BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

8/9/2019 BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

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587

J. Phycol.35, 587598 (1999)

BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLASENSU LATO (CHLOROPHYTA)1

Volker A. R. Huss,2 Carola Frank, Elke C. Hartmann, Monika Hirmer, Annette Kloboucek,Barbara M. Seidel, Petra Wenzeler, and Erich Kessler

Institut fur Botanik und Pharmazeutische Biologie der Universitat, Staudtstrasse 5, D-91058 Erlangen, Germany

A multimethod approach was used to characterizeunicellular green algae that were traditionally as-signed to the genus ChlorellaBeijerinck and to re-solve their phylogenetic relationships within theChlorophyta. Biochemical, physiological, and ultra-structural characters, together with molecular datasuch as DNA base composition and DNA hybridiza-tion values, were compared with a molecular phy-logeny based on complete 18S rRNA sequences.Our results show thatChlorella taxa are dispersedover two classes of chlorophytes, the Trebouxiophy-ceae and the Chlorophyceae. We propose that only

four species should be kept in the genus Chlorella(Chlorophyta, Trebouxiophyceae): C. vulgaris Beijer-inck, C. lobophoraAndreyeva,C. sorokinianaShih. etKrauss, and C. kessleri Fott et Novakova. Commoncharacteristics of these taxa are glucosamine as adominant cell wall component and the presence ofa double thylakoid bisecting the pyrenoid matrix.Norspermine, norspermidine, and secondary carot-enoids are never produced. Other Chlorella spe-cies belong to different taxa within the Trebouxio-

phyceae (C. protothecoides Auxenochlorella proto-thecoides [Kruger] Kalina et Puncocharova, C. el-lipsoidea, C. mirabilis, C. saccharophila,and C.luteoviridis) and Chlorophyceae (C. zofingiensisand

C. homosphaera Mychonastes homosphaeraKalinaet Puncocharova). The latter taxa can easily be rec-ognized by the production of secondary carotenoidsunder nitrogen-deficient conditions.

Key index words: 18S rRNA; chemotaxonomy;Chlo-rella; Chlorophyta; DNA base composition; DNA/DNA hybridization; molecular systematics;Muriella;

phylogeny; Prototheca; Scenedesmus

Members of the genus Chlorella Beijerinck areamong the best-studied unicellular green algae.Since Warburg (1919) introduced mass cultures of

Chlorellafor basic research, these algae have servedas model organisms for pioneering plant physiolog-ical and biochemical studies of, for example, pho-tosynthesis and nitrate reduction (Warburg and Ne-gelein 1920, Pirson 1937, Kessler 1953, Falkowskiand Raven 1997). In agriculture and biotechnology,they are extensively used in some countries for feeds

1 Received 17 June 1998. Accepted 15 January 1999.2Author for reprint requests; e-mail [email protected]

erlangen.de.

(single cell protein) or for human consumption asprotein-rich health food, in waste treatment and

water recovery, as a gas exchange system, and formicrobial energy production (Golueke and Oswald1964, Fogg 1971, Soeder 1976, 1980, Abbott andCheney 1982 and references therein, Dunahay et al.1992).Chlorellaextracts have even been ascribed topossess antitumor (Konishi et al. 1985, Miyazawa etal. 1988) and antimicrobial effectiveness (Pratt andSpoehr 1944, Tanaka et al. 1986).

The lack of obvious morphological characterscombined with an exclusively asexual reproductive

cycle by means of autospores has caused consider-able problems in the taxonomic description andidentification ofChlorellaspecies (Kessler and Huss1992). However, the choice of suitable strains orspecies is crucial for many of the previously men-tioned applications (Kessler 1986, 1992). In thepast, numerous methods have been applied to thetaxonomy ofChlorella. Many of them were based onnutritional requirements of Chlorella strains whencultivated under autotrophic or heterotrophic con-ditions (Shrift and Sproul 1963, Shihira and Krauss1965). Also, numerical classification strategies havebeen applied for the evaluation of taxonomic con-clusions (Cullimore 1969, DaSilva and Gyllenberg1972). In their monograph of the genus Chlorella,Fott and Novakova (1969) combined morphologicaland structural features with some physiological char-acteristics, resulting in the description of nine spe-cies and six varieties. Others have used serologicalcross-reactions (Sanders et al. 1971, Maruyama 1977,Kummel and Kessler 1980) for the identificationand classification of Chlorella strains. A third cate-gorization has been based on the ultrastructure andchemical composition of the cell wall (Atkinson etal. 1972, Conte and Pore 1973, Yamada and Saka-guchi 1982, Blumreisinger et al. 1983, Takeda 1991,1996a) and pyrenoid ultrastructure (Ikeda and Tak-

eda 1995). The most acknowledged and practicablesystem for species delimitation inChlorellaproved tobe a chemotaxonomical classification scheme com-bining biochemical and physiological characters(Kessler and Soeder 1962, Kessler 1982, 1984,1992). This work led to the characterization of 19taxa, some of which exhibited striking differences(Table 1). In contrast, other important genera ofgreen algae, such as Scenedesmusand Ankistrodesmus/Monoraphidium, appeared physiologically and bio-chemically much more uniform (Hellmann and Kes-


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588 VOLKER A. R. HUSS ET AL.

TABLE1. Biochemical and physiological characters of 19Chlorellataxa (including three taxa belonging to the genusScenedesmus). Hydr. hydrogenase, Sec. car. secondary carotenoids, NO nitrate reduction, Thiam. thamine requirement, B12 vitamin B12require-3ment, Mann. growth on mannitol, pH limit of growth at pH, %NaCl limit of growth, C upper limit of growth, %GC mol%GC of the DNA.

Species

No.of

Strains Hydr.Sec.car. NO3 Thiam. B12 Mann. pH

%NaCl C %GC

C. vulgaris

C. lobophoraC. sorokiniana

C. spp. (paramecii)C. spp. 21118C. kessleri

20

117

31

10

()

3.54.5

4.03.55.0

5.55.0

2.53.0

34

113

11

12

2832

303642

263028

3436

5863

616268, 7375

6667515457

C. minutissima 2 5.5 1 32 46C. protothecoides( Auxenochlorella

protothecoides)C.ellipsoideaC.mirabilisC.luteoviridisC.saccharophilaC.saccharophila211-9b

( Watanabea reniformis)

16

23661

3.54.0

2.03.04.03.0

2.03.03.0

34

211

3546

3

2834

28302628

282630

26

5862

565756584445495249

C.homosphaera

( Mychonastes homosphaera)C.zofingiensisC.zofingiensisC-1.2.1 ( SAG 4.80)

(Muriella aurantiaca)

1

31

6.0

5.05.54.5

1

11

28

2828

73

5063

C.fuscavar. fusca( Scenedesmus abundans)

1 4.0 2 3455

C.fuscavar. rubescens( Scenedesmus rubescens)

1 4.5 3 30 57

C.fuscavar. vacuolata( Scenedesmus vacuolatus)

11 3.03.5 3 3236 5052

sler 1974b, Kessler 1982; Kessler et al. 1997). Theseresults suggested substantial heterogeneity within

the genus Chlorella.Comparative studies of nucleic acids are indis-pensable for tracing the phylogenetic relationshipsof these algae. Previous work on the DNA base com-position revealed an amazingly broad range (44%75%) of the molar guanosine cytidine (GC) con-tent within the genusChlorella(Fig. 1, Table 1). Thisheterogeneity was subsequently confirmed by exten-sive quantitative DNA/DNA hybridization studies(Kerfin and Kessler 1978, Huss et al. 1986, 1987a,b, 1988, 1989a, b). Under optimal DNA reasso-ciation conditions, no significant DNA hybridizationcould be detected between any species and, in somecases, not between strains of a single species. Ex-

tended studies using relaxed reassociation con-ditions (Huss et al. 1989a) revealed an interspecificrelationship between C. vulgarisand C. sorokiniana,a species formerly called C. vulgaris forma tertiabyFott and Novakova (1969). Both species appear mor-phologically identical, and C. sorokiniana could beseparated from C. vulgarisonly by its possession ofhydrogenase activity and thermophily (Table 1; Kes-sler 1982). More surprisingly, a similar close rela-tionship could be shown for the three varieties ofC.fusca and species of the morphologically different

genus Scenedesmus. A possible relationship betweenC. fuscaand Scenedesmushas been proposed before

on evidence of similarity of sterols (Patterson 1974),ribosomal proteins (Gotz and Arnold 1980a, b), andcytochrome c-553 (Kummel and Kessler 1980). Onthe basis of submicroscopical structures of the cell

wall, Fott et al. (1975) recognized onlyC. fuscavar.fuscaas a unicellular member of the genus Scenedes-mus(later described asS. abundansby Hegewald andSchnepf 1991), whereas such a relation was not ac-knowledged for the varieties vacuolataand rubescens.

The resolution of DNA/DNA reassociation stud-ies allows only the detection of closely related spe-cies of a given genus (cf. Schleifer and Stackebrandt1983). To reveal the relationships between all Chlo-rellaspecies and to determine their phylogenetic po-

sition within the Chlorophyta, comparative se-quence analyses of conserved genes, such as the 18Sribosomal RNA genes, are especially useful. Previous

work based on that gene supported the proposedheterogeneity of the genus Chlorella(Huss and So-gin 1990) and showed that the three former varie-ties of C. fuscaare different species of the genusScenedesmus(Kessler et al. 1997). Here we present acomprehensive 18S rRNA based phylogeny of thegenus Chlorella sensu lato, including species ofPro-totheca, Muriella,and Scenedesmus.


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589CHLORELLA PHYLOGENY

FIG. 1. Summary of the nuclear DNAbase composition in the genusChlorellasen-su lato. The bars represent the range of mo-lar guanosine cytidine (GC) contents de-termined for the number of strains shownin brackets. Modified after Huss and Jahnke(1994).

MATERIALS AND METHODS

All organisms used in this investigation, their strain numbers,origins, and the GenBank/EMBL accession numbers for their 18SrRNA gene sequences are listed in Table 2. The sequences of 17strains ofChlorellaand related taxa that were determined in thisstudy are indicated by an asterisk. Other organisms taken for ref-erence were chosen to show the relationships ofChlorellaspeciesto other genera in the Trebouxiophyceae/Chlorophyceae. Theulvophycean algaeUlothrix zonataandGloeotilopsis planctonicawereused as outgroups in the phylogenetic analyses.

Mass culturing of algae, DNA isolation and purification, anddetermination of the DNA base composition (GC content) have

been described previously (Huss et al. 1986). DNA reassociationkinetics for the quantitative determination of DNA similarities

were determined optically in a UV/VIS spectrophotometer (Gil-ford Response) equipped with a thermoprogrammer as de-scribed by Huss et al. (1987b). Nucleic acid similarities of heter-ologous DNAs, expressed as degree of binding (%D), were cal-culated using the equation of De Ley et al. (1970). Mean values

were taken from at least three independent determinations of thethermal melting points and DNA similarities.

The 18S rRNA genes were amplified from total genomic DNAor from isolated nuclear DNA (Huss et al. 1988) by the polymer-ase chain reaction (PCR) as described by Huss and Sogin (1990)

with eukaryote-specific synthetic oligonucleotide amplificationprimers (Table 3). The amplified DNA fragments were eithercloned into bacteriophages M13mp18 and M13mp19 (Medlin etal. 1988) or directly sequenced taking advantage of the Dyna-beads-280 streptavidin system (Hultman et al. 1991). Sequences

of the coding and noncoding strand were determined by the di-deoxynucleotide chain-terminating sequencing method (Sangeret al. 1977) with oligonucleotide primers that are complementaryto evolutionary conserved regions of the 18S rRNA gene (Table3).

All sequences used in this work were manually aligned on aMicroVAX computer with the sequence editor program distrib-uted by G. Olsen (Olsen et al. 1992). For the phylogenetic ana-lyses, the sequences for organisms listed in Table 2 were extractedfrom a larger alignment that included about 250 sequences ofgreen algae as well as several sequences from land plants andother groups of organisms. To improve the alignment of the dataset, secondary structure models that combined features of the

models proposed by Huss and Sogin (1990) and Neefs and DeWachter (1990) were constructed for all sequences. Highly vari-able regions that could not be aligned unambiguously for all se-quences used were excluded from the analyses, resulting in a totalof 1751 positions. The alignment is available from the authors onrequest.

Phylogenetic trees were inferred from the aligned sequencedata by the neighbor-joining (NJ), the maximum parsimony(MP), and the maximum likelihood (ML) method. Neighbor-

joining and MP bootstrap analyses (Felsenstein 1985) were con-ducted with the PHYLIP package 3.572c of Felsenstein (1995) ona Silicon Graphics Indy computer. For the NJ analysis (Saitou and

Nei 1987), the correction of Kimura (1980) was used to convertpairwise sequence similarities into evolutionary distances, the ad-dition of taxa was jumbled, and a transition/transversion ratio of2.0 was selected. The same ratio was used for the MP analysis withrandom addition of taxa repeated three times for each individualbootstrap replication. A total of 1000 bootstrap resamplings wasused for each method.

For the ML analyses, the fastDNAml program of Olsen et al.(1994) was used to infer the tree topology shown in Figure 2.This topology has the largest Ln likelihood that could be achievedin 10 independent analyses using the generalized two-parametermodel of evolution (Kishino and Hasegawa 1989), empirical basefrequencies, and random addition of taxa. In addition, 100 boot-strap replications were carried out with a varying input order oftaxa until the best Ln likelihood score was reached three timesin a maximum of eight independent searches within each boot-strap replication.

RESULTS

Table 1 shows a chemotaxonomic classificationsystem that allows assignment of unidentified strainsofChlorella-like algae to the indicated taxa. Data aregathered from the work of Kessler (cf. Kessler andHuss 1992) with new information added. The ad-

vantage of this system is that most of the biochem-ical and physiological characters considered can eas-ily be determined without special equipment. In ad-dition to the DNA base compositions of more than


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TABLE2. List of organisms used in the analyses, their origin, and their GenBank/EMBL accession numbers for 18S rRNA gene sequenc-es. ALCP LAlgotheque, Laboratoire de Cryptogamie, Paris; Andreyeva V.M. Andreyeva, St. Petersburg, Russia; Baslerova M.Baslerova, Praha, Czech Republic; Bethesda Culture Collection at Bethesda, Maryland, USA; CCAP Culture Centre (now Collection)of Algae and Protozoa, Cambridge (now Ambleside), UK; CCHU Culture Collection Hiroshima University; CBS Carolina BiologicalSupply; IB Culture Collection of the Botanical Institute at Innsbruck, Austria; Pore R.S. Pore, Morgantown, West Virginia, USA;SAG Sammlung von Algenkulturen der Universitat Gottingen, Germany (Schlosser 1994); UTEX University of Texas Culture Col-lection (Starr and Zeikus 1993).

Taxonomicposition Species Strain

GenBank/EMBL

acc. no.Refer-encea

Chlorophyta

Chlorophyceaeb Ankistrodesmus stipitatus (Chodat) Komarkova-LegnerovaCharacium hindakiiLee et BoldChlamydomonas humicolaLuckschChlamydomonas reinhardtiiDangeardChlorella homosphaeraSkuja ( Mychonastes homosphaera[Skuja]

Kalina et Puncocharova)

SAG 202-5UTEX 2098c

UTEX 225UnknownCCAP 211/8ec

X56100M63000U13984M32703X73996

ABCD*

Chlorella minutissima Fott et Novakova ( Mychonastes homos-phaera[Skuja] Kalina et Puncocharova)

Lefevre ALCP no. 87 Y13761 *

Chlorella zofingiensisDonzChlorella zofingiensisDonz ( Muriella aurantiacaVischer)

SAG 211-14c

Bethesda C-1.2.1X74004X74005

**

Hydrodictyon reticulatum (Linne) LagerheimMuriella aurantiacaVischerNeochloris aquaticaStarrPediastrum duplexMeyen

Scenedesmus abundans(Kirchn.) Chod. ( Chlorella fuscavar. fus-ca Shih. et Krauss)

CBSSAG 249-1c

UTEX 138Isolated by L.W.W.

UTEX 343

M74497X91268M62861M62997

X73995

E*BB

F

Scenedesmus costato-granulatusSkujaScenedesmus obliquus(Turpin) KutzingScenedesmus producto-capitatusSchmulaScenedesmus rubescens(Dangeard) Kessler et al. ( Chlorella fusca

var.rubescens [Dangeard] Kessler et al.

SAG 18.81SAG 276-3aSAG 21.81CCAP 232/1c

X91265X56103X91266X74002

FAFF

Scenedesmus vacuolatus(Shih. et Krauss) Kessler et al. ( Chlorel-la fuscavar. vacuolata Shih. et Krauss)

SAG 211-8bc X56104 F

Spermatozopsis similisPreisig et MelkonianVolvox carterif. nagariensis

SAG 1.85UTEX 1885

X65557X53904

GH

Trebouxiophyceae b Chlorella ellipsoideaGerneckChlorella kessleriFott et NovakovaChlorella lobophoraAndreyevaChlorella luteoviridisChodatChlorella minutissimaFott et NovakovaChlorella mirabilisAndreyevaChlorella protothecoidesKruger ( Auxenochlorella protothecoides

[Kruger] Kalina et Puncocharova)

SAG 211-1ac

SAG 211-11gc

Andreyeva 750-Ic

SAG 211-2ac

Bethesda C-1.1.9Andreyeva 748-Ic

SAG 211-7ac

X63520X56105X63504X73997X56102X74000X56101

*A**

A*

A

Chlorella saccharophila(Kruger) MigulaChlorella saccharophila(Kruger) Migula ( Watanabea reniformis

Hanagata et al.)

SAG 211-9ac

SAG 211-9bX63505X73991

**

Chlorella sorokinianaShihira et Krauss

Chlorellaspp.Chlorella vulgarisBeijerinckDictyochloropsis reticulata(Tschermak-Woess) Tschermak-Woess

SAG 211-8kc

SAG 211-40aBaslerova Prag A14SAG 211-18SAG 211-11bc

CCHU 5616

X62441X73993X74001X73992X13688Z47207

****

AI

Leptosira terrestris(Fritsch et John) FriedlMyrmecia bisectaReisiglNanochlorum eucaryotumMenzel et WildPrototheca wickerhamiiTubaki et Soneda

Prototheca zopfiiKrugerTrebouxia impressaAhmadjian

SAG 463-3IB T74SAG 55.87SAG 263-11Pore 1283

SAG 263-1UTEX 892

Z28973Z47209X06425X74003X56099

X63519Z21551

J, KIL*

A

*J

Ulvophyceae Gloeotilopsis planctonicaIyengar et PhiliposeUlothrix zonata(Weber et Mohr) Kutzing

SAG 29.93SAG 38.86

Z28970Z47999

JK

a *: This work; A: Huss and Sogin (1990); B: Lewis et al. (1992); C: Gordon et al. (1995); D: Gunderson et al. (1987); E: Wilcox et al.(1992); F: Kessler et al. (1997); G: Sensen et al. (1992); H: Rausch et al. (1989); I: Friedl (1995); J: Friedl and Zeltner (1994); K: Friedl(1996); L: Sargent et al. (1988).

b sensuFriedl (1995).c Type species.


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TABLE 3. Oligonucleotide primers used for PCR and sequencing reactions of SSU rRNA genes of green algae.

Primer name Annealing sitea Sequenceb Reference

PCR primers

5-PCRc

5-PCRe

3-PCRc

3-PCRe

121121

1774179817741798

CCGAATTCGTCGACAACCTGGTTGATCCTGCCAGTd

WACCTGGTTGATCCTGCCAGTf

CCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACd

GATCCTTCYGCAGGTTCACCTAC f

Medlin et al. 1988This workMedlin et al. 1988This work

Sequencing primersg

M13 Universal1

82300528690920960

105512001400

42084100

371387576591899913

1132114811871202126912831428144316291645

GTAAAACGACGGCCAGT

CTGGTTGATCCTGCCAGh

GAAACTGCGAATGGCTC

AGGGTTCGATTCCGGAG

CGGTAATTCCAGCTCC

YAGAGGTGAAATTCT

GAAACTTAAAKGAATTG

TTTGACTCAACACGGG

GGTGGTGCATGGCCG

CAGGTCTGTGATGCCC

TGYACACACCGCCCGTC

Pharmacia, USBGunderson et al. 1986M.L. Sogin, pers. comm.Elwood et al. 1985Gunderson et al. 1986Elwood et al. 1985Elwood et al. 1985Gunderson et al. 1986M.L. Sogin, pers. comm.Modified after Gunderson et al. 1986Elwood et al. 1985

108300536690

92010551200140014901520

11096398382584567913899

114611321283126914431428164416301767174917891774

CTGATTTAATGAGCC

TCAGGCTCCCTCTCCGG

GWATTACCGCGGCKGCTG

AGAATTTCACCTCTG

ATTCCTTTRAGTTTC

CGGCCATGCACCACC

GGGCATCACAGACCTG

ACGGGCGGTGTGTRC

CTTGTTACGACTTCTCCh

CYGCAGGTTCACCTACh

Modified after Gunderson et al. 1986Elwood et al. 1985Gunderson et al. 1986Elwood et al. 1985

Elwood et al. 1985Elwood et al. 1985Gunderson et al. 1986Elwood et al. 1985This workGunderson et al. 1986

aAnnealing positions refer to the SSU rRNA sequence ofChlorella vulgaris(Huss and Sogin 1989).bAmbiguous nucleotides are abbreviated according to the IUB-standard: K G/T, R A/G, W A/T, Y C/T.c PCR primers used for ligating PCR-fragments into M13 vectors.d Primer contains additional restriction sites at the 5 -end to facilitate ligation into M13 vectors.e PCR primers used for the Dynabeads-280 streptavidin sequencing system.f Primer is biotinylated at the 5-end to allow binding of strepavidin covalently linked to the surface of the Dynabeads.g Designation of the sequencing primers refer to approximate annealing sites ofEscherichia coliSSU rRNA. Forward () primers are

complementary to the coding, and reverse () primers to the noncoding DNA strand.h Optional sequencing primers close to the 5- or 3-end of the SSU rRNA gene that were not routinely used.

100 Chlorellastrains indicated in Table 1 and sum-marized in Figure 1, we determined the nuclear GCcontent from Chlorella homosphaera CCAP 211/8eand C. minutissima Lefevre ALCP 87 as 72.0 mol%each and fromMuriella aurantiacaSAG 249-1 as 64.1mol%.

We have reconfirmed DNA hybridization betweenPrototheca wickerhamii strains SAG 263-11 and Pore1283 and determined a DNA similarity of 83%D 3.5%D (mean SD, n 12). This value indicatesconspecificity of both strains and is consistent withthe finding that the 18S rRNA genes of both strainsare identical. The DNA similarity of only 6%D er-

roneously reported earlier for the same strains(Huss et al. 1988) was probably caused by confusionof DNA samples.

A partial 18S rRNA gene sequence of 1164 nucle-otides from Chlorella minutissima strain Lefevre thatincluded the most variable regions differed by onlyone nucleotide compared with the type strain CCAP211/8e of C. homosphaera. The DNA similarity be-tween both strains was determined as 90.2%D 14.0%D (mean SD, n 8), thus confirming con-specificity. As a control, 28.8%D 13.1%D (mean

SD, n 8) were obtained betweenC. minutissimastrain Lefevre and C. sorokinianaPrag A14, whichhas a similar GC content.

The phylogenetic tree in Figure 2 contains theinformation of four independent analyses (see leg-end to Fig. 2). It demonstrates the problematic stateof Chlorella taxonomy. Chlorella species are distrib-uted over two classes of green algae: the Treboux-iophyceae and the Chlorophyceae. Each class is sup-ported by high bootstrap values, confirming that theTrebouxiophyceae are a sister class to the Chloro-phyceae (Friedl 1995). The type species ofChlorella,C. vulgaris, belongs to the Trebouxiophyceae and is

closely associated with C. lobophora, C. sorokiniana,andC. kessleri. The next most closely related clusterscontain C. protothecoidestogether with species of thegenus Prototheca, and C. minutissima together withNanochlorum eucaryotum.However, the branching or-der of these groups could not be resolved.

The heterotrophic genusPrototheca(includingC.protothecoides) is characterized by an accelerated mu-tation rate of its 18S rRNA gene. This is most obvi-ous for P. zopfiiwith its unusually long branch. TheP. zopfii sequence contains several base substitutions


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FIG. 2. Phylogenetic tree inferred from 18S rRNA gene sequences showing the polyphyly of the genusChlorellawithin the Treboux-iophyceae/Chlorophyceae. Taxa that were traditionally assigned to Chlorellaare circled. Sequences determined in this study are typed inboldface. The tree topology is derived from a maximum likelihood analysis (based on 1751 positions) that yielded the best Ln likelihood(11625.86) in 10 independent searches with jumbled taxon addition. Bootstrap values are shown at the internal nodes for maximumlikelihood (ML; 100 replications), neighbor joining (NJ; 1000 replications), and maximum parsimony (MP; 1000 replications), respec-tively, if the node is supported by at least two bootstrap values of 50% or above. Branch lengths correspond to evolutionary distances. Adistance of 0.01 is indicated by the scale.


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at positions that are otherwise conserved within thegreen algae, and the 18S rRNA gene ofC. protothe-coidescontains a unique insertion in the variable he-lix E21-1 (Huss and Sogin 1990, Huss et al. 1994).

A second group within the Trebouxiophyceae issupported only by very low bootstrap values of 45%to 59% and might actually consist of several inde-

pendent lineages. It contains zoospore-forming coc-coid green algae that are often found as phycobiontsof lichens, such as species ofTrebouxiade Puymaly,Myrmecia Printz, and DictyochloropsisGeitler (Friedl1995). SeveralChlorellaspecies fall within this group,and some of them are also known to be symbionts.Strain HHG ofC. saccharophila (Huss et al. 1987b)is an endosymbiont of Heterostegina depressa (Fora-minifera; Lee et al. 1982), and strain SAG 3.80 ofC.ellipsoidea (Kessler 1987) was isolated from the li-chen Trapelia coarctata(Tschermak-Woess 1978). Aclose relationship of C. ellipsoidea with C. mirabilisand ofC. saccharophilawith C. luteoviridis is support-ed by high bootstrap values. In contrast, strain SAG

211-9b of C. saccharophilais more distinct and takesa position ancestral to both C. saccharophilaand C.luteoviridis.

The remainingChlorellaspecies studied belong toa different class, the Chlorophyceae (Fig. 2). Thisclass comprises several independent lineages whoserelationships cannot be resolved reliably by the 18SrRNA data. One of these lineages is represented bythe Chlamydomonadales (sensu Melkonian 1990).The genus Scenedesmus represents another lineagethat contains the former varieties of Chlorella fusca,namely, var. vacuolata ( Scenedesmus vacuolatus),

var. fusca ( S. abundans), and var. rubescens ( S.rubescens) (Kessler et al. 1997). Other lineages in-

clude the Hydrodictyaceae plus Characiaceae, theAnkistrodesmaceae represented by Ankistrodesmusstipitatus; C. homosphaeratogether with strain LefevreofC. minutissima;and C. zofingiensis.However, strainC-1.2.1 ofC. zofingiensis is conspecific with Muriellaaurantiaca, and both algae form an unresolved lin-eage within the Chlorophyceae as well.

The taxonomic conclusions that follow from thisand a similar study by Hanagata and Chihara (1997;Hanagata, pers. comm.) are being considered byHanagata et al. (pers. comm.).

DISCUSSION

Tracing the evolutionary history of a group of or-

ganisms with such few morphological and ultrastruc-tural features as those found in the genus Chlorellais not feasible on the basis of morphological criteriaalone. Therefore, we combined several phenotypicand genotypic features to characterizeChlorellataxa

with respect to their interspecific relationships. Al-though the phenotypic characters listed in Table 1are not sufficient alone for deriving phylogenetic re-lationships, it is possible to deduce their phyloge-netic significance by comparing these data with themolecular phylogeny. Whenever necessary and ap-

plicable, such a strategy could solve a problem thatarose when molecular trees took an increasingly pre-dominant role in systematics: We frequently are con-fronted with the fact that the molecular and theclassical morphology-based systematics are notcongruent for a given group of organisms. The iden-tification of phylogenetically significant phenotypic

characters and their application to the systematicsof such critical groups might allow the assignmentof strains into a natural system without the need fordetermining a molecular sequence. For the genusChlorella, this was clearly not possible before. In ad-dition to its systematic value, the knowledge of bio-chemical and physiological properties might be ofpractical importance in basic research or biotech-nology (Kessler 1986, 1992).

Concerning the molecular part of such an ap-proach, several methods are available with differingresolution that can be used for different taxonomiclevels. From the methods that we have applied tothe systematics of the genus Chlorella, the determi-

nation of the DNA base composition is indicative ofthe heterogeneity of the organisms studied. Asshown in Figure 1, the nuclear base composition

within the genusChlorellaextends over a wide range,covering almost the entire GC spectrum found ineukaryotes. This criterion alone shows thatChlorellain its present definition cannot represent a naturalgenus. De Ley (1969) has estimated that prokaryotesseparated by 16 mol% GC can share at most 4% oftheir nucleotide sequences. The situation is morecomplicated with eukaryotes, as the different quan-tity of repetitive sequences could have an influenceon the nuclear base composition. Likewise, DNAbase modifications can influence the thermal melt-

ing point of DNA, which is obtained for calculatingthe GC content (cf. Ehrlich et al. 1975, Rae andSteele 1978). However, both factors are likely notresponsible for the observed heterogeneity in basecomposition in Chlorella because large differences

were observed neither in the amount of repetitivesequences nor in DNA base modification (Dorr andHuss 1990, Huss and Jahnke 1994).

It is especially intriguing that within strains of asingle species, C. sorokiniana, the DNA base com-position varies by up to 15 mol% GC. Despite thesedifferences and despite some heterogeneity also inbiochemical and physiological properties (Table 1),our rRNA sequence analyses in Figure 2 as well as

DNA/DNA hybridization studies (Huss et al. 1986,1989a) show that the different strains ofC. sorokini-anawith RNA homologies of 99.5% to 99.8% haveto be regarded at least as closely related species.Such a large discrepancy between genomic basecomposition and rRNA similarity is unprecedentedas far as we know. An extreme bias in the codonusage of the different strains could provide an ex-planation but has not yet been studied.

Although DNA/DNA hybridizations, in contrastto sequence comparisons, are of only semiquantita-


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tive value, they provide a superior method for delim-iting species and identifying closely related species

within a genus. In a comparison of 16S rRNA ho-mologies and respective DNA hybridization values inprokaryotes, several groups of organisms could beidentified that share almost identical 16S rRNA se-quences but in which DNA hybridization may be as

low as 25%, thus indicating that they represent in-dividual species (Stackebrandt and Goebel 1994).Above an RNA homology of about 97.5%, DNA re-association values can either be low or as high as100%. Each method is strong in those areas of re-lationships in which the other method fails to depictsuch relationships reliably. On the basis of our ex-tensive DNA hybridization studies in the genusChlo-rella(Huss et al. 1986, 1987a, b, 1988, 1989a, b), itis therefore unnecessary to determine rRNA se-quences of strains with more than about 40% DNAsimilarity to a reference strain.

Since the publication of the first 18S rRNA treethat included Chlorella species (Huss and Sogin

1990), numerous sequences of different chloro-phyte lineages have become available, allowing ob-servation of the heterogeneity of these algae in amuch broader phylogenetic context. On the basis ofrRNA sequence analyses, Friedl (1995) establishedthe new class Trebouxiophyceae as a sister group tothe Chlorophyceae. Chlorella species are scatteredover both classes with the type species C. vulgaristogether with the most closely related taxa, C. lobo-phora, C. sorokiniana,and C. kessleri,belonging to theTrebouxiophyceae. (C. pyrenoidosa[Chick 1903],a taxon often mentioned in the literature, could notbe recognized according to morphological charac-ters [Shihira and Krauss 1965, Fott and Novakova

1969]. Most of the strains so labeled seem to belongto C. fusca,which was assigned to the genus Sce-nedesmus[Kessler et al. 1997].) According to the mo-lecular data it seems appropriate to restrict the ge-nus Chlorella to these species. C. protothecoides(Auxenochlorella protothecoides[Kruger] Kalina et Pun-cocharova) is more closely related to the heterotro-phic genus Protothecaand already displays a consid-erable evolutionary distance toC. vulgaris.However,it must be taken into account that the long branchesleading to C. protothecoidesand especiallyP. zopfiiindicate a much higher mutation rate of their 18SrDNAs compared to the other algae (cf. Huss andSogin 1990). This tachytelic behavior complicates es-

timating the time that passed after these algae sep-arated from the C. vulgarisgroup and obscures thereal relationships. The same applies for the relation-ship between C. protothecoides and Prototheca. Al-though the rRNA tree indicates a closer relationshipof C. protothecoideswith P. zopfiiinstead of cluster-ing the twoPrototheca species, the bootstrap supportis relatively poor, and long branch attraction (Fel-senstein 1988) could be responsible for this topol-ogy. With respect to the complete loss of autotrophyin the genus Prototheca, it would be more parsimo-

nious if C. protothecoides,which is auxotrophic andmesotrophic (Table 1), were ancestral to both Pro-totheca species. Then, the ability to synthesize chlo-rophyll could have been lost only once, whereas oth-erwise the reappearance of autotrophy has to bepostulated for C. protothecoides, or a twofold inde-pendent loss for P. wickerhamii and P. zopfii must

have occurred.The microalgae C. minutissima and Nanochlorumeucaryotum,which are placed in our phylogeny intoa moderately supported common cluster, are char-acterized by their small cell size of about 2 m (Fottand Novakova 1969, Wilhelm et al. 1982) and by agenome size that is among the smallest found so far

within eukaryotes (Wilhelm et al. 1982, Zahn 1984,Dorr and Huss 1990). The taxonomic status of bothalgae is rather confusing. Following a comparativeultrastructural study with autospore-forming Nan-nochloris species including N. coccoides SAG 251-1,Nanochlorum eucaryotumwas transferred by Menzeland Wild (1989) to the genus Nannochloris. This

transfer is incorrect in two respects. First, N. eucary-otumwith a GC content of 48 mol% (Zahn 1984) isnot related to N. coccoides(66 mol% GC; Huss and

Jahnke 1994) according to the rRNA phylogeny(Krienitz et al. 1996). Second, according to the orig-inal description by Naumann (1921), Nannochlorispropagates by binary division and not by autospo-rulation and belongs to the Ulotrichales (Ulvophy-ceae). Therefore, the autospore-forming N. coccoi-des has been transferred to the genus Choricystis(Skuja) Fott as C. minor(Krienitz et al. 1996). Therevised name Nannochloris eucaryotum (Wilhelm etal.) Menzel et Wild is untenable, as would be a trans-fer to the genusChoricystis.We suggest maintenance

of the original genus name Nanochlorum Wilhelm,Eisenbeis, Wild et Zahn, to avoid further confusion.On the other hand, the description ofChlorella min-utissimaby Fott and Novakova (1969) was based onstrain Lefevre no. 87 of the Paris collection as thetype culture and included, among others, strain C-1.1.9 of the Cambridge collection. Our moleculardata, in concordance with observations made by Kal-ina et Puncocharova (1987), show that the type cul-ture ofC. minutissimais conspecific withC. hom-osphaeraSkuja 1948 (Mychonastes homosphaeraKal-ina et Puncocharova), a species name that has to begiven priority over C. minutissimaFott et Novakova1969. For strain C-1.1.9, which is unrelated to C.

homosphaera(Fig. 2), we propose keeping the nameC. minutissimaprovisionally until more informationbecomes available.

For a long time, Chlorella saccharophilaand C. ellip-soideawere not distinguishable according to the che-motaxonomic criteria of Kessler and were combinedinto a single species, C. saccharophila(Kessler 1967).Differences in the DNA base composition (Hell-mann and Kessler 1974a), salt tolerance (Kessler1974a), and fermentation products (Vinayakumarand Kessler 1975) later allowed the separation of


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some strains. The observation that these algae arefurther characterized by an extreme cadmium sen-sitivity and by the inability to grow on mannitol as acarbon source in the dark led to the designation ofthese strains as C. saccharophila var. ellipsoidea (Kes-sler 1986, 1987) according to Fott and Novakova(1969; but see Puncocharova 1994). DNA hybridiza-

tion studies by Huss et al. (1987b, 1989a) finally ledto a complete delimitation of both taxa into two spe-cies: C. saccharophila and C. ellipsoidea(Kessler andHuss 1992). The rRNA phylogeny not only confirmsthis separation but also justifies the transfer into twodistinct genera apart from Chlorella(Hanagata andChihara, pers. comm.). Moreover, strain SAG 211-9b of C. saccharophilais also distinct in several re-spects from the type culture SAG 211-9a. Acid andsalt tolerance is less pronounced (Kessler 1965,1974a), DNA base composition is slightly lower, andDNA hybridiziations between both taxa showed nosignificant values (Huss et al. 1987b, 1989a). Ac-cordingly, the phylogenetic tree in Figure 2 shows a

separate position ofC. saccharophila SAG 211-9b( Watanabea reniformis Hanagata, Karube, Chiharaet Silva; Hanagata et al. 1998) more distant thanC. luteoviridis. The close relationship of C. luteo-viridiswith C. saccharophilais further substantiatedby the ability to grow on mannitol (Kessler 1987),by a high acid and salt tolerance (Table 1), and bya similar cell wall composition (Takeda 1991, 1993a,1996a). On the other hand,C. ellipsoideais closelyrelated to C. mirabilis.

Chlorella fusca, C. homosphaera, and C. zofin-giensis belong to a different class, the Chlorophy-ceae. The varieties of C. fuscahave already beentransferred to the genus Scenedesmuson the basis of

not only the high rRNA similarities but also bio-chemical, physiological, serological, and moleculardata (Kessler et al. 1997).Chlorella homosphaera(Mychonastes homosphaera [Skuja] Kalina et Punco-charova) has always been an isolated taxon withinthe genus Chlorellawith no apparent affinity to anyother species (Table 1). The 18S rRNA phylogenyconfirms this view and indicates an independent lin-eage of C. homosphaerawithin the Chlorophyceae.

Another independent lineage is represented byC.zofingiensis.This alga has been transferred to the ge-nusMuriella(Boye-Peters) Vischer by Hindak (1982)and later to the genus MychonastesSimpson et Van

Valkenburg by Kalina and Puncocharova (1987) on

the basis of morphological studies, but neither trans-fer is supported by our analyses. Only strain C-1.2.1of C. zofingiensis, which could be discerned fromthis taxon only after molecular methods were ap-plied (Huss et al. 1989b), is related to the genusMuriellaand conspecific withM. aurantiaca(Fig. 2).However, the type culture of C. zofingiensis(strainSAG 211-14) represents an independent lineage

within the Chlorophyceae. Further studies have toshow whether C. zofingiensiscan be assigned to anexisting genus (such as strain C-1.2.1) or whether it

has to be described as a new taxon. Therefore,C.zofingiensis is a prominent example demonstratingthe problems and pitfalls in the systematics of chlo-rococcalean algae when based on phenotypic crite-ria alone.

The close relationship between the autosporicand zoosporic taxa included in this study can be ex-

plained by multiple complete loss of motility in dif-ferent chlorophycean lineages (Wilcox et al. 1992);therefore, autosporulation cannot be used as a phy-logenetic marker. Likewise, the almost complete 18SrRNA identity of the unicellular C. fuscavar.rubes-cens(Scenedesmus rubescens) with the coenobialS.obliquusand other examples within the genus Sce-nedesmus show that coenobial versus unicellular isnot a reliable characteristic. Therefore, the molec-ular phylogeny can be used to recognize phyloge-netically relevant and unreliable nonmolecularproperties. This recognition is useful for the modi-fication of existing phenotypically based classifica-tion schemes toward a natural system.

When the molecular phylogeny is compared withthe chemotaxonomical classification scheme ofChlo-rella in Table 1, many features, although in theircombination very useful for species delimitation, arenot relevant in a phylogenetic sense per se. For ex-ample, the presence or absence of hydrogenase ac-tivity, the first criterion that allowed discriminationbetween the closely related species C. vulgarisandC. sorokiniana (Kessler and Soeder 1962), is foundnot only in the Trebouxiophyceae but also in theChlorophyceae. As hydrogenase is active only understrictly anaerobic conditions, it might represent arelic from the early, anaerobic phase of life on earth(Kessler 1974b). It is unlikely to play a functional

role in algae and apparently was independently lostseveral times during evolution. Other features haveproved to be phylogenetically more reliable markersfor systematic purposes. Thermophily withinChlorel-laand Chlorella-like algae is restricted so far exclu-sively to strains ofC. sorokiniana(Table 1). Growthon mannitol in the dark is indicative for the relatedtaxa C. saccharophilaand C. luteoviridisand cad-mium sensitivity for C. ellipsoidea(Kessler 1987).Norspermine is produced only byC. saccharophila,C. luteoviridis, C. ellipsoidea, andC. mirabilis,allmembers of one of the two subgroups within theTrebouxiophyceae, whereas norspermidine wasfound inScenedesmus, C. zofingiensis,and C. hom-

osphaera,all members of the Chlorophyceae (Hege-wald and Kneifel 1982). Members of the second sub-group of the Trebouxiophyceae, which contains thetrueChlorellaspecies as well as C. minutissimaandC. protothecoides,produce neither norspermine nornorspermidine.

Perhaps the most significant and easily deter-mined feature of phylogenetic relevance is the pro-duction of secondary carotenoids. These ketocaro-tenoids, mainly astaxanthin, canthaxanthin, andechinenone, are produced under conditions of ex-


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treme nitrogen deficiency in most members of theChlorophyceae but so far have never been observedin the Trebouxiophyceae (cf. Czygan 1968, 1982).The production of secondary carotenoids seems tobe related to the ability to synthesize sporopolleninand to the development of the outer, trilaminar cell

wall found in these algae (Atkinson et al. 1972; but

see Burczyk et al. 1995). The sugar composition ofthe cell wall is another character that has beenshown to be useful for the systematics ofChlorellabythe work of Takeda (1991, 1993a, b, 1996a, b).Here, the true Chlorella species C. vulgaris, C. lo-bophora, C. sorokiniana, and C. kessleriare character-ized by glucosamine as the exclusive component ofthe rigid cell wall, making them completely differentfrom all other Chlorella species. Similarly, in astudy of pyrenoid ultrastructure, the same speciescould be discerned from others by the presence ofa double thylakoid bisecting the pyrenoid matrix(Ikeda and Takeda 1995).

For a century, the taxonomy of the genus Chlorella

has been problematic and the phylogenetic positionof different species within the chlorophytes was un-known. Molecular data as well as a combination ofphysiological, biochemical, and ultrastructural char-acters now make it possible to identifyChlorella-likestrains and place them into a natural system. Obvi-ously, however, characters that have proved to be ofphylogenetic significance inChlorellamight not nec-essarily have the same importance in other groupsof algae and have to be determined independentlyfor each group.

We wish to thank Mrs. C. Holweg, Mrs. G. Steingraber, and Mrs.E. Weitemeyer for excellent technical assistance. We are also

grateful to Drs. N. Hanagata and M. Chihara for providing theirnew classification system ofChlorellaprior to publication. Thiswork was supported by the Deutsche Forschungsgemeinschaftand the Universitatsbund Erlangen-Nurnberg.

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BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

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