392 BUCHHEIM ET AL.mically independent sources of molecular phylogenetic (Buchheim et al., 1990). New sequence data for Chla-mydomonas monadina, Chlamydomonas mutabilis,evidence for a parallel set of taxa.Chlamydomonas nivalis, Chlamydomonas radiata,and Chlorococcum echinozygotum were obtained byMATERIALS AND METHODS amplifying genomic DNA using the polymerase chainreaction (PCR). Genomic DNA was obtained using ex-Taxon Selection traction protocols described previously (Buchheim andChapman, 1992). The ﬂanking primers used to amplifyA total of 31 ﬂagellate taxa were included in the phy-the nuclear SSU rRNA gene, NS-1 and ITS-2, are de-logenetic analyses (Table 1). Twenty-three of these taxascribed by White et al. (1990). These two primers am-consist of Chlamydomonas species representing all ofplify a ca. 2.2-kb product that includes most of the SSUthe 15 sporangial wall autolysin groups sensu Schlo¨sserrRNA gene. DNA sequences were obtained with end-(1984) and eight of the nine morphological groupslabeled primer ([33P]dATP, NEN DuPont) and double-(Hauptgruppen sensu Ettl, 1976; 1983; see Table 1).stranded ampliﬁcation product utilizing the protocolsMany of the isolates of Chlamydomonas currentlyand reagents for cycle-sequencing that accompany themaintained by the Sammlung von Algenkulturen Go¨t-AmpliTaq Cycle Sequencing kit (Perkin–Elmer). Se-tingen (SAG) have undergone a reexamination culmi-quencing primers for the cycle-sequencing protocol arenating in name changes for a few of the holdingsidentical to the sequencing primers used in the RTase(Schlo¨sser, 1994; personal communication; see alsoreactions (see Buchheim et al., 1990; Buchheim andBuchheim et al., 1994). Any changes that affect theChapman, 1991, 1992).taxa in this study are listed in Table 2. Because thestudies of nuclear and chloroplast data were conductedChloroplast Sequence Dataindependently, the issue of comparable taxon samplingSequence data from the chloroplast-encoded LSUmust be addressed. The nuclear and chloroplast datarRNA gene for 13 of 31 ingroup taxa are from previousfrom all but 9 of the 33 taxa (31 ingroup and 2 outgroupwork (see Table 1). These published sequences were ob-taxa) are derived from the same culture isolate (Tabletained using the T7 DNA polymerase (Pharmacia) in1). Of these nine pairs of isolates that differ, ﬁve pairsthe presence of recombinant plasmid DNA clones repre-are regarded as cultures related by source of isolatesenting the entire LSU rRNA gene (Turmel et al.,(Starr and Zeikus, 1993; Schlo¨sser, 1994). The sources1993). New sequence data for each of the remainingof taxa differ between the nuclear and chloroplast data18 taxa were generated from three overlapping PCR-for only Scenedesmus obliquus (outgroup taxon), Chla-ampliﬁed fragments using the dsDNA cycle sequenc-mydomonas monadina, Dunaliella parva, and Ste-ing system from Life Technologies and a collection ofphanosphaera pluvialis.32P-labeled primers that are complementary to highlyNuclear Sequence Data conserved regions of the LSU rRNA gene. These prim-ers allowed us to sequence the entire coding regions ofSequence data from the nuclear-encoded SSU rRNAgene for 20 of 31 ingroup taxa are from previous investi- the PCR-ampliﬁed fragments and part of the intronsthat were found. For most taxa, the 5′ and 3′ ends ofgations (Table 1). These published nuclear sequenceswere obtained using either rRNA templates (Buchheim the LSU rRNA gene segment ampliﬁed correspond topositions 38 and 2598, respectively, in the Escherichiaet al., 1990; Buchheim and Chapman, 1991, 1992) orDNA templates (Gunderson et al., 1987; Huss and coli 23S rRNA (Gutell et al., 1994). The PCR ampliﬁca-tions were carried out using genomic DNA and previ-Sogin, 1990; Lewis et al., 1992). Of those taxa for whichonly partial rRNA sequences were obtained, an average ously described conditions (Turmel et al., 1994). Geno-mic DNA from each taxon was prepared as follows.of 250 bases per taxon (except Chlamydomonas callosa,Carteria olivieri, and Chlorogonium elongatum, for Cells from a 500-ml culture that was grown in modiﬁedVolvox medium (McCracken et al., 1980) were har-which template was no longer available) were added forthis investigation. These sequence additions are de- vested by centrifugation, washed twice in 500 µl ofbuffer A (10 mM Tris–HCl, pH 8.0, 10 mM EDTA, 10rived from the use of the 18P universal eukaryoticprimer (Hamby et al., 1988) as a sequencing primer in mM NaCl), resuspended in 250 µl of buffer A, andground with liquid nitrogen with the aid of a mortarthe RTase reactions. The sequence additions roughlycorrespond to positions 650 through 950 of the pub- and pestle. As grinding proceeded, the mortar wasslowly warmed; immediately after all of the liquid ni-lished Chlamydomonas reinhardtii sequence (Gun-derson et al., 1987). New sequence data for Chla- trogen had evaporated, 15 µl of a 1 mg/ml proteinaseK solution and 15 µl of 10% (w/v) sodium dodecyl sul-mydomonas algoe¨formis, Chlamydomonas gigantea,Chlamydomonas pallidostigmatica, Chlamydomonas fate were added. The resulting mixture was transferredinto a 1.5-ml microtube, incubated at 50°C for 2 h, andpseudopertusa, Chlamydomonas starrii, and Chla-mydomonas sp. SAG 66.72 were obtained using then extracted successively with 250 µl of phenol, phe-nol:chloroform:isoamyl alcohol (25:24:1), and chloro-rRNA templates and RTase as described previously
PHYLOGENY OF CHLAMYDOMONADALES 393TABLE 1List of Taxa Examined in This StudySourcecAutolysin MorphologicalgroupagroupbNuclear ChloroplastOutgroup taxonAnkistrodesmus Cordastipitatus (Chodat) Korma´rkova´ Legnerova´ NAdNA SAG 202-5eSAG 202-5Scenedesmus Meyenobliquus (Turp.) Ku¨tz. NA NA SAG 276-3aeUTEX 393Ingroup taxonCarteria Diesingcrucifera Korshikov NA Pseudagloe¨ UTEX 432fUTEX 432olivieri West NA Eucarteria UTEX LB 1032fUTEX LB 1032radiosa Korshikov NA Eucarteria UTEX LB 835fUTEX LB 835Chlamydomonas Ehrenbergagloe¨formis Pascher NA Pseudagloe¨ UTEX 231 UTEX 231callosa Gerloff 6 Euchlamydomonas UTEX 624gSAG 9.72eugametos Moewus 12 Chlamydella UTEX 9gUTEX 9kfrankii Pascher 9 Euchlamydomonas SAG 19.72gSAG 19.72kgeitleri Ettl 14 Chlorogoniella SAG 6.73gSAG 6.73kgelatinosa Korshikov 11 Euchlamydomonas SAG 69.72gSAG 69.72kgigantea Dill NA Pleiochloris UTEX LB 848 UTEX LB 848humicola Lucksch 7 Chlorogoniella SAG 11-9gSAG 11-9kiyengarii Mitra 5 Euchlamydomonas SAG 25.72hSAG 25.72kkomma Skuja 2 Euchlamydomonas SAG 26.72gSAG 26.72kmexicana Lewin 4 Chlorogoniella SAG 11-60agSAG 11-60akmonadina Stein NA Chlamydella SAG 31.72 SAG 55.72mutabilis Gerloff NA Pseudagloe¨ UTEX 578 SAG 34.72nivalis (Bauer) Wille NA Sphaerella UTEX LB 1969 UTEX LB 1969pallidostigmatica King 10 Chlamydella SAG 9.83 SAG 9.83kpeterﬁi Gerloff 3 Chlamydella SAG 70.72gSAG 70.72kpitschmannii Ettl 13 Chlorogoniella SAG 14.73gSAG 14.73kpseudopertusa Ettl NA Amphichloris SAG 42.72 SAG 42.72radiata Deason et Bold NA Agloe¨ UTEX 966 UTEX 966reinhardtii Dangeard 1 Euchlamydomonas CC-400iSAG 11-32bkstarrii Ettl 12 Chlorogoniella SAG 3.73 SAG 3.73sp. 66.72 8 NA SAG 66.72 SAG 66.72kzebra Korshikov 15 Euchlamydomonas SAG 10.83gSAG 10.83kChlorococcum Meneghiniechinozygotum Starr NA NA UTEX 118 SAG 213-5Chlorogonium Ehrenbergelongatum (Dang.) Dang. NA NA UTEX 11gUTEX 11Dunaliella Teodorescuparva Lerche NA NA UTEX LB 1983jSAG 19(1)Haematococcus C. A. Agardhlacustris (Girod-Chantras) Rostaﬁnski. NA NA UTEX 16hSAG 34-1bStephanosphaera Cohnpluvialis Cohn NA NA UTEX LB 771hSAG 78-1aaSporangial wall autolysin group sensu Schlo¨sser (1984).bMorphological group sensu Ettl [1976, 1983 (Chlamydomonas taxa); 1979, 1983 (Carteria taxa)].cSource of taxa used to generate the data (nuclear or chloroplast): CC, from the Chlamydomonas Genetics Center at Duke University;SAG, from the Sammlung von Algenkulturen Go¨ttingen; UTEX, from the Culture Collection at the University of Texas at Austin.dNot applicable or information not available.ePartial sequence extracted from published sequence data of Huss and Sogin (1990).fIncludes published sequence data of Buchheim and Chapman (1992).gIncludes published sequence data of Buchheim et al. (1990).hIncludes published sequence data of Buchheim and Chapman (1990).iPartial sequence extracted from published sequence data of Gunderson et al. (1987).jPartial sequence extracted from published sequence data of Lewis et al. (1992).kPublished sequence data of Turmel et al. (1993).
394 BUCHHEIM ET AL.TABLE 2 tively. The alignments are available from the authorsupon request.List of Nomenclatural and Taxonomic Changesamong SAG Isolates of Chlamydomonas Data AnalysesOriginal Reason Three methods of phylogenetic reconstruction, maxi-designation New designation for changemum parsimony, neighbor-joining (NJ), and maximumlikelihood (ML), were employed in a comparison of theChlamydomonasagloe¨formis Chlamydomonas debaryana Goroschankin Reexaminationatwo independent molecular data sets. All parsimonyeugametos Chlamydomonas moewusii Gerloff Synonymybanalyses were conducted using the program PAUP,frankii Chlamydomonas culleus Ettl Reexaminationaversion 3.1.1 (Swofford, 1993) mounted on a Macin-geitleri Chlamydomonas noctigama Korshikov Reexaminationagelatinosa Sphaerellopsis aulata (Pascher) Gerloff Reexaminationatosh Quadra (800 or 950) machine. Bootstrap valueshumicola Chlamydomonas applanata Pringsheim Reexaminationc(Felsenstein, 1985) from 100 resamplings were calcu-iyengarii Chlamydomonas proboscigera Korshikov Synonymyblated for each set of data. Decay indices (Bremer, 1988;komma Chlamydomonas debaryana Goroschankin Reexaminationamexicana Chlamydomonas oblonga Pringsheim ReexaminationaMishler et al., 1991) were also calculated and mappednivalis Chlamydomonas augustae Skuja Reexaminationato cladograms or consensus trees reconstructed frompeterﬁi Chlamydomonas asymmetrica Korshikov Reexaminationaeach of the data sets. Tree searches were conductedpallidostigmatica Chlamydomonas segnis Ettl Reexaminationasp. SAG 66.72 Chlorococcum novae-angliae Archibald et Bold Reexaminationaheuristically using the TBR option with MULPARS.To increase the probability of ﬁnding all islands ofaSchlo¨sser (1994, personal communication).most parsimonious trees, the order of taxon additionbEttl (1983).was randomized 50 times. All trees were rooted usingcEttl and Schlo¨sser (1992).the outgroup method. Characters were optimized tobranches under the assumption of accelerated transfor-mation (ACCTRAN). All characters were regarded asunordered (Fitch, 1971).form:isoamyl alcohol (24:1). Following precipitationFor the NJ analyses (Saitou and Nei, 1987) of thewith ethanol in the presence of 0.2 M NaCl, nucleicnuclear and chloroplast data sets, 1000 bootstrap repli-acids were dissolved in 100 µl of TE buffer (10 mM Tris–cates of each data set were generated using SEQBOOT,HCl, pH 8.0, 1 mM EDTA), 2 µl of a 10 mg/ml RNaseand pairwise distances were calculated with DNADISTA solution was added, and the resulting mixture wasusing the Kimura (1980) two-parameter model of nucle-incubated at 37°C for 30 min. The DNA was precipi-otide change. Topologies were reconstructed from thetated three times with ethanol in the presence of 2.5 Mdistance matrices using NEIGHBOR. Majority-ruleammonium acetate and was ﬁnally dissolved in 10 µlconsensus trees were produced using CONSENSE, andTE.branch lengths were estimated using FITCH with theSequence Alignments user tree option. All of these analyses were carried outusing the PHYLIP package, version 3.5c (Felsenstein,Previous work (Buchheim et al., 1990; Turmel et al.,1993) served as the starting point for all alignments. 1993) mounted on a Sun SPARCstation 10 Model 40.Each ML analysis was conducted using the fast-Both sets of sequences, nuclear and chloroplast, weremanually aligned on the basis of conserved primary DNAml software (Olsen et al., 1994) mounted on a SunSPARCstation 10 and the fastDNAml boot script pro-and secondary structure models. The alignments of nu-clear sequences were completed on a VAX-6320 com- vided with this software. Bootstrap values from 100 re-samplings were calculated for each data set. Majority-puter (University of Oklahoma Genetic ComputerGroup) using the LINEUP program from the GCG se- rule consensus trees were produced using CONSENSE,and branch lengths were estimated using fastDNAmlquence analysis package (Genetics Computer Group,1991), version 7.3, whereas the chloroplast sequences with the user tree option.S. obliquus was used as an outgroup taxon to root thewere aligned on a Sun SPARCstation 10 Model 40 us-ing the Genetic Development Environment program trees calculated with the NJ and ML methods, whereasboth S. obliquus and Ankistrodesmus stipitatus were(Stephen Smith, previously of the Harvard GenomeLaboratory). The secondary structure model for Volvox employed as outgroup taxa to root the trees generatedwith the parsimony method. These two autosporiccarteri (Rausch et al., 1989) was used to assist inaligning the partial nuclear rRNA sequences. The sec- (non-zoospore-producing) taxa are classiﬁed in thegreen algal order Chlorococcales by most algal taxono-ondary structures of all chloroplast rRNA sequenceswere modeled using the program XRNA (B. Wieser, un- mists. The Chlorococcales represent one of seven or-ders, including the Chlamydomonadales, within thepublished results). For the phylogenetic analyses, re-gions not clearly alignable for all taxa were excluded. Chlorophyceae (sensu Mattox and Stewart, 1984); thus,morphological criteria indicate that these two taxa areThe nuclear and chloroplast data sets used in our studyconsist of 990 and 2426 aligned nucleotides, respec- not within the group of interest.
PHYLOGENY OF CHLAMYDOMONADALES 395TABLE 3 mannii, C. geitleri, C. pseudopertusa, Chlamydomonassp. SAG 66.72, and Clc. echinozygotum), a ‘‘C. gigantea’’Comparison of the Primary Structure of the Nuclearlineage (C. gigantea, C. frankii, and C. pallidostigma-and Chloroplast Sequence Datatica), a ‘‘C. radiata’’ lineage (C. radiata, C. nivalis, andC. mutabilis), and a Carteria lineage (Car. olivieri andNuclear ChloroplastCategory of comparison data data Carteria crucifera). Although congruent regardingsome of the major divergences (see Strict, Fig. 1), theTotal number of sites 990 2426results from the three distinct methods of phylogeneticTotal variable sites 220 (22.2%) 865 (35.7%)reconstruction differ from one another in some re-Total binary transitions 106 (10.7%) 345 (14.2%)Total A ↔ G transitions 51 (5.2%) 182 (7.5%) spects. The relative positions of Carteria radiosa andTotal C ↔ T transitions 55 (5.5%) 163 (6.7%) Chlamydomonas monadina are especially labile acrossTotal binary transversions 61 (6.2%) 201 (8.3%)the three methods. The former ranges from an allianceTotal A ↔ C transversions 8 (0.8%) 39 (1.6%)with the Haematococcus lineage (Pars and NJ, Fig. 1)Total G ↔ T transversions 17 (1.7%) 66 (2.7%)to a position as the sister group to the C. radiata lin-Total A ↔ T transversions 19 (1.9%) 75 (3.1%)Total G ↔ C transversions 17 (1.7%) 21 (0.9%) eage (ML, Fig. 1). The position of C. monadina variesTotal multiple-state sites 53 (5.4%) 309 (12.7%) from the sister group to the Haematococcus lineageInformative sites 126 (12.7%) 586 (24.2%)(Pars and NJ, Fig. 1) to the sister group of the C. euga-Informative binary transitions 65 (6.6%) 210 (8.7%)metos lineage (ML, Fig. 1). In addition, the position ofInformative A ↔ G transitions 28 (2.8%) 106 (4.4%)the C. radiata lineage relative to the HaematococcusInformative C ↔ T transitions 37 (3.7%) 104 (4.3%)Informative binary transversions 18 (1.8%) 102 (4.2%) and C. eugametos lineages is either ambiguous (ParsInformative A ↔ C transversions 2 (0.2%) 18 (0.8%) and NJ, Fig. 1) or is weakly resolved (ML, Fig. 1). Last,Informative G ↔ T transversions 7 (0.7%) 31 (1.3%)the C. gigantea lineage varies from a position as theInformative A ↔ T transversions 7 (0.7%) 44 (1.8%)sister group of the Euchlamydomonas lineage (ParsInformative G ↔ C transversions 2 (0.2%) 9 (0.4%)and ML, Fig. 1) to a position as the sister group to aInformative multiple-state sites 43 (4.2%) 266 (11.0%)clade that includes the two Carteria taxa and is basalto the Haematococcus and C. eugametos lineages (NJ,Fig. 1).RESULTSPhylogenetic Analysis of Chloroplast DataStructure of the Sequence Data Parsimony analysis of the chloroplast data set re-sulted in a single minimal length tree (L ϭ 2464, CI ϭA summary of the primary structure of the two in-0.418, RI ϭ 0.591; see Fig. 2). The results from NJ anddependent data sets is presented in Table 3. TheML analysis of the chloroplast data are also presentedsummary includes a comparison of variable and in-in Fig. 2. The chloroplast data consistently resolve theformative sites, transitions and transversions, andsame seven lineages determined from the nuclear datamultistate sites. The chloroplast data are more variable(i.e., ‘‘Euchlamydomonas’’ lineage, ‘‘C. mexicana’’ lin-than the nuclear data in terms of both number and per-eage, ‘‘Haematococcus’’ lineage, ‘‘C. eugametos’’ lineage,centage of variable sites as well as number and percent-‘‘C. gigantea’’ lineage, ‘‘C. radiata’’ lineage, and a ‘‘Car-age of informative sites (see discussion below).teria’’ lineage). In addition, the chloroplast data arePhylogenetic Analysis of Nuclear Data more robust across methods of phylogenetic reconstruc-tion than the nuclear data. The results from the threeParsimony analysis of the nuclear data set resultedanalyses (Fig. 2) differ only in (1) the position of D.in 24 minimal length trees [L ϭ 326, CI ϭ 0.518 (Klugeparva and H. lacustris within the Haematococcus lin-and Farris, 1969), RI ϭ 0.682 (Farris, 1989)]. A stricteage, (2) the relative positions of C. gelatinosa, C. cal-consensus analysis (Rohlf, 1982) illustrates the taxo-losa, C. reinhardtii, and C. zebra within the Euchlamy-nomic congruence between the competing hypothesesdomonas lineage, and (3) the position of Car. radiosa.(Fig. 1). Results from NJ and ML analysis of the nu-clear data are also presented in Fig. 1. The nuclear dataconsistently resolve seven lineages that include aDISCUSSION‘‘Euchlamydomonas’’ lineage (C. reinhardtii, Chlamy-domonas komma, C. starrii, C. callosa, Chlamydo-Strength and Quality of Phylogenetic Signalmonas iyengarii, and Chlamydomonas zebra), a ‘‘Chla-mydomonas mexicana’’ lineage (C. mexicana and C. The chloroplast phylogenies are clearly more robustthan the nuclear phylogenies. This difference is best ex-peterﬁi), a ‘‘Haematococcus’’ lineage (Haematococcuslacustris, C. agloe¨formis, C. humicola, Chlorogonium plained by an examination of the structure of the twodata sets. One possible factor in this difference betweenelongatum, S. pluvialis, and D. parva), a ‘‘Chlamydo-monas eugametos’’ lineage (C. eugametos, C. pitsch- data sets may be the use of partial nuclear sequences
396 BUCHHEIM ET AL.FIG. 1. Analyses of nuclear-encoded SSU rRNA sequence data using the methods of maximum parsimony, NJ, and ML. The parsimony(Pars) tree is a strict consensus analysis of 24 minimal length trees. Bootstrap values for all unambiguously resolved nodes are includedabove each internode; corresponding DI values for the parsimony tree are included below each internode. Note that bootstrap values below50 are not reported for the parsimony tree. A strict consensus tree of the parsimony, NJ, and ML trees is also shown.chloroplast data have nearly twice the number (as ain comparison with the complete chloroplast sequences.In addition, the nuclear data have more ambiguous percentage of the total number of sites) of informativesites compared to that in the nuclear data set (Tablesites (a consequence of sequences derived from reversetranscriptase sequencing) than the chloroplast data. 3). Moreover, the number of informative transversionsin the chloroplast data set is nearly quadruple that inAlthough the differences in the structure of variationbetween nuclear and chloroplast data sets are less dra- the nuclear data set (Table 3). These two observa-tions are consistent with the results of analyses of thematic than the difference in size (990 sites vs 2426sites, respectively; see Table 3), the chloroplast data two independent data sets in which the branches ontrees derived from the chloroplast data generally ex-generally have greater percentages of binary transi-tions, binary transversions, and multistate sites than hibit more robust character support than comparablebranches on trees derived from analysis of the nuclearthe nuclear data. The more important observation, atleast in terms of the parsimony analyses, is that the data. In other words, the chloroplast data are not only
PHYLOGENY OF CHLAMYDOMONADALES 397FIG. 2. Analyses of chloroplast-encoded LSU rRNA sequence data using the methods of maximum parsimony, NJ, and ML. The parsi-mony (Pars) tree is the single minimal length tree found. Bootstrap values for all unambiguously resolved nodes are included above eachinternode; corresponding DI values for the parsimony tree are included below each internode. A strict consensus tree of the parsimony, NJ,and ML trees is also shown.comprising biﬂagellate unicells. Ettl (1976, 1983) hasmore variable, but also have a greater density of phylo-genetically informative sites than the nuclear data. As organized the members of the genus into nine morpho-logical Hauptgruppen. The nine Hauptgruppen differwould be predicted from these observations, the chloro-plast data (CI ϭ 0.418, RI ϭ 0.591) exhibit greater lev- from one another primarily in chloroplast shape, pyre-noid position, pyrenoid number, and papillum size andels of detected homoplasy than the nuclear data (CI ϭ0.518, RI ϭ 0.682). We have attempted to minimize shape. Results from analyses of both molecular datasets strongly support the conclusion that the genusundetected homoplasy in both data sets through theuse of relatively extensive taxon sampling. Chlamydomonas is not monophyletic and, in addition,they suggest that the morphological criteria used to de-Taxonomic and Phylogenetic Implicationslimit the nine Hauptgruppen (sensu Ettl, 1976, 1983)Chlamydomonas. Chlamydomonas is a speciose (ca. should be reexamined. In general, the topologies from450 species sensu Ettl, 1976, 1983), green algal genus both data sets are inconsistent with the morphological
398 BUCHHEIM ET AL.groups. The only morphological alliance that demon- can be demonstrated between ﬂagellate genera. For ex-ample, both the nuclear and chloroplast data indicatestrates any degree of cohesiveness when tested againstthe molecular data is the Euchlamydomonas Haupt- a close alliance between Clc. echinozygotum and eitherC. eugametos and/or C. pitschmannii. Are the autoly-gruppe. A number of taxa ascribed to this group, includ-ing C. reinhardtii, form a monophyletic assemblage sins of either of these Chlamydomonas taxa similar tothat of Clc. echinozygotum? If autolysin data are to(Figs. 1 and 2). However, the alliance of C. starrii (Chlo-rogoniella sensu Ettl, 1976, 1983) among these euchla- have relevance for green algal systematics in general,then the scope of comparison must be broadened be-mydomonads (Figs. 1 and 2) represents an exception tothis generalization. Assuming that the two molecular yond the genus Chlamydomonas given the compellingevidence for nonmonophyly within the genus.data sets are accurately recovering historical informa-tion, the inconsistencies between molecular-based phy-logenies and morphological-based classiﬁcations may Is Carteria monophyletic? Although not nearly asspeciose, the quadriﬂagellate, unicellular genus Car-be due to homoplasy in the morphological data. Giventhat Ettl’s (1976) classiﬁcation is based extensively on teria parallels Chlamydomonas in morphological vari-ability (Ettl, 1979, 1983). Lembi (1975) has demon-chloroplast morphology, one could conclude that vari-ability in this character may be plastic or exhibit non- strated that at least two distinct, ultrastructurallydeﬁned lineages exist among species of Carteria. Thehomology. However, it must also be noted that the in-consistencies between molecules and morphology may two lineages have recently been examined using nu-clear-encoded rRNA sequence data (Buchheim andsimply be a consequence of comparing a classiﬁcationbased on a subjective evaluation of evidence (i.e., mor- Chapman, 1992). These molecular data corroborate theultrastructural interpretation and, furthermore, theyphological) with a phylogenetic interpretation of evi-dence (i.e., molecular). The morphological criteria need indicate that these lineages are not monophyletic(Buchheim and Chapman, 1992). Results of analysesto be reexamined to identify potential plasticity or non-homology of characters. Furthermore, these morpho- from the present investigation support the distinctionof two Carteria lineages (Car. olivieri ϩ Car. cruciferalogical data need to be interpreted within a phyloge-netic framework. vs Car. radiosa). The nuclear data remain consistentin failing to support monophyly of the genus CarteriaDiversity within the genus Chlamydomonas has alsobeen demonstrated at the biochemical level. Schlo¨sser (Fig. 1), whereas the chloroplast data are ambiguousregarding the question of Carteria monophyly. The par-(1984) presented evidence for the existence of 15 dis-tinct lineages within the genus Chlamydomonas based simony and ML analyses of the chloroplast data sup-port a monophyletic Carteria (88 and 52% bootstrapon differences in enzymatic speciﬁcity of sporangialwall autolysins. Because the taxon sampling scheme values, respectively), while NJ analysis supports aparaphyletic Carteria (54% bootstrap value; see Fig. 2).for both the nuclear and chloroplast data sets was pri-marily based on autolysin diversity, the phylogenetic Although error due to limited taxon sampling in thegenus Carteria may be inﬂuencing the results of analy-analyses do not provide the opportunity to thoroughlytest the autolysin classiﬁcation. However, both sets of ses from both data sets, the inclusion of sequence datafor an additional Carteria taxon (Car. lunzensis SAG 8-molecular data presented here are consistent with twoinferences from the autolysin evidence and inconsis- 3) in the chloroplast data set does not resolve the issue.The parsimony and ML analysis support monophyly,tent with another. For example, C. peterﬁi and C. mexi-cana produce distinct sporangial wall autolysins (types whereas the NJ analysis supports paraphyly (data notshown). While failing to support Carteria monophyly,three and four, respectively); however, Schlo¨sser (1984)demonstrated that the type three autolysin is capable the nuclear data are ambiguous, under different meth-ods of phylogeny reconstruction, regarding the place-of lysing the type four sporangial wall. In addition, thetype 14 autolysin (e.g., C. geitleri) is capable of lysing ment of Car. radiosa (Fig. 1). This observation suggeststhat the nuclear data may be inﬂuenced by inadequatethe type 8 sporangium (Chlamydomonas sp. SAG66.72). These autolysin cross-reactivities are sugges- taxon sampling in the genus Carteria.Aside from the apparent differences between molecu-tive of a phylogenetic link that is corroborated by mo-lecular evidence (Figs. 1 and 2). In contrast, while C. lar data sets regarding the Carteria question, both arecongruent in supporting a basal lineage within the or-starrii and C. eugametos are reported to share the type12 autolysin (Schlo¨sser, 1984), molecular data do not der Chlamydomonadales that includes the quadriﬂag-ellates Car. olivieri and Car. crucifera. In the absencesupport the alliance inferred from the biochemical evi-dence (Figs. 1 and 2). Clearly, C. starrii and C. euga- of any other information, one would conclude that thequadriﬂagellate condition is the ancestral state for themetos are candidates for a reexamination of autolysindata. Another new line of investigation is suggested by Chlamydomonadales (see also Buchheim and Chap-man, 1992). Thus, the quadriﬂagellate condition, whichthe observation that at least some of the distinct autol-ysin groups appear to have allies other than Chlamydo- appears to be the single synapomorphy for the genusCarteria, may be plesiomorphic and, therefore, cannotmonas. Speciﬁcally, it would be of interest to green al-gal phylogeneticists to determine if autolysin similarity be used to diagnose a taxon (see also Buchheim and
PHYLOGENY OF CHLAMYDOMONADALES 399Chapman, 1992). Despite what are compelling hen- halophilic, unicellular, biﬂagellate green algae thathave been characterized as wall-less (e.g., Smith,nigian arguments, the limited taxon sampling forces usto conclude that the issue of Carteria monophyly re- 1950). However, careful examination has shown thatDunaliella exhibits an extracellular, wall-like layermains unresolved.(Oliveira et al., 1980, Melkonian and Preisig, 1984).Chlorococcum and the class Chlamydophyceae.Like Chlorococcum, Dunaliella has been the subject ofBoth molecular data sets include sequences from twodebate regarding its phylogenetic position. Ettl (1981,members of the genus Chlorococcum, Clc. novae-1983) considered the absence of a typical cell wall asangliae (originally identiﬁed as Chlamydomonas sp.evidence against the inclusion of Dunaliella with otherSAG 66.72, see Table 2) and Clc. echinozygotum. Thisanteriorly biﬂagellate unicells that he placed in thegenus has been the subject of some debate among algalChlamydophyceae (sensu Ettl, 1981; 1983). Conse-systematists. Chlorococcum is characterized by a lifequently, Ettl separated Dunaliella from the walledhistory that includes a zoospore (motile) stage and aﬂagellates (i.e., order Chlamydomonadales, class Chla-unicellular, nonmotile, vegetative stage. Both the zoo-mydophyceae, sensu Ettl, 1981) into the order Dunali-spore and the nonmotile vegetative stage possess cellellales (class Chlorophyceae sensu Ettl, 1981, 1983). Al-walls. Although the zoospore stage is strongly reminis-though Melkonian (1990) did not adopt the broadcent of a chlamydomonad vegetative cell (which areconcept of the classes Chlorophyceae and Chlamydo-normally motile), the nonmotile stage has been cited asphyceae, he did recognize an order Dunaliellales as sep-evidence of a link with other nonmotile, unicellar algaearate from the Chlamydomonadales, within a classwhich lack any motile stages. Traditionally, unicellularChlorophyceae (sensu Melkonian, 1990), arguing thatorganisms with a nonmotile, walled, vegetative stageultrastructural differences indicate that Dunaliella ishave been placed in the order Chlorococcales by algalnot a wall-less equivalent of Chlamydomonas. In con-systematists (e.g., Smith, 1950; Prescott, 1951; Mattoxtrast, Floyd (1978), Mattox and Stewart (1984), andand Stewart, 1984; Bold and Wynne, 1985; Melkonian,Chappell et al. (1989), citing similarities in cell division1990). However, the observation that the zoosporesand ﬂagellar apparatus architecture and interpretingfrom some members of the Chlorococcales (e.g., Chlo-the absence of a typical cell wall as loss or extreme mod-rococcum) exhibit a glycoprotein cell wall like that ob-iﬁcation, argued for an alliance of Dunaliella with otherserved on the motile vegetative cells of the Chlamydo-chlamydomonadalean ﬂagellates. Using complete nu-monadales (Miller, 1978) has been acknowledged to beclear SSU rRNA sequences, Lewis et al. (1992) showedof potential phylogenetic signiﬁcance (Melkonian,that D. parva is allied with Characium vacuolatum and1990). Both Clc. novae-angliae (Ettl and Ga¨rtner, 1988)Ettlia minuta, both of which are chlorococcalean taxaand Clc. echinozygotum (Deason, 1983) have beenthat produce walled zoospores. The D. parva sequenceshown to exhibit cell wall features similar to those ofpublished by Lewis et al. (1992) was used in the nuclearthe Chlamydomonadales. Ettl (1981) has argued thatdata set of the present investigation. Our results,the presence of a glycoprotein cell wall on the motilewhich corroborate the ﬁndings of Lewis et al. (1992)stages of Chlamydomonas, Chlorococcum, and otherand support the conclusions of Floyd (1978), Mattoxunicellular green algae is an important, class-level phy-and Stewart (1984), and Chappell et al. (1989), demon-logenetic marker. Speciﬁcally, Ettl has erected a newstrate that D. parva is allied with the chlamydomona-class of green algae, the Chlamydophyceae, that in-dalean (walled) ﬂagellates. Thus, in the case of Duna-cludes many taxa that produce zoospores possessing aliella, the molecular data are inconsistent with theglycoprotein cell wall. Although Ettl’s classiﬁcation hasconcept of the classes Chlamydophyceae and Chloro-been criticized for being inconsistent in applying diag-phyceae (sensu Ettl, 1981, 1983) and do not support thenostic criteria (Mattox and Stewart, 1984), our molec-concept of a separate order Dunaliellales (sensu Mel-ular data suggest that at least two species of Chloro-konian, 1990). If we assume that D. parva is typical ofcoccum have chlamydomonadalean sister taxa. As athe genus (see Lerche, 1937) and if the cell surface coatconsequence, the results from analyses of molecularof Dunaliella is indeed glycoprotein in nature (Oliveiradata offer two alternatives for the status of the genuset al., 1980), then the results from analyses of molecu-Chlorococcum. Either Chlorococcum does not compriselar evidence presented here lead us to conclude that thea natural group or the genus should not be allied withextracellular layer in Dunaliella is modiﬁed from theother chlorococcalean taxa (sensu Smith, 1950; Mattoxglycoprotein cell wall of an ancestral chlamydomonada-and Stewart, 1984; Bold and Wynne, 1985; Melkonian,lean taxon.1990), but rather should be allied with the Chlamydo-monadales. Only additional taxon sampling within theThe Haematococcus lineage. Of the core taxagenus Chlorococcum will allow us to resolve this issue.within the Haematococcus lineage, four (Dunaliella,Nonetheless, the results from the present investigationHaematococcus, Chlorogonium, and Stephanosphaera)have profound implications for our concept of ordinalpossess morphological features that may represent syn-classiﬁcation within the green algae.apomorphies of the group or for a subset of taxa withinthe group. Speciﬁcally, all four exhibit a persistence ofDunaliella. Members of the genus Dunaliella are
400 BUCHHEIM ET AL.motility or sporangial ﬂagella during sporulation of the C. radiata, C. eugametos, and Haematococcus lin-eages, and the other major clade composed of the Eu-(Droop, 1956a, b; Pocock, 1960; Ettl, 1983). In addition,Chlorogonium, Haematococcus, and Stephanosphaera chlamydomonas and C. gigantea lineages (Fig. 2). Thenuclear data are ambiguous, under different methodsexhibit multiple (Ͼ2) contractile vacuoles (CVs) scat-tered throughout the cytoplasm. Two apical CVs is the of phylogeny reconstruction, regarding the position ofthe C. radiata lineage within the major clade (Fig. 1),typical condition observed for virtually all other chla-mydomonadalean taxa (Ettl, 1983; Melkonian, 1990). whereas the chloroplast data consistently place the C.radiata lineage at the base of the clade (Fig. 2). BothBecause both multiple and scattered CVs and persis-tent motility/ﬂagella during cell division appear to rep- data sets strongly suggest that the C. mexicana lineageis the sister group to the Euchlamydomonas lineage.resent apomorphic states, it follows that these char-acters represent evidence of congruence with the Under all three methods of phylogeny reconstruction,the chloroplast data strongly suggest that the C. gigan-molecular data. However, both data sets indicate thatC. agloe¨formis and C. humicola are allied within the tea clade is the sister group to the Euchlamydomonasplus C. mexicana clade. Parsimony and ML analysis ofHaematococcus lineage. Neither of these taxa are re-ported to exhibit either persistence of motility/ﬂagella the nuclear data also support this alliance.during cell division or multiple, scattered CVs (Ettl,Summary and Conclusions1976, 1979, 1983). In fact, C. humicola and several re-The two data sets are congruent for those aspects oflated isolates in autolysin group seven have been re-the respective topologies that are relatively robust andcently reexamined (Ettl and Schlo¨sser, 1992) anddiffer for those parts of the topologies that are not. Bothshown to exhibit two apical CVs and ﬂagellar resorp-data sets fail to support a monophyletic Chlamydomo-tion prior to the onset of zoosporogenesis. Thus, C. hu-nas, resolving at least six lineages that include speciesmicola represents an exception to the morphologicalof this genus. Neither data set is consistent with thetrends exhibited by other taxa in the HaematococcusHauptgruppen scheme for the genus Chlamydomonaslineage. One explanation of this observation is to raise(sensu Ettl, 1976, 1983). The issue of monophyly/pa-the possibility that some of the putative synapomor-raphyly of the genus Carteria has not been resolved.phies may not be homologous across taxa. For example,The nuclear data set fails to support a monophyleticthe form of persistent ﬂagella that characterizes Duna-Carteria, whereas the chloroplast data set is ambigu-liella may be fundamentally different from that in theous regarding the question of monophyly. The allianceother taxa in the Haematococcus lineage. Although Du-of Chlorococcum among the Chlamydomonadales notnaliella is not truly wall-less, it exhibits binary ﬁssiononly represents support for concluding that the Chloro-which is typical of other wall-less ﬂagellates. In con-coccales is not a natural group (Fritsch, 1945; Mattoxtrast, multiple ﬁssion is typical of most chlamydomo-and Stewart, 1984; Melkonian, 1990), but also chal-nad ﬂagellates. Thus, it is logical to assume that binarylenges the traditional concepts of both the Chlorococ-ﬁssion in Dunaliella is coupled with the loss of a typicalcales and Chlamydomonadales. Both data sets supportchlamydomonad cell wall. Nonetheless, these morpho-the halophilic Dunaliella as a member of the Chlamy-logical differences indicate we must be cautious in ourdomonadales, suggesting that the unusual cell surfaceinterpretation of character homology. At the very least,coating of Dunaliella is modiﬁed from the typical gly-these observations indicate that persistent motility/coprotein wall of other chlamydomonadalean taxa.ﬂagella take different forms within the HaematococcusWithin the Haematococcus lineage, evidence of congru-lineage. Expanded taxon sampling may help resolveence between molecular and morphological data issome of these issues, but a careful comparison of cellfound in the case of taxa that exhibit multiple and scat-division characteristics will be needed to reconcile thetered CVs (Haematococcus, Chlorogonium, and Ste-apparent differences between our phylogenetic analy-phanosphaera) and possess ﬂagellated division stagessis of molecular data and the interpretation of morpho-(Dunaliella, Haematococcus, Chlorogonium, and Ste-logical evidence presented above.phanosphaera). However, C. agloe¨formis and C. hu-Relationships among the Lineages micola represent exceptions to this list of congruences.Despite differences in detail and robustness, the twoThe nuclear and chloroplast data sets are generallyindependent data sets exhibit considerable taxonomiccongruent in resolving six lineages that include Chla-congruence regarding the relationships of ﬂagellatemydomonas taxa as well as a basal Carteria lineage.taxa representing the order Chlamydomonadales.Furthermore, the two data sets are congruent in recog-nizing a clade that includes the C. radiata, C. euga-metos, and Haematococcus lineages. The chloroplastACKNOWLEDGMENTSdata are consistent under different methods of phylog-eny reconstruction in support of a fundamental dichot- We thank U. G. Schlo¨sser for his generous gift of green algalomy among the six chlamydomonad lineages. This di- strains and for sharing unpublished results. The Oklahoma Univer-sity Genetic Computer Group is supported by grants from the Centerchotomy is characterized by one major clade composed
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