. sp. — Sybil Sybil AY391363 AY391366 AY391369

Galdieria daedala

IPPAS P508

AY541283 AY541291 AY541302

Galdieria maxima

IPPAS P507 AY391364 AY391367 AY391370

Galdieria partita

IPPAS P500

AY541284 AY541292

AB18008

Galdieria sulphuraria

-A SAG 108.79 AY119695 AY119731 AY119767UTEX 2393

AY541285

X52758 AF233069DBV 011 CEMD

AY541286 AY541293 AY541303

DBV 018 CNASC

AY541287 AY541294 AY541304

DBV 015 NAFG — — AY541305DBV 017 NASF — — AY541306DBV 021 MEVU — — AY541307DBV 074 JAVA — — AY541308DBV 135 AZUF — — AY541309


-B DBV 009 VTNE AY119696 AY119732 AY119768DBV 012 BNTE

AY541288 AY541295 AY541310

DBV 063 AGCS AY119697 AY119733 AY119769DBV 002 NAPS — —

AY541311

Environmental sample Pisciarelli-A1 — —

AY541312

Pisciarelli-A12 — —

AY541313

Pisciarelli-B15 — —

AY541314


AY541315


AY541316

Pisciarelli-C1 — —

AY541317


AY541318


AY541319

Pisciarelli-D1 — —

AY541320


AY541321


AY541322

Pisciarelli-E10 — —

AY541323


AY541324


AY541325

Porphyridiales

Bangiopsis subsimplex

PR21 AY119700 AY119736 AY119772

Dixoniella grisea

SAG 39.94 AY119702 AY119738 AY119773

Flintiella sanguinaria

SAG 40.94 AY119704 AY119740 AY119774

Porphyridium aerugineum

SAG 1380–2 AY119705 AY119741 AY119775

Rhodella violacea

SAG 115.79 AY119706 AY119742 AY119776

Rhodosorus marinus

SAG 116.79 AY119708 AY119744 AY119778

Stylonema alsidii

SAG 2.94 AY119709 AY119745 AY119779

1830

C . C I N I G L I A

E T A L .


Molecular Ecology

,

13

, 1827–1838

Rhodochaetales

Rhodochaete parvula

UTEX LB 2715 AY119707 AY119743 AY119777

Florideophycidae

Chondrus crispus

Nova Scotia, Canada AY119710 AY119746 U02984

Palmaria palmata

Maine, USA AY119711 U28165 U28421

Thorea violacea

SAG 51.94 AY119712 AY119747 U28421

Chlorophyta

Mesostigma viride

GenBank NC_002186 NC_002186 NA

Nephroselmis olivacea


Glaucophyta

Cyanophora paradoxa


NA: the

rbc

L gene of the green and glaucophyte algae are of a cyanobacterial origin, whereas those in the red algae and red algal-derived plastids are of proteobacterial origin [e.g. Valentin, Zetsche (1990)].

Taxa Source

psa

A

psb

A

rbc

L

Culture Collection of the Dipartimento di Biologia vegetale,Naples (DBV, Italy), the Culture Collection of Microalgaeof the Institute of Plant Physiology, Saint Petersburg [IPPAS,Russia (http://wdcm.nig.ac.jp/CCINFO/CCINFO.xml?596)] and the Sammlung von Algenkulturen, Pflanzenphy-siologisches Institut der Universität Göttingen [SAG,Germany (http://wwwuser.gwdg.de/

∼

epsag/phykologia/epsag.html)] were used in the study. We have includedthree isolates of

Cyanidium caldarium

and two represent-atives of


, from both Italian and Asiansites, two mesophilic strains of

Cyanidium

sp. collectedin two Italian caves (Sybil cave at Cumar, Naples andMonte Rotaro, Ischia), and 10 strains of

Galdieria

collectedfrom different sites around the world. These latter isolatescomprise three Russian species (

Galdieria maxima

,

Galdieriadaedala

, and

Galdieria partita

) with the remainder beingstrains of


.Algal tissue was ground with glass beads by using a

Mini-BeadBeater (Biospec Products, Inc., Bartlesville, OK,USA). Total genomic DNA was extracted using the DNeasyPlant Mini Kit (Qiagen, Santa Clarita, CA, USA). PCR wereconducted using specific primers for the plastid genes psaAand psbA (Yoon et al. 2002a; 2002b). Three novel degenerateprimers were used to amplify the rbcL gene: rC475F; 5′-AAAACTTTCCAAGGRCCWGC-3′, rC910r; 5′-TTWCCT-GCTCTRTGTAARTG-3′, rCR; 5′-GCWGTTGGTGTYTCHACWAAATC-3′. These primers were designed on thebasis of sequence comparisons of the plastid gene inCyanidium and in other red algae. The PCR products werepurified with the QIAquick PCR purification kit (Qiagen)and used for direct sequencing using the BigDyeTMTerminator Cycle Sequencing Kit (PE-Applied Biosystems,Norwalk, CT, USA), and an ABI-3100 at the Center forComparative Genomics at the University of Iowa.

Field sampling and environmental PCR

We sampled biomats of Cyanidiales from five differenthabitats at Pisciarelli (Naples, Italy), which is a hydrothermalarea located in the central part of the Quaternary volcaniccomplex of the Phlegrean Fields. These sites had distinctenvironmental regimes and were named Sites A–E (seeFig. 1B). Site A had a population of Cyanidiales growingon the rocks surrounding a hot sulphur spring where thesulphur steams were intense and the temperature rangedfrom 45 to 55 °C. Site B was a dry, crypto-endolithic site inwhich the biomat grew inside the layers of the alunite opalrocks and the temperature ranged from 18 to 30 °C. SiteC was a relatively dry, interlithic site in which the Cyani-diales grew within a fissure that was enveloped in sulphuremissions and was covered by a thin and crumbly sulphatecrystal layer. Site D was a humid but low temperatureregion where the Cyanidiales lived on muddy soil flankingthe stream that flows from the hot spring. The temperaturegradually decreased at Site D, reaching around 25–40 °C.Finally, at Site E, the biomat grew on hot rocks lying on thesoil behind the hot springs.

In each case, environmental material was collected witha spatula near the middle of the particular site. The algalmaterial was transported to the laboratory in 50 mL Falcontubes. The Cyanidiales cells are extremely stable and didnot require any further handling prior to DNA extraction.Between 10 and 20 mg of material was used for each DNApreparation as described above. For Site B, the surface rockwas first removed and the crypto-endolithic cells werecollected. After genomic DNA was extracted from the cellsin these natural populations, the rbcL gene was amplifiedusing PCR. The amplification products were cloned intothe pGEM-T vector (Promega). A total of 90 clones were

Table 1 Continued

C Y A N I D I A L E S B I O D I V E R S I T Y 1831

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1827–1838

picked (up to 20 per site) and characterized by the patternsproduced by restriction enzyme digests (i.e. using, DraIIand RsaI). Three isolates from the DBV collection thatrepresented the known genera (Cyanidium, Cyanidioschyzonand Galdieria) of Cyanidiales were used as positive con-trols. One to two clones representing each distinct bandingpattern were sequenced from these environmentalsamples and added to the existing rbcL data set. All culturedCyanidiales from the DBV collection in Naples are availablefrom G. P. Although we do not have vouchers of eachenvironmental sample used in this work, additional collec-tions from these sites are also available upon requestfrom G. P.

Phylogenetic analyses

Sequences were aligned manually using SeqPup (Gilbert1995). The alignment used in the phylogenetic analyses isavailable upon request from D. B. We analysed two differentdata sets. In the first analysis, we concatenated partialsequences of the three plastid genes psaA (1395 nt), psbA(957 nt) and rbcL (1215 nt) found in 17 Cyanidiales, 15 non-Cyanidiales red algae, two green algae and a glaucophyte asthe outgroup. In the second analysis of the rbcL data set, weincluded 27 representatives of the Cyanidiales from thecultured strains and GenBank of a worldwide distribution(three American, four Asian and three Russian) and 17local populations from Italy. We added 14 environmentalsamples from Pisciarelli. We used non-Cyanidiales redalgae as the outgroup in this analysis because the rbcL geneof the green and glaucophyte algae are of cyanobacterialorigin, whereas those in the red algae and red algal-derivedplastids are of proteobacterial origin (Valentin & Zetsche1990). We excluded third codon positions from the rbcLdata set to reduce the possible misleading effects of muta-tional saturation in the DNA sequences (for details, seePinto et al. 2003).

Phylogenetic trees were inferred with minimum evolu-tion (ME), Bayesian inference and maximum likelihood(ML) methods. In the ME analyses of the three-gene dataset, we generated distance matrices using LogDet transfor-mation and the general time reversible model (Rodriguezet al. 1990) with estimations of nucleotide frequencies, theshape parameter of the gamma distribution to accommo-date rate variations across sites and the proportion ofinvariant sites (GTR + I + Γ model) with the paup* compu-ter program (Swofford 2002). The parameter estimatesfor the GTR + Γ + I model were estimated using paup* anda starting ME tree was built with HKY-85 distances. Tenheuristic searches with random-addition-sequence start-ing trees and tree-bisection–reconnection (TBR) branchrearrangements were performed to find the optimal MEtrees. Best scoring trees were held at each step. To test thestability of monophyletic groups in the ME analyses, 2000

bootstrap replicates (Felsenstein 1985) were analysed usingthe ME-LogDet and ME-GTR approaches. In the Bayesianinference of the DNA data (mrbayes version 3.0b4,Huelsenbeck & Ronquist 2001), we used the site-specificGTR + Γ (ssGTR) model with separate model parameterestimates for the three data partitions (i.e. 1st, 2nd and3rd codon positions). Metropolis-coupled Markov chainMonte Carlo (MCMCMC) from a random starting tree wasinitiated in the Bayesian inference and run for 2000 000generations. Trees were sampled each 1000 cycles. Four chainswere run simultaneously of which three were heated andone was cold, with the initial 200 000 cycles (200 trees)being discarded as the ‘burn-in’. Stationarity of the loglikelihoods was monitored to verify convergence by 200 000cycles (results not shown). A consensus tree was madewith the remaining 1800 phylogenies to determine theposterior probabilities at the different nodes. For the analysisof rbcL, the same settings were implemented in theBayesian inference as described above, except for the useof a two-partition evolutionary model (i.e. 1st and 2ndcodon positions).

In addition to the DNA analyses, phylogenetic inferenceusing protein data was also done for the three-gene andrbcL data sets. An optimal ML tree (ML-protein) was inferredin each case using ‘proml’ (phylip version 3.6, Felsenstein2002) and the JTT evolutionary model with 10 random-sequence version additions and global rearrangements.Bootstrap analysis was performed with these data usingthe JTT model (2000 replicates for the three-protein dataset, 100 replicates for the rbcL data set) as described above.

To assess the stability of the monophyletic groups iden-tified in the three-gene and rbcL plastid trees, we generatedalternate trees in which critical branches in the ME-LogDettree were rearranged and compared to the likelihood (underthe Tamura–Nei + Γ model) of the ‘best’ tree (Tamura &Nei 1993). Phylogenetic support for these trees was assessedusing the one-sided Kishino–Hasegawa test (Kishino &Hasegawa 1989; Goldman et al. 2000) implemented in tree-puzzle version 5.1 (Schmidt et al. 2002).

Results

Phylogeny of the concatenated three-gene data set

A total of 3567 nt and 1189 aa from the three plastid genespsaA, psbA, and rbcL in 17 representatives of the Cyanidiales(for a total of 45 novel sequences) and representatives ofthe major lineages of Bangiophycidae red algae (Mülleret al. 2001; Yoon et al. 2002b) were used to infer the phylo-genetic relationships of the Cyanidiales. The ME-LogDettree of the concatenated sequences shows strong supportfor the monophyly of the Cyanidiales, with these taxaforming a sister group to the rest of the red algae (ME-LogDetbootstrap = 100%, ML-protein bootstrap = 91% and

1832 C . C I N I G L I A E T A L .


posterior probability = 1.0, Fig. 2). Within the Cyanidiales,four lineages are resolved with strong support. The first isthe Galdieria spp. lineage (excluding Galdieria maxima), whichconsists of two subclades. The second lineage is composedof the Cyanidium caldarium strains, the third is a new cladeformed by mesophilic Cyanidium sp., and the fourth includesCyanidioschyzon merolae, which groups with Galdieria maximawith strong bootstrap support. The interrelationshipof these four distinct lineages is poorly resolved in Fig. 2,with only the early branching of the Galdieria lineage havingmoderate bootstrap support (ME-LogDet = 78%, ML-protein= 64%). The ME-GTR and ML-protein trees are generally

consistent with the ME-LogDet tree shown in Fig. 2, exceptfor the branching order of the non-Cyanidiales red algae(trees not shown).

rbcL phylogeny

The ME-LogDet tree of rbcL sequences was inferredfrom a data set of 810 nt (excluding 3rd codon positions)and 405 aa from 41 Cyanidiales, including 14 representativesof environmental samples. This tree (Fig. 3) shows a verysimilar topology to the three-gene tree. The four groups areagain resolved. The Galdieria lineage is demarcated clearly

Fig. 2 Phylogeny of the Cyanidiales inferred from minimum evolution (ME) analysis using the LogDet transformation of the combinedplastid DNA sequences of psaA, psbA, and rbcL. Results of a ME-LogDet bootstrap analysis are shown above the branches, whereas thebootstrap values from a protein maximum likelihood analysis using the JTT evolutionary model are shown below the branches. Onlybootstrap values > 60% are shown. The thick nodes represent > 95% Bayesian posterior probability for clades using the site-specific GTR model.



into two subgroups, Galdieria-A and Galdieria-B. The repre-sentatives of Galdieria-A have a worldwide distribution(i.e. Italy, United States, Mexico, Indonesia, and Russia),whereas members of the Galdieria-B lineage are restricted

to Italy. The Cyanidium caldarium sequences and themesophilic-Cyanidium sp. lineages maintain a sister-grouprelationship [now with more support (LogDet = 85%,posterior probability = 0.95)] with the addition of more

Fig. 3 Phylogeny of the Cyanidiales inferred from a minimum evolution (ME) analysis using the LogDet transformation of the rbcLsequences. Results of a ME-LogDet bootstrap analysis are shown above the branches, whereas the bootstrap values from a proteinmaximum likelihood analysis using the JTT evolutionary model are shown below the branches. Only bootstrap values > 60% are shown.The thick nodes represent > 95% Bayesian posterior probability for clades using the site-specific GTR model. Sequences from theenvironmental survey are represented by ‘Pisciarelli-[followed by] collection site’ and are marked with asterisks.

1834 C . C I N I G L I A E T A L .


sequences. Cyanidioschyzon merolae, Galdieria maxima andone of the environmental samples (C16) are part of a weaklysupported lineage.

Distribution of Cyanidiales in the environmental samples

The results of the analysis of environmental samples aresummarized in Fig. 4. We surveyed a total of 90 clonesfrom five sites. Members of the Galdieria-A lineage werefound at Sites A (2/20 clones), D (5/19 clones) and E (10/13 clones), all of which were relatively humid conditions,whereas members of Galdieria-B were found only at Sites B(19/19) and C (8/20), which are relatively dry. It is interestingthat all the representatives of Site B, which is a crypto-endolithic habitat, was only of the Galdieria-B type. Cyanidio-schyzon merolae coexisted with Galdieria-A (i.e. Sites A, Dand E) and with Cyanidium caldarium at Site C (11/20).

Discussion

Biodiversity of the extremophilic Cyanidiales

Our study was aimed primarily at assessing the extentof biodiversity of Cyanidiales at different environmentalhabitats in the Phlegrean Fields and to incorporate thisassessment of biodiversity into a broadly sampled androbust phylogenetic framework. As a result of our approach,we have identified four well-supported lineages of Cyani-diales in addition to establishing the phylogenetic positionsof the three traditionally recognized genera Cyanidium,Cyanidioschyzon, and Galdieria. The four lineages are

(1) Galdieria-A and Galdieria-B (excluding Galdieria maxima);(2) Cyanidium caldarium; (3) mesophilic Cyanidium spp.;and (4) Cyanidioschyzon merolae and Galdieria maxima (seeFigs 2 and 3). Morphology is normally a valuable tool insystematics; however, in the case of the unicellular Cyani-diales, it appears to not be sufficiently informative toestablish a robust taxonomy (e.g. Pinto et al. 2003). Belowwe discuss in more detail each of the four lineages we haveidentified in our study, their constituent taxa and theenvironmental conditions in which they were found.

Galdieria-A and -B lineages

The systematics of the genus Galdieria is yet to be definedclearly. A recent study that utilized a combined morpholo-gical and physiological approach revealed substantialcomplexity in this genus, with critical characters such ascell size and number and shape of plastids providing noobjective basis for discriminating among the four speciesGaldieria sulphuraria, Galdieria daedala, Galdieria partita andGaldieria maxima. In addition, high levels of rbcL sequencedivergence within the genus made it difficult to refute orconfirm the existing taxonomy of Galdieria (Pinto et al. 2003).In this study, we have gained a much broader perspectiveon this genus and can now separate it clearly into twosubclades, Galdieria-A and Galdieria-B, with Galdieria maximadistantly related to these species (see Fig. 2 and below).There are, however, no distinctive morphological orultrastructural characters that distinguish these taxa(for details, see Pinto et al. 2003).

It is intriguing that all of the samples from Site B in ourenvironmental survey were positioned in the Galdieria-Blineage (see Figs 2 and 3). The Galdieira-B population at SiteB grows beneath the surface of rocks (see Fig. 1B), wherethere is presumably a moderate humidity. Eight sequencesfrom Site C were also positioned in the Galdieria-B lineage.The Site C biomat grows in a relatively dry area betweenthe fissures of rocks that are exposed to sulphur emissions.These results suggest that Galdieria-B may be adaptedto dry conditions. Although we have no direct evidence forthis hypothesis, it is interesting that Galdieria sulphurariaDBV 002 differs markedly from studied members of theGaldieria-A clade (e.g. DBV 074, SAG 107–79 and SAG 107–89), in that it can exist at extreme salt concentrations (8–10% NaCl) and is unable to utilize nitrate (Pinto et al. 2003).Perhaps these traits are adaptations to its crypto-endolithichabitat. In general, ecophysiological studies show thatdesiccation tolerance is a restrictive factor for the distributionof the Cyanidiales (Gross 1999). Taken together, our resultssuggest that the Galdieria-B lineage may be adapted speci-fically to dry environments and dominates this ecophysio-logical niche in the crypto-endolithic site. Unlike theGaldieria-B lineages, the Galdieria-A strains were collectedat the hydrothermal areas (e.g. hot springs) and established

Fig. 4 Results of the environmental survey showing thedistribution of Cyanidiales species/lineages clones with respect tothe eco-physiological conditions at Pisciarelli, Italy. Up to 20clones were sampled from each site. In this figure, G-A is Galdieria-A, G-B is Galdieria-B, Cy is Cyanidium, Cz is Cyanidioschyzon andunid. is unidentified taxa.



in culture collections [i.e. DBV, IPPAS, SAG, UTEX (http://www.bio.utexas.edu/research/utex/)]. The finding ofGaldieria-B at only the Italian sites should not necessarilybe taken to indicate an endemic species, because we mayfind members of this group if we sample dry thermophilicenvironments in other regions of the world.

An interesting feature of the Galdieria-A lineage is theclear sorting of the lineages based on geographical origin.The taxa collected from Italy, the United States, Mexico,Russia (except Galdieria maxima, but see below) and Indo-nesia each form a distinct monophyletic clade in Fig. 3.This suggests that long-distance dispersal may be limitedin Galdieria-A, or alternatively that our sampling was notcomplete enough at the different sites to discover morerare migrants. Support for the first idea comes from theintriguing distribution pattern of the Cyanidiales. Thesealgae are scattered throughout the world; however, theirhabitat is locally restricted to thermal and acidic sites.Geographic isolation may therefore be expected among thesetaxa due to their inability to undergo long-distance migra-tion caused by reduced tolerance to desiccation andmesophilic conditions, such as low temperature and neutralpH (Brock 1978; Gross 1999). Combined with the apparentabsence of resting spores in Cyanidiales, members ofGaldieria-A may undergo rapid interpopulation divergenceculminating in some cases in speciation events. This would,of course, not hold for the mesophilic Cyanidium sp. (seebelow), about which we currently know very little, bothwith regard to phylogenetic breadth and distribution atother mesophilic sites. For the remaining acido-thermophilictaxa (i.e. Cyanidium caldarium, Cyanidioschyzon merolae,Galdieria-B and other species in this genus), the prevailingview is that these cells are dispersed infrequently via wind,streams and oceanic currents (see Discussion in Brock 1978;Gross et al. 2001).

Cyanidium caldarium and the mesophilic lineages

The Cyanidium caldarium lineage forms a strongly supportedgroup that is surprisingly broadly distributed. In starkcontrast to Galdieria-A, Cyanidium caldarium includes closelyrelated isolates from Indonesia (DBV 182, Java), Italy (DBV019, Siena; DBV 020, Acqua Santa; C2, Pisciarelli) andJapan (RK1), suggesting that these taxa have an effectivedispersal mechanism(s). Another intriguing result of ouranalysis of Cyanidium spp. is the identification of a novelmonophyletic lineage of mesophilic taxa. We collected thesemesophilic strains from the Sybil cave at Cuma and fromMonte Rotaro, where the habitats are nonacidic (pH 7.0–7.2) and nonthermal. The mesophilic Cyanidium sp. growson the bottom of the walls on the shaded side of the Sybilcave. Mesophilic Cyanidiales were first described bySchwabe as a new algal species, C. chilense, from a cave onthe Chilean coast (Schwabe 1936). Even though some of the

mesophilic isolates were reported with morphologicaland ecophysiological descriptions (Hoffmann 1994), theirsystematic position remained unknown. Our study is thefirst to demonstrate the phylogenetic relationship of meso-philic Cyanidium sp. within the Cyanidiales. The concaten-ated three-gene and rbcL trees both support a sisterrelationship of the mesophilic and extremophilic (Cyanidiumcaldarium) Cyanidium lineages and their distinct phylo-genetic position with regard to the acido-thermophilicGaldieria-A and -B lineages (see Figs 2 and 3). This result is,however, without ML bootstrap support in the three-genetree (Fig. 2).

In another study from our laboratory (Yoon et al. 2004),the 16S rRNA and the first and second codon positionsof five plastid genes (psaA, psaB, psbA, rbcL and tufA) wereanalysed in a combined data set of 5177 nt. This tree includedseven Cyanidiales representing the four lineages identifiedhere plus 14 non-Cyanidiales red algae, chromists, greenand glaucophyte algae for a taxonomically broad sampleof 46 plastid genomes. The trees inferred from this dataset robustly support (bootstrap with ME-GTR + I + Γ = 100%and unweighted maximum parsimony = 97%, posteriorprobability = 1.0) the monophyly of the mesophilic Cyanid-ium spp. and the existence of a super-clade comprised ofCyanidium caldarium RK1 and Cyanidioschyzon merolae DBV201 + Galdieria maxima. The Galdieria lineage is therefore theearliest divergence in the Cyanidiales in this analysis. Thislatter result is also found in Figs 2 and 3, albeit with poorersupport. We describe in some detail the results of thesix-gene analysis because the phylogenetic position of themesophilic Cyanidium spp. is a critical issue in Cyanidialesevolution. This group potentially holds the key for identi-fying the ancestral condition of both the Cyanidiales and ofall red algae. The results presented here, and in particularto those of Yoon et al. (2004) resolve this issue by providingsupport for the idea that mesophily is a derived characterin the ancestrally thermo-acidophilic Cyanidiales.

To assess alternative hypotheses about Cyanidialesevolution, we rearranged the position of the Cyanidialeslineages relative to each other in the three-gene and rbcLME-LogDet trees (Figs 2 and 3) and in the six-gene tree (treeof highest likelihood in the Bayesian posterior distribution)in Yoon et al. (2004) and compared the log-likelihoodsusing the one-sided KH-test. In these rearrangements, themesophilic taxa were moved to the branch uniting allthe red algae, the Cyanidiales or the Galdieria lineage (seeFigs 2 and 3). Results of the KH-test for these rearrange-ments are summarized in Table 2 and show that positioningthe mesophilic Cyanidium spp. at the base of the red algae isrejected with the three-gene and six-gene data sets, whereasa basal position in the Cyanidiales is strongly rejected bythe latter data set and has a low probability (albeit nonsig-nificant) in the three-gene data set (P = 0.074). Movementof the mesophilic clade to the base of the Galdieria lineage

1836 C . C I N I G L I A E T A L .


results in a significantly worse tree using the six-gene data.The rbcL data set does not significantly reject theserearrangements, but this presumably reflects the lowerresolving power in a single-gene analysis. Given the resultsof the KH-test, in particular using the most informative six-gene data set, and the bootstrap and Bayesian results for thethree-gene tree (Fig. 2), we suggest that the mesophilicCyanidium spp. are not a basal divergence in either the entirered algal clade or in the Cyanidiales. This is consistent withthe idea that mesophily is a derived condition in the ances-trally thermo-acidophilic Cyanidiales. Our data do notresolve, however, the issue of whether the first red algaewere mesophiles or extremophiles, although we currentlyfavour mesophily because the early divergences in thesister groups of the reds, the green and glaucophyte algae(e.g. Friedl et al. 2000; Moreira et al. 2000) are all mesophilic.This hypothesis could potentially be overturned if futurebiodiversity sampling shows the Cyanidiales to be para-phyletic with extremophilic members that branch at thebase of the mesophilic red algae.

Cyanidioschyzon and Galdieria maxima lineages

Cyanidioschyzon merolae is distinct from Cyanidium andGaldieria with regard to the following morphologicalcharacters: the cells of Cyanidioschyzon merolae have a moreelliptical shape, they lack cell walls or vacuoles and theydivide through binary fission rather than through endo-spores, as is the case for Cyanidium caldarium and Galdieriasulphuraria (Albertano et al. 2000). Our analyses showed,however, a sister group relationship between the morpho-logically distinct Cyanidioschyzon merolae and Galdieriamaxima. This result is consistent with previous reports (Gross

et al. 2001; Yoon et al. 2002b; Pinto et al. 2003), although theenigmatic relationship is hard to explain using traditionaltaxonomic criteria that rely on morphological and ecophy-siological characters. Galdieria maxima, for example, has alarger cell dimension (10–16 mm), is spherical in shapewith one multilobed plastid, contains several vacuoles andcell division occurs through the formation of sporangiawith 4–16 spores, rather than by binary fission as inCyanidioschyzon merolae. In addition, Galdieria maxima isable to live mixotrophically (i.e. the simultaneous capacityfor photosynthesis and for the uptake of particulate and/or dissolved organic compounds) and heterotrophically,unlike Cyanidioschyzon merolae which is autotrophic andrestricted to acido-thermophilic sites (Pinto et al. 2003).Results of the KH-test, however, robustly reject [P < 0.001(except for P = 0.024 with rbcL), see Table 2] the forcedmonophyly of the Russian Galdieria species (i.e. Galdieriadaedala, Galdieria maxima and Galdieria partita) or the posit-ioning of Galdieria maxima at the base of the Galdierialineage. Although our data argue convincingly for a distantphylogenetic relationship of Galdieria maxima to the otherRussian Galdieria species, an alternative explanation forour results (until now unsubstantiated) is that the Galdieriamaxima clone may represent a contaminant in the ‘typical’Galdieria maxima culture. Light microscopic analyses donot, however, show obvious cell size heterogeneity in thisIPPAS culture. Finally, one of the environmental sampleswe have identified (C16) is also a member of the Cyani-dioschyzon + Galdieria maxima lineage (Fig. 3), although wehave no morphological data for this taxon. In summary,our results potentially reveal the traditional systematicscheme to have grossly underestimated the biodiversityof Cyanidiales. More extensive sampling from different

Rearrangement Tree ∆ log-like. KH-prob.

ME-LogDet Tree (Fig. 2) Three-gene Best 1.000Meso. >> base of all reds Three-gene 79.25 < 0.001**Meso. >> base of Cyanid. Three-gene 21.57 0.074Meso. >> base of Galdi. Three-gene 21.23 0.065G. max. >> Russian Three-gene 715.69 < 0.001**G. max. >> base of Galdi. Three-gene 261.37 < 0.001**ME-LogDet Tree (Fig. 3) rbcL Best 1.000Meso. >> base of all reds rbcL 3.74 0.265Meso. >> base of Cyanid. rbcL 3.74 0.272Meso. >> base of Galdi. rbcL 3.90 0.260G. max. >> Russian rbcL 130.59 < 0.001**G. max. >> base of Galdi. rbcL 22.79 0.024*Best Bayesian Tree (from Yoon et al. unpubl.) Six-gene Best 1.000Meso. >> base of all reds Six-gene 109.72 < 0.001**Meso. >> base of Cyanid. Six-gene 68.83 < 0.001**Meso. >> base of Galdi. Six-gene 70.26 < 0.001**G. max. >> Russian Six-gene 677.89 < 0.001**G. max. >> base of Galdi. Six-gene 212.37 < 0.001**

Table 2 Results of the one-sided KH-test ofrearranged trees inferred from the plastidthree-gene, rbcL and six-gene data sets. Meso.,Galdi., Cyanid., G. max. and Russian are meso-philic Cyanidium spp., the Galdieria-A and -Bclades, Cyanidiales, Galdieria maxima, and theRussian isolates of Galdieria daedala andGaldieria partita, respectively. The asterisksindicate significance at *P < 0.050 and **P <0.010, ∆ log-like. indicates the differencein log-likelihood between the best andrearranged trees and >> denotes the move-ment of taxa to different positions in thetrees



natural habitats will be required to elucidate in greaterdetail the biodiversity and phylogeny of this group.

Environmental survey

The environmental PCR and the restriction enzymeapproach allowed us to survey efficiently the populationstructure of Cyanidiales, thereby leading to the identificationof novel lineages such as Galdieria-B and the establishmentof previously unrecognized species relationships. In thelatter case we found, for example, that Galdieria-A andCyanidioschyzon merolae coexist at all the humid sites (i.e.Sites A, D and E). Within the populations, Cyanidioschyzonmerolae was more abundant than Galdieria-A at the hydro-thermal habitats (e.g. 90% at Site A, 74% at Site D), whereasGaldieria-A dominated Site E (77%), where the sulphurfumes arose from the rocks. In stark contrast, Galdieria-Bpreferred relatively dry habitats (i.e. Sites B and C). Takentogether, the environmental survey appears to be a usefultool for ecophysiological and phylogenetic studies. Ourdemonstration of significant intraspecific variation in Cyani-diales shows the potential of this technique for studyinggenetic differentiation on a geographical scale probablydown to the level of within-population spatial structure.

Conclusions

In conclusion, we provide a robust phylogeny of theCyanidiales using a concatenated data set of three plastidgenes and an environmental PCR survey. We establish theexistence of at least four distinct Cyanidiales lineagesthat incorporate the three traditional genera. These arethe Galdieria spp. lineage, the Cyanidium caldarium lineage, themesophilic Cyanidium spp. lineage, and the Cyanidioschyzonmerolae plus Galdieria maxima lineage. Although we havemaintained the traditional species concepts in our analyses,inspection of the branch lengths that define within-lineagediversity suggest that the Cyanidiales could plausibly besplit into many more species than is currently the case.Within-lineage (often species) sequence divergence in theCyanidiales is comparable, for example, to between-orderdivergences in the non-Cyanidiales red algae [e.g. compareCompsopogon coeruleus (Compsopogonales) to Rhodochaeteparvula (Rhodochaetales)]. It is possible, however, that thehigh divergence rates in the Cyanidiales are explained byan elevated mutation rate in these taxa, resulting poten-tially from DNA damage in their extreme environments.Our analyses also reject the notion of a mesophilic ancestryof the Cyanidiales and suggest that this lineage wasancestrally thermo-acidotolerant. The environmental PCRsurvey was a powerful approach for discovering Cyani-diales biodiversity and was easy to implement with theseterrestrial algae. We predict that future analyses using thisapproach at many other, until now poorly sampled, extre-

mophilic sites will further broaden our understanding ofCyanidiales taxon diversity and phylogeny.

Acknowledgements

This work was supported by grants awarded to D. B. by theNational Science Foundation (DEB 01–07754, MCB 02–36631).This paper is dedicated respectfully to Wolfgang Gross, who wasan active member of the phycology and Cyanidiales community.

References

Albertano P, Ciniglia C, Pinto G, Pollio A (2000) The taxonomicposition of Cyanidium, Cyanidioschyzon and Galdieria: an update.Hydrobiologia, 433, 137–143.

Amaral Zettler LA, Gomez F, Zettler E et al. (2002) Microbiology:eukaryotic diversity in Spain’s River of Fire. Nature, 417, 137.

Brock TD (1978) The genus Cyanidium. In: Thermophilic Microorgan-isms and Life at High Temperatures (ed. Starr PM), pp. 255–301.Springer-Verlag, New York.

Brock TD (1997) The value of basic research: discovery of Thermusaquaticus and other extreme thermophiles. Genetics, 146,1207–1210.

Butterfield NJ (2000) Bangiomorpha pubescens n. gen., n. sp.: impli-cations for the evolution of sex, multicellularity, and the Meso-proterozoic/Neoproterozoic radiation of eukaryotes. Paleobiology,26, 386–404.

DeLong EF, Pace NR (2001) Environmental diversity of bacteriaand archaea. Systematic Biology, 50, 470–478.

Felsenstein J (1985) Confidence limits on phylogenies: an approachusing the bootstrap. Evolution, 39, 783–791.

Felsenstein J (2002) PHYLIP 3.6. Distributed by the author. Depart-ment of Genetics, University of Washington, Seattle.

Friedl T, Besendahl A, Pfeiffer P, Bhattacharya D (2000) The distri-bution of group I introns in lichen algae suggests that licheniza-tion facilitates intron lateral transfer. Molecular Phylogenetics andEvolution, 14, 342–352.

Gilbert DG (1995) SEQPUP, a Biological Sequence Editor and AnalysisProgram for the Macintosh Computer. Indiana University, Bloom-ington, IN.

Goldman N, Anderson JP, Rodrigo AG (2000) Likelihood-basedtests of topologies in phylogenetics. Systematic Biology, 49,652–670.

Gross W (1999) Revision of comparative traits for the acido- andthermophilic red algae Cyanidium and Galdieria. Kluwer AcademicPublishers, London.

Gross W, Heilmann I, Lenze D, Schnarrenberger C (2001)Biogeography of the Cyanidiaceae (Rhodophyta) based on 18Sribosomal RNA sequence data. European Journal of Phycology,36, 275–280.

Helmchen TA, Bhattacharya D, Melkonian M (1995) Analysesof ribosomal RNA sequences from glaucocystophyte cyanellesprovide new insights into the evolutionary relationships ofplastids. Journal of Molecular Evolution, 41, 203–210.

Hoffmann L (1994) Cyanidium-like algae from caves. In: Evolu-tionary Pathways and Enigmatic Algae: Cyanidium Caldarium(Rhodophyta) and Related Cells (ed. Seckbach J), pp. 175–182.Kluwer Academic Publishers, London.

Hough DW, Danson MJ (1999) Extremozymes. Current Opinion inChemical Biology, 3, 39–46.

1838 C . C I N I G L I A E T A L .


Huelsenbeck JP, Ronquist F (2001) mrbayes: Bayesian inference ofphylogenetic trees. Bioinformatics, 17, 754–755.

Kishino H, Hasegawa M (1989) Evaluation of the maximumlikelihood estimate of the evolutionary tree topologies from DNAsequence data, and the branching order in Hominoidea. Journalof Molecular Evolution, 29, 170–179.

Lopez-Garcia P, Rodriguez-Valera F, Pedros-Alio C, Moreira D(2001) Unexpected diversity of small eukaryotes in deep-seaAntarctic plankton. Nature, 409, 603–607.

Merola A, Castaldo R, Gambardella P, Musachio R, Taddei R(1981) Revision of Cyanidium caldarium: three species of acido-philic alage. Giornale Botanico Italiano, 115, 189–195.

Moon-van der Staay SY, De Wachter R, Vaulot D (2001) Oceanic18S rDNA sequences from picoplankton reveal unsuspectedeukaryotic diversity. Nature, 409, 607–610.

Moreira D, Le Guyader H, Phillippe H (2000) The origin of redalgae and the evolution of chloroplasts. Nature, 405, 69–72.

Müller KM, Oliveira MC, Sheath RG, Bhattacharya D (2001) Ribos-omal DNA phylogeny of the Bangiophycidae (Rhodophyta)and the origin of secondary plastids. American Journal of Botany,88, 1390–1400.

Ott FD, Seckbach J (1994) New classification for the genus Cyanid-ium Geitler 1933. In: Evolutionary Pathways and Enigmatic Algae:Cyanidium Caldarium (Rhodophyta) and Related Cells (ed. Seckbach J),pp. 145–152. Kluwer Academic Publishers, London.

Pinto G, Albertano P, Ciniglia C et al. (2003) Comparativeapproaches to the taxonomy of the genus Galdieria merola(Cyanidiales, Rhodophyta). Cryptogamie Algologie, 24, 13–32.

Rodriguez F, Oliver JL, Marin A, Medina JR (1990) The generalstochastic model of nucleotide substitution. Journal of TheoreticalBiology, 142, 485–501.

Rothschild LJ, Mancinelli RL (2001) Life in extreme environments.Nature, 409, 1092–1101.

Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002)tree-puzzle: maximum likelihood phylogenetic analysisusing quartets and parallel computing. Bioinformatics, 18,502–504.

Schwabe GH (1936) Über einige Blaualgen aus dem mittleren undsüdlichen Chile. Verhandlungen des Deutschen WissenschaftlichenVereins zu Santiago de Chile, 3, 113–174.

Sentsova OY (1991) Diversity of acido-thermophilic unicellularalgae of the genus Galdieria (Rhodophyta, Cyanidiophyceae).Botanicheskii Zhurnal, 76, 69–79.

Swofford DL (2002) PAUP*: Phylogenetic Analysis Using Parsimony(*and Other Methods) 4.0b8. Sinauer, Sunderland, MA.

Tamura K, Nei M (1993) Estimation of the number of nucleotidesubstitutions in the control region of mitochondrial DNA inhumans and chimpanzees. Molecular Biology and Evolution, 10,512–526.

Valentin K, Zetsche K (1990) Rubisco genes indicate a closephylogenetic relation between the plastids of Chromophyta andRhodophyta. Plant Molecular Biology, 15, 575–584.

Yoon HS, Hackett JD, Bhattacharya D (2002a) A single origin of theperidinin- and fucoxanthin-containing plastids in dinoflagellatesthrough tertiary endosymbiosis. Proceedings of the NationalAcademy of SciencesUSA, 99, 11724–11729.

Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D, (2004)A molecular timeline for the origin of photosynthetic eukaryotes.Molecular Biology and Evolution, 21, 809–818.

Yoon HS, Hackett JD, Pinto G, Bhattacharya D (2002b) The single,ancient origin of chromist plastids. Proceedings of the NationalAcademy of Sciences USA, 99, 15507–15512.

hidden biodiversity of the extremophilic cyanidiales red...

Documents