J Mol Evol (1998) 47:420–430

© Springer-Verlag New York Inc. 1998

Phylogenetic Position of the Within the Chlamydophyceae as Revealed by Analysis of 18S rDNA and rbcL Sequences

D. Hepperle,1 H. Nozaki,2 S. Hohenberger,3 V.A.R. Huss,3 E. Morita,2 L. Krienitz1

1 Institute of Freshwater Ecology & Inland Fisheries, Department of Limnology of Stratified Lakes, Alte Fischerhu¨tte 2, D-16775 Neuglobsow, Germany 2 University of Tokyo, Department of Biological Sciences, Graduate School of Science, Hongo, Bunkyo, Tokyo 113, Japan 3 University of Erlangen, Institute of Botany and Pharmaceutical Biology, Staudtstr. 5, D-91058 Erlangen, Germany

Received: 9 June 1997 / Accepted: 17 October 1997

Abstract. Four genera of the Phacotaceae (, and ഛ86.6% in the rbcL gene. It showed major similari- , Wislouchiella, ), a family ties to the 18S rDNA of Dunaliella salina, with 95.3%, of loricated green algal flagellates within the Volvocales, and to the rbcL sequence of Chlamydomonas tetragama, were investigated by means of transmission electron mi- with 90.3% sequence homology. Additionally, the croscopy and analysis of the nuclear encoded small- Phacotaceae sensu stricto exclusively shared 10 (rbcL: 4) subunit ribosomal RNA (18S rRNA) genes and the plas- characters which were present neither in other Chlam- tid-encoded rbcL genes. Additionally, the 18S rDNA of ydomonadales nor in Dysmorphococcus globosus. Dif- pluvialis and the rbcL sequences of ferent phylogenetic analysis methods confirmed the hy- elongatum, C. euchlorum, Dunaliella pothesis that the Phacotaceae are polyphyletic. The parva, Chloromonas serbinowii, Chlamydomonas ra- Phacotaceae sensu stricto form a stable cluster with af- diata, and C. tetragama were determined. Analysis of finities to the Dunaliellaes and possibly Haematococcus ultrastructural data justified the separation of the Phaco- pluvialis. Dysmorphococcus globosus represented an in- taceae into two groups. Phacotus, Pteromonas, and Wis- dependent lineage that is possibly related to Chlamydom- louchiella generally shared the following characters: onas moewusii and C. tetragama. egg-shaped protoplasts, a single pyrenoid with planar thylakoid double-lamellae, three-layered lorica, flagellar Key words: Phacotaceae — Volvocales — 18S rDNA channels as part of the central lorica layer, mitochondria — Phylogeny — Lorica-bearing flagellates — Green al- located in the central cytoplasm, lorica development that gae occurs in mucilaginous zoosporangia that are to be lysed, and no acid-resistant cell walls. Dysmorphococcus was clearly different in each of the characters mentioned. Direct comparison of sequences of Phacotus lenticularis, Introduction Pteromonas sp., Pteromonas protracta, and Wislouch- iella planctonica revealed DNA sequence homologies of Ettl (1983) classified the flagellated members of the ജ98.0% within the 18S gene and 93.9% within the rbcL green algae with respect to their cell walls and or- gene. D. globosus was quite different from these species, ganization into three classes: (1) Prasinophyceae— with a maximum of 92.9% homology in the 18S rRNA scale-bearing taxa (Polyblepharidales), cyst-forming Prasinophytes (Halosphaerales), and thecate taxa (Tetraselmidales); (2) —taxa without cell walls (Dunaliellales); and (3) Chlamydophyceae— Correspondence to: D. Hepperle; e-mail: [email protected] (‘‘beha¨utete’’) flagellates that have cell walls and appear 421 as unicellular () or multicellular perle et al. 1994; Hepperle and Krienitz 1996). This in- taxa (coenobial, ‘‘Volvocales’’). But he also mentioned dicates that the family might be polyphyletic and thus an in a previous study that Chlamydomonas seemed to be an artificial conglomerate. Although the loricae were stud- artificial genus that unified different phylogenetic lin- ied almost excessively, ultrastructural data on internal eages and had a more provisional character (Ettl 1976). morphology are scarce. This stimulated reanalyses of ul- However, this system is not consistent with analyses trastructure from different members of the Phacotaceae. based upon comparison of characteristics of the flagellar Concerning the of the Phacotaceae it is of apparatus and molecular markers, such as 18S rDNA. interest that the genus Dysmorphococcus (Takeda 1916) Now, it is well accepted that the Prasinophytes represent was at first arranged within the ‘‘Coccomonadineae’’ at least two independent lineages of green algae and that together with Coccomonas and Pedinopera (Pascher the Tetraselmidales are to be included in the Pleurastro- 1927). At that time, Phacotus and Pteromonas were the phyceae (Steinko¨tter et al. 1994). The remaining two only members of the ‘‘Phacoteae.’’ But later the two groups, Chlamydomonadales and Volvocales, were also groups were united in a single family termed ‘‘Phacota- treated as independent lineages within the Chlorophy- ceae’’ (Bold and Starr 1953; Huber-Pestalozzi 1961; ceae by Mattox and Stewart (1984). Bourrelly 1966; Prescott 1970; Iyengar and Desikachary Studies of the nuclear-encoded small-subunit ribo- 1981). As indicated by Ettl (1983), the Phacotaceae somal RNA (18S rRNA) genes revealed that multicellu- might be divided into two groups with different lorica lar taxa (except ) had a close relation- morphologies: one group is characterized by a lorica ship to some members of the Chlamydomonadales consisting of a single, uniform piece and the other by (especially to Chlamydomonas reinhardtii), whereas bivalved loricae that consist of two shell-like halves. other taxa were quite apart from these (Rausch et al. Here we present the first study on the phylogenetic relationships of the Phacotaceae based on nucleotide se- 1989; McAuley et al. 1990; Buchheim and Chapman quence and ultrastructural data from various phacotacean 1991). Analysis based on morphological and/or molecu- algae. lar data contributed to the idea that a set of coccal- organized chlorophytes also belong to the Chlamydophy- ceae, i.e., Characium vacuolatum (Wilcox et al. 1992), and Botryococcus braunii (Sawayama et al. 1995). Lewis Materials and Methods et al. (1992) compared morphological and molecular data and showed that chlorococcalean species that were char- Culture. Unialgal cultures of Pteromonas protracta (UTEX LB 647), Dysmorphococcus globosus (SAG 20-1), Wislouchiella planctonica acterized by a clockwise orientation of the basal bodies (UTEX LB 1030), Haematococcus lacustris (SAG 34-1b), Chlorogo- were also phylogenetically closely related to the nium elonagatum (IAM C-293), C. euchlorum (CCAP12/3), and Chlamydophyceae. Chlamydomonas tetragama (NIES-446) were obtained from either the The Phacotaceae were regarded as a monophyletic Culture Collection at the University of Texas (UTEX; Austin, TX, USA), the Culture Collection of Algae and Protozoa (CCAP; Cumbria, group of flagellated green algae within the Volvocales. UK), the Culture Collection of the Institute of Applied Microbiology They comprise about 70 species in 15 genera (Ettl 1983). (IAM; Tokyo, Japan), the Microbial Culture Collection, National In- The family was proposed on the presence of one single, stitute for Environmental Studies (NIES; Tsukuba, Japan), or the uniting criterion: the lorica. According to Preisig et al. Sammlung von Algenkulturen (SAG; Go¨ttingen, Germany). Phacotus (1994) loricae are considered to be cell walls fitting lenticularis (KR 91/1) was isolated by L. Krienitz from Lake Tollense and Pteromonas strains (KR 91/2, KR 91/3) derived from an oxidation loosely over the body proper of the organism and not pond in Neuglobsow (Brandenburg, Germany). All algae were cultured exhibiting defined chemical compositions. At least the in 3,000-ml batch cultures and medium 7 (Schlo¨sser 1982). organic compounds of the loricae of the Phacotaceae are supposed to be of dictyosomal origin and seem to as- Ultrastructure. Cells were fixed simultaneously in a mixture of semble on the cell surface after their excretion (Walne 1.25% glutaraldehyde, 1% osmium tetroxide, and 30 mM HEPES, pH and Dunlap 1994, 1995; Hepperle and Krienitz 1996). A 7.2, at 4°C (modified from McFadden and Melkonian 1986). After two subsequent, unknown mechanism leads to mineralization rinses in distilled water at 4°C the samples were dehydrated at room temperature in an ascending acetone series. After infiltration of the of the loricae with metallic compounds, such as calcium cells in Spurr’s low-viscosity resin:acetone (2:1) for 12 h and evapo- carbonate in the case of Phacotus (Hepperle and Krienitz ration of the acetone, the samples were transferred to pure resin for 4 1997) or iron salts in Dysmorphococcus (Porcella and h (Spurr 1969). Subsequently, the samples were centrifuged and poly- Walne 1980). This process usually results in a more or merized at 60°C for 48 h. less rigid cell wall exhibiting a species- and/or genus- specific structure and shape. Most authors consider the DNA Extraction, Polymerase Chain Reaction (PCR), and Sequenc- lorica of the Phacotaceae to be a conserved, apomorphic ing of 18S rDNA. Total DNA of cells harvested by centrifugation was characteristic (e.g., Walne and Dunlap 1994). Neverthe- obtained either by detergent treatment in 20% sodium dodecyl sulfate (SDS) or, in the case of W. planctonica, by cell fracture in a nitrogen- less, a comparison of lorica morphology and metal com- cooled mortar and extraction in CTAB at 60°C (Doyle and Doyle position reveals extreme differences between the genera 1990). After treatment with RNase A (final concentration, 50 ␮g/ml; 30 (Dunlap and Walne 1993; Walne and Dunlap 1994; Hep- min at 37°C) the 18S rDNA genes were amplified by use of PCR (Saiki 422 et al. 1988; Medlin et al. 1988). The sequence of the complete 18S gene Table 1. GenBank accession numbers or identification numbers of was determined after cloning into the M13 vector or by direct sequenc- culture collections ing following the dideoxy method (Sanger et al. 1977) using universal eukaryotic primers (Gunderson et al. 1986; Elwood et al. 1985). Accession Taxon No.

DNA Extraction, PCR, and Sequencing of 18S rDNA. Methods for 18S outgroup processing DNA for rbcL genes were described in previous studies Mantoniella squamata X73999 (Nozaki et al. 1995, 1997). The resulting sequence data corresponded Nephroselmis olivacea X74754 to positions 31–1,158 of the Chlorella ellipsoidea rbcL gene (Yoshi- Chlorella fusca var. rubescensa X74002 naga et al. 1988). Chlorella vulgaris X13688 18S ingroup Asteromonas gracilis M95614 Phylogenetic Analysis. Manual alignment and homology analysis Botryococcus braunii X78276 were carried out by use of an IBM-compatible personal computer and Carteria crucifera D86501 the Windows-based multisequence alignment editor of Hepperle Carteria radiosa D86500 (1995). The sequences were aligned according to conservation patterns Characium vacuolatum M63001 of both primary and secondary structures (Gutell et al. 1985). Those Chlamydomonas asymmetrica U70788 parts of the sequences where a proper alignment was not possible were Chlamydomonas baca U70781 excluded from the phylogenetic analysis. Distance matrix calculation, Chlamydomonas bipapillata U70783 bootstrap analysis, neighbor joining (NJ) (Saitou and Nei 1987) and Chlamydomonas dysosmos U13985 maximum-parsimony (MP) analysis and construction of consensus Chlamydomonas fimbriata U70784 trees were carried out by means of PHYLIP Version 3.5c (Felsenstein Chlamydomonas humicola U13984 1995). Chlamydomonas macrostellata U70785 For calculation of distance matrices, the Kimura (1980) Chlamydomonas moewusii U41174 two-parameter model and, for maximum-likelihood analysis, the Chlamydomonas moewusii U70786 fastDNAml algorithm were used (Felsenstein 1981; Olsen et al. 1994). Chlamydomonas monadina U57694 All analyses were combined with a 100-fold bootstrap analysis except Chlamydomonas nivalis U57696 in the case of rbcL sequences, where 1,000 replicates were calculated. Chlamydomonas noctigama U70782 Outgroups and ingroups may be deduced from Table 1. After construc- Chlamydomonas noctigama U70787 tion of phylogenetic trees, group-specific sites were identified by direct Chlamydomonas pitschmannii U70789 comparison of sequences. As Chlamydomonas moewusii and C. euga- Chlamydomonas pulsatilla AB001037 metos were shown to be conspecific, we use the term C. moewusii here Chlamydomonas pulsatilla AB001038 (Gowans 1963, 1976; Thomas and Delcarpio 1971; Turmel et al. 1993). Chlamydomonas pulsatilla AB001039 C. dysosmos and C. humicola are now united in C. applanata (Ettl and Chlamydomonas radiata U57697 Schlo¨sser 1992; Rumpf et al. 1996). Chlamydomonas rapa U70790 Chlamydomonas reinhardtii M32703 Chlamydomonas sp. AB001374 Results Chlamydomonas zebra U70792 Chlorococcopsis minuta M62996 Chlorococcum echinozygotum U57698 As the morphologies of some phacotacean taxa have Chlorococcum hypnosporum U41173 been presented in several previous studies (Belcher and Chlorococcum oleofaciens U41176 Swale 1967; Gerard and Walne 1979; Dunlap and Walne Chlorococcum sp. U41178 1993; Hepperle et al. 1994; Walne and Dunlap 1994, Chloromonas clathrata U70791 Chloromonas oogama U70793 1995; Hepperle and Krienitz 1996, 1997), we limit the Chloromonas perforata U70794 presentation of ultrastructural observations to a mini- Chloromonas rosae U70796 mum and focus on characters that are considered to be Chloromonas serbinowii U70795 diacritic, i.e., gross morphology of the protoplast, ultra- Dunaliella parva M62998 structure of the loricae and pyrenoids, and location of Dunaliella salina M84320 Dysmorphococcus globosus X91629, this study mitochondria. For information on the ultrastructure of Gongrosira papuasica U18503 Haematococcus see Dodge (1973), Fritsch (1961), Joyon SAG34-1b, this study (1963, 1965), Sprey (1970), and Wygasch (1963, 1964). Haematococcus zimbabwiensis U70797 Phacotus lenticularis X91628, this study Pleurastrum insigne Z28972 Size and Shape of Cells. Cells of Phacotus lenticularis Polytoma anomale U22931 (Fig. 1a), Pteromonas protracta (Fig. 1d), and Wislouch- Polytoma difficile U22931 iella planctonica (Fig. 1g) were usually more or less Protosiphon botryoides U41177 egg-shaped and exhibited lengths of 10–16 ␮m and di- Pteromonas protracta X91627, this study ameters of 6–10 ␮m. The size of protoplasts ranged be- Spermatozopsis similis X65557 ␮ Stephanosphaera sp. U70798 tween 4–10 and 5–9 m. Dysmorphococcus globosus Tetracystis aeria U41175 cells were more voluminous (28 × 26 ␮m; Fig. 1j) and Volvox carteri X53904 spherical. Usually, the lorica was also nearly spherical Wislouchiella planctonica UTEX 1030, this study and the smaller protoplast (diameter, ∼16 ␮m) was lo- cated apically. At the basal pole the protoplast bore a 423 Table 1. Continued observed at other locations. The flagellar channels and pores exhibited a substructure more complicated than Accession those of Phacotus. All layers also developed within the Taxon No. sporangium (not shown). The ultrastructure of W. planc- rbcL outgroup tonica loricae is still unknown and could not be studied, Chlorella ellipsoidea D10997 as cultured algae did not readily develop cell walls ex- rcL ingroup cept that sometimes an (1) inner mucilaginous sheath and Carteria cerasiformias D89768 Carteria crucifera D63431 (2) an electron-opaque, woolly outer layer were observed Carteria eugametos UTEX 233 (Fig. 1h). The loricae of these three genera were known Carteria obtusa D89769 to share a common characteristic in exhibiting a rota- Carteria radiosa D89770 tional symmetry that bears a resemblance to a bilateral Chlamydomonas debaryana D86838 symmetry: In Phacotus, the lens-shaped lorica exists of Chlamydomonas moewusii M15842 Chlamydomonas radiata UTEX 966, this study two shell-like halves: in Pteromonas, the loricae are Chlamydomonas reinhardtii J01399 compressed and bear a wing-like projection; and Wis- Chlamydomonas tetragama NIES 446, this study louchiella loricae are also compressed, with a broad Chlorogonium elongatum IAM C-293, this study winglike expansion that bears two rounded horns (also Chlorogonium euchlorum CCAP 12/3, this study cf. Ettl 1983). Chloromonas serbinowii UTEX 492, this study Dunaleilla parva UTEX 1983, this study In D. globosus, the loricae consisted of two layers: (1) Dysmorphococcus globosus SAG 20-1, this study an inner mucilaginous sheath and (2) an electron-opaque Eudorina illinoisensis D63433 woolly outer layer that was mineralized with granular Gonium pectorale D63437 components (Fig. 1k). These granular mineralizations Paulschulzia pseudovolvox D86837 also accompanied the outer layer that extended inward Phacotus lenticularis KR 91/1, this study Phacotus lenticularis SAG 61/1, this study around the flagellar shafts. An apical cell wall papilla Pteromonas protracta UTEX 647, this study was present. Pteromonas angulosa KR 91/2, this study Pteromonas angulosa KR 91/3, this study Structure and Location of Pyrenoids. P. lenticularis, Volvox dissipatrix D63447 Pt. protracta, and W. planctonica contained single pyre- Volvox rousseletii D63448 noids that were located in the basal part of the more or a According to Kessler et al. (1997). less cup-shaped chloroplast. In Phacotus, the pyrenoid matrix was traversed by a single thylakoid double- footlike appendix that extended through the mucilagi- lamella (Fig. 1c). The inner thylakoid membranes that nous sheath of the lorica. touch each other were undulating. Starch was formed in two slightly curved plates on the pyrenoid. In principle, Location of Mitochondria. In P. lenticularis, Pt. pro- the pyrenoids of Pteromonas and Wislouchiella exhib- tracta, and W. planctonica, mitochondrial profiles were ited similar arrangements except they were traversed by found mainly in the central cytoplasm between the chlo- several or many thylakoid double-lamellae. Starch was roplast and the anterior cell pole (Figs. 1a,d,g). In the present in several slightly curved plates on the pyrenoid case of D. globosus, mitochondrial profiles were usually matrix (Figs. 1f,i). located in the peripheral cytoplasm, i.e., in the space In contrast, the chloroplast of D. globosus, which was between the plasmalemma and the chloroplast (Fig. 1j). sometimes more netlike than cup-shaped, contained sev- It remained unclear if there was just one ramified mito- eral pyrenoids. These were not fixed in position. Starch chondrium or several small ones. was developed in the form of a hollow sphere on the pyrenoid matrix. Several single thylakoids that were tu- Structure of Loricae. The lorica of P. lenticularis con- bular in shape traversed the starch and the pyrenoid ma- sisted of three layers: (1) an inner mucilaginous sheath, trix (Fig. 1l). (2) an electron-opaque layer, and (3) a calcified layer with electron-opaque rods (Fig. 1b). These layers are 18S rDNA. The determined 18S rDNA sequences of known to develop during sporulation within the zoospo- phacotacean species and Haematococcus pluvialis did rangium (cf. Hepperle and Krienitz 1996). At the apical not include any introns. The alignment is available from cell pole, the central layer extended inward and formed the authors upon request. Comparison of sequences the flagellar channels. Pt. protracta also developed lori- based on secondary structures revealed some hypervari- cae consisting of three layers: (1) an inner mucilaginous able regions (loop regions) that were excluded from the sheath, (2) a central, slightly electron-opaque layer, and alignment. The number of aligned positions used for the (3) an electron-opaque, outer layer (Fig. 1e). The latter phylogenetic analysis was 1,613 sites and included 643 was locally thickened such that a hexagonal network was variable sites and 4 gap-only sites. Exclusion of the out- present on the cell wall surface. The central layer was groups yielded 594 sites that were variable. thickened at the flagellar channels and could hardly be Direct comparison of the complete 18S rDNA se- 424 425

Table 2. Pairwise comparison of a 18S rDNA sequences and b rbcL sequences of Phacotaceae and of some chlamydophycean taxa that proved to be more closely relateda a 18S C. rein. C. moew. Dy. glob. D. sal. H. pluv. H. zimb. S. pluv. P. lent. Pt. prot. W. planct.

C. reinharadtii — 91.1 94.2 94.3 94 93.5 90.8 92.8 93.1 92.9 C. moewusii 141/1591 — 91.5 91.7 91.5 90.9 88.6 90.6 91 91.1 Dy. globosus UTEX 855 93/1591 136/1593 — 95.9 95 94.6 91.7 93.3 93.5 93.6 D. salina 91/1590 132/1592 65/1590 — 96.9 96.3 93.5 95.2 95.6 95.7 H. pluvialis SAG 34-1b 95/1590 135/1592 79/1592 50/1591 — 95.8 93.1 95 95.3 95.3 H. zimbabwiensis 103/1589 144/1591 86/1591 59/1590 66/1590 — 96.5 94.1 94.7 94.7 S. pluvialis 146/1589 182/1591 132/1591 103/1590 109/1590 56/1588 — 91.6 92.2 92.2 P. lenticularis KR991/1 114/1589 149/1591 107/1591 76/1590 80/1590 94/1589 133/1589 — 99 98.7 Pt. protracta UTEX 647 110/1589 143/1591 103/1591 70/1590 74/1590 85/1589 124/1589 16/1588 — 99.6 W. planct. UTEX 1030 113/1589 142/1591 102/1591 69/1590 74/1590 85/1589 124/1589 20/1588 6/1588 —

P. lent. P. lent. Pt. prot. Pt. ang. Pt. ang. b rbcL C. rein. C. moew. C. tetr. Dy. glob. Ch. elon. 91/1 61/1 647 91/2 91/3

C. reinhardtii — 89.3 91.2 86.6 91.7 89.5 91.3 90.3 90.2 90.2 C. moewusii 121/1128 — 90.8 87.9 88.4 87.5 88.3 88.4 88.5 88.5 C. tetragama 99/1128 104/1128 — 90.3 91.9 88.9 90.2 90.4 90.3 90.3 Dy. globosus SAG 20-1 151/1128 136/1128 109/1128 — 88.3 86.6 86.9 87.4 87.2 87.2 Ch. elongatum IC293 94/1128 131/1128 91/1128 132/1128 — 89.5 91.2 89.9 90.0 90.0 P. lenticularis KR 91/1 119/1128 141/1128 125/1128 151/1128 119/1128 — 96.2 94.0 93.9 93.9 P. lenticularis SAG 61/1 98/1128 132/1128 111/1128 148/1128 99/1128 43/1128 — 92.7 92.6 92.6 Pt. protracta UTEX 647 109/1128 131/1128 108/1128 142/1128 114/1128 68/1128 82/1128 — 99.4 99.4 Pt. angulosa KR 91/2 110/1128 130/1128 109/1128 144/1128 113/1128 69/1128 83/1128 7/1128 — 100.0 Pt. angulosa KR 91/3 110/1128 130/1128 109/1128 144/1128 113/1128 69/1128 83/1128 7/1128 0/1128 — a Only sites that could be aligned unequivocally were compared. For comparison, the sequences of Chlamydomonas reinhardtii, C. moewusii, and * identical characters/length of alignment ס) Dunaliella salina were included in the analysis. Numbers above the diagonal indicate the homology 100); pairs (x/y) in the triangle below the diagonal indicate the number of different sites (x)/number of sites that were compared (y). Number of sites compared: a 1612 sites and b 1128. quences (Table 2) revealed that, within the Phacotaceae, 1,803). In the following these species are termed the highest similarity existed between Pt. protracta and ‘‘Phacotaceae sensu stricto’’ because the 18S rDNA of W. planctonica (99%; 18 of 1,792 sites were different). Dysmorphococcus globosus was quite different from P. lenticularis was closely related to W. planctonica these three species [e.g., 91.7% (149/1,803) to P. len- (97.9%; 38/1,794) and Pt. protracta (97.5%; 44/1,795). ticularis]. The rDNA exhibiting the fewest differences The most closely related Chlamydophyceae were Chlam- from D. globosus was also that of D. salina (in the re- ydomonas dysosmos (e.g., to W. planctonica, 93.9%; duced data set: 93.8%; 112/1,810). 109/1,793) and Dunaliella salina (e.g., to 93.9%; 121/ Consensus trees constructed by either NJ or parsi-

< Fig. 1. Ultrastructure of phacotaceen species. a–c Phacotus lenticu- sists of three layers: an inner mucilaginous sheath, a central electron- laris; d–f Pteromonas protracta; g–i Wislouchiella planctonica; j–l opaque layer that contributes to the flagellar channels (arrowhead), and Dysmorphococcus globosus. a Cell of P. lenticularis. Note that cul- an outer electron-opaque layer. The flagellar shaft was shedded distal tured cells usually do not calcify. The presence of two pyrenoids in the to the flagellar transition zone. f Pyrenoids of the same species are distinctly cup-shaped plastid indicates that this cell is short before cell traversed by several thylakoid double-lamellae and exhibit several division. Two dictyosomes are located apically of the nucleus and the starch plates. g Oblique section through a zoospore of W. planctonica. mitochondria are located centrally (arrows). b Apical cell pole of a P. Note that only one large pyrenoid is present and that the mitochondria lenticularis cell with a calcified lorica. The latter one consists of three are more or less in the central cytoplasm (arrows). h Detail of presum- distinct layers: an inner mucilaginous sheath, an electron-opaque cen- able lorica. Here only two layers were observed. i Detail of W. planc- tral layer (arrowhead), and an outer layer that is mineralized with tonica pyrenoid showing many thylakoid double-lamellae within the calcite crystals that dissolve during preparation. Note that the flagellar electron-opaque matrix. Note the undulation of the central thylakoid channels are thickened parts of the central electron-opaque layer. c The membranes. j The cells of D. globosus are about two times larger than pyrenoid of P. lenticularis is traversed by a single double-thylakoid. those of the other species. Note that the mitochondrial profiles are Starch develops in two bended plates on the pyrenoid matrix. d Zoo- located peripherally in the space between the chloroplast and the plas- spore of Pt. protracta. Note the presence of a wing-like lorica projec- malemma (arrows) and the presence of an apical papilla (arrowhead). tion that is seen as a taillike appendage in longitudinal sections. The k Detail of the apical cell pole. The flagellar channels are part of the chloroplast bears a single pyrenoid. Mitochondrial profiles are located outer lorica layer. l Pyrenoid traversed by tubular thylakoids and sur- centrally (arrow). e Apical cell pole of Pt. protracta. Note the com- rounded by a homogeneous starch sheet. Scale bars: first column, 5 plicated structure of the flagellar channels and pores. The lorica con- ␮m; second column, 0.5 ␮m; third column, 1 ␮m. 426

Fig. 2. Consensus tree obtained from a NJ analysis of the 18S rDNA drawn through indicate branches that are supported by at least one of genes. The number pairs (x/y) at the forks indicate the number of times the two algorithms. Dotted lines indicate clusters with bootstrap values the group consisting of the species which are to the right of that fork below 50. Stable groups are also marked by brackets. One thousand six occurred among 100 calculated NJ (x) or parsimony (y) trees. Lines hundred twelve sites of the 18S rDNA gene were analyzed. mony algorithms revealed similar or identical tree to- Determination of characters that were present exclu- pologies (Fig. 2): a multifurcation into at least 13 inde- sively in highly supported clusters revealed that a set of pendent clades was observed. Six of these lineages were characters was shared only by the Phacotaceae sensu represented by only a single species. Neither deletion of stricto. These species exhibited 18 sites (1 gap; A, 7; C, single species from the alignment nor exclusion of more 2; G, 4; U, 4) that were different from all other variable regions affected the principal topology of trees. chlamydophycean 18S rDNAs. D. globosus and the other The Phacotaceae distributed into two groups that Phacotaceae did share any exclusive characters that were never clustered together. The first group contained P. not found in other chlamydophycean species. lenticularis, Pt. protracta, and W. planctonica; the sec- ond, D. globosus. The latter one represented a branch rbcL. The alignment of rbcL sequences was 1,128 that contained only this species. It exhibited no affiliation sites long and contained 399 variable sites that contrib- to either the other phacotacean species or to Haemato- uted to the phylogenetic analysis (Table 2b). Two iso- coccus pluvialis, which was found to have a weak affili- lates of Pteromonas angulosa (KR 91/2 and KR 91/3) ation to a cluster containing mainly coccal green algae had exactly the same sequence. and (H. zimbabwiensis and Stepha- The topology of consensus trees derived from maxi- nosphaera sp.). mum-likelihood, NJ, and parsimony analysis varied 427

Fig. 3. Strict consensus trees as derived from three statistical analy- was supported by higher bootstrap values (ജ50%; lines drawn ses of rbcL gene sequences. Length of the alignment was 1,128 sites. through) or that bootstrap support was not sufficient (dotted lines). a Neighbor-joining analysis (NJ). b Maximum-parsimony analysis Arrows point to those bootstrap values that indicated statistical support (MP). c Maximum-likelihood analysis (ML). Numbers at the forks for a separation of D. globosus from the other Phacotacean species. indicate the bootstrap support for the clusters that are to the right of that Note that the two Carteria groups cluster in two subsequent branches furcation. NJ and MP analyses were combined with a 1,000-fold boot- and that D. globosus is clearly separated from the other Phacotacean strap analysis, whereas ML analysis derived from 100 bootstrap repli- species in trees from NJ and MP analysis. cates. The drawing style of cluster lines indicates either that a group mainly in the arrangement of the basal groups (Figs. Discussion 3a–c). Five of the six investigated phacotaceen species comprising the genera Phacotus and Pteromonas clus- This study demonstrates that the Phacotaceae must be tered in a stable branch (bootstrap values ജ94.4). They considered as a polyphyletic family that should be split had sequence homologies of more than 96.2%. The two into at least two groups. The first group contains Phaco- Phacotus strains differed in 43 characters, whereas all tus, Pteromonas, and Wislouchiella and possibly more Pteromonas strains had a maximum of 7 differences taxa and forms an independent lineage within the (Table 2). Chlamydophyceae. We termed this group ‘‘Phacotaceae According to NJ and MP analysis, this ‘‘Phacotaceae sensu stricto,’’ as they contain the type genus Phacotus. sensu stricto’’ cluster was related to two other clusters, These taxa are closely related to each other as shown by the Volvocales branch (including Chlamydomonas rein- analysis of molecular data. The members within this hardtii) and a weakly supported branch that contained group also share a set of morphological characters. (1) Dunaliella and Chlorogonium. The bootstrap value for The protoplast is usually more or less egg-shaped and this trifurcated group was 74 and 60%, respectively contains two apical vacuoles. (2) The loricae consist, as (Figs. 3a,b). Although ML analysis yielded a similar to- far as investigated, of three layers: an inner mucilaginous pology, the bootstrap support was only 46% (Fig. 3c). sheath, an electron-opaque, clearly delineated central As already shown for the 18S rDNA genes, D. glo- layer, and a prominent surface layer. In the case of bosus did not cluster with the other species, but accord- Phacotus the latter one is calcified, whereas in Pteromo- ing to NJ and ML analysis it was found to be loosely nas it exhibits a hexagonal pattern. (3) The flagellar associated with C. moewusii and C. tetragama. MP channels are not accompanied by the outermost but by yielded a consensus tree where D. globosus could rep- the central lorica layer. (4) Mitochondria are usually lo- resent its own lineage. The bootstrap values mentioned cated in the central cytoplasm. However, a circadian above indicate that, at least in NJ and MP analysis, Dys- change in positions of the mitochondria as known from morphococcus is clearly separated from the other Phaco- other Chlamydophyceae may exist (Blank and Arnold tacean species. Direct comparison of sequences showed 1980; Blank et al. 1980; Gaffal and Schneider 1978; that this species had the highest similarity to C. tetra- Grobe and Arnold 1975; Osafune et al. 1972, 1975). The gama (90.3%; 109 of 1,128 sites were different), whereas chloroplast is typically (5) cup-shaped and contains (6) a the other Phacotacean species had at least 142 different single pyrenoid that is penetrated by one single or several characters (homologies ഛ87.4%). (7) planar thylakoids. (8) Vegetative multiplication oc- 428 curs in mucilaginous sporangia that are formed between the development of papillae, the occurrence of several parts of the mother lorica. Release of the daughter zoo- pyrenoids per chloroplast, the formation of tubular and spores occurs after (9) complete formation of the lorica lamellar thylakoid systems that traverse the pyrenoid ma- by (10) lysis of the sporangium (Hepperle and Krienitz trix, and the formation of cellular appendages that extend 1996). (11) These species overcome environmental limi- into the cell wall as present in D. globosus but also in the tations by formation of palmellae. (12) Sporopollenin- Haematococcaceae. The formation of sporopollenin-like like substances could not be detected. compounds in the cell wall also developed independently Analysis of 18S rDNA and rbcL sequence data sug- in different lineages, e.g., in Haematococcus and D. glo- gests that D. globosus represents an independent lineage bosus (Good and Chapman 1979). However, the statis- that is separated from the other phacotacean taxa, al- tical support for the separation of D. globosus from the though its phylogenetic affiliation remains unclear. Phacotaceae sensu stricto was resolved only in the phy- This second group, which possibly also includes logenetic trees based on rbcL gene sequences. In general, Cephalomonas granulata (see Hepperle et al. 1994), is the observed ‘‘terminal groups’’ such as the Phacotaceae characterized by (1) a protoplast that bears a basal foot- sensu stricto were well supported in both the rbcL and like appendage and (2) a lorica consisting of an inner the 18S rRNA gene trees, and the phylogenetic relation- mucilaginous and an outer electron-opaque, woolly ships between the major clades could not be resolved layer; (3) the flagellar channels are part of the outer readily. This was indicated by the low bootstrap values lorica layer extending inward. (4) Mitochondrial profiles and difference in tree topology between the two kinds of are located mainly in the peripheral cytoplasm. (5) The gene trees. We suppose that this may be due to rapid chloroplast is frequently netlike rather than cup-shaped evolution of the major chlamydophycean lineages within and (6) up to several pyrenoids are present in this species a short radiation period. (Bold and Starr 1953; Dawson and Harris 1987). In other Dysmorphococcus species no pyrenoid or a few pyren- oids were observed (Dawson and Harris 1979; Min-Juan References et al. 1991; Shyam 1981). (7) Each pyrenoid is pen- etrated by several tubular thylakoids that do not occur Belcher JH, Swale EMF (1967) Observations on Pteromonas tenuis sp. nov. and P. angulosa (CARTER)LEMMERMANN (Chlorophyceae, pairwise as in the other Phacotaceae. (8) At least in the Volvocales) by light and electron microscopy. 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