J. Phycol. 37, 852–865 (2001) TRADITIONAL GENERIC CONCEPTS VERSUS 18S rRNA GENE PHYLOGENY IN THE GREEN ALGAL FAMILY SELENASTRACEAE (CHLOROPHYCEAE, CHLOROPHYTA) 1 Lothar Krienitz2 Institut für Gewässerökologie und Binnenfischerei, D-16775 Stechlin, Neuglobsow, Germany Iana Ustinova Institut für Botanik und Pharmazeutische Biologie der Universität, Staudtstrasse 5, D-91058 Erlangen, Germany Thomas Friedl Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Universität Göttingen, Untere Karspüle 2, D-37037 Göttingen, Germany and Volker A. R. Huss Institut für Botanik und Pharmazeutische Biologie der Universität, Staudtstrasse 5, D-91058 Erlangen, Germany Coccoid green algae of the Selenastraceae were in- few diacritic characteristics and that contain only a vestigated by means of light microscopy, TEM, and small number of species) and to reestablish “large” 18S rRNA analyses to evaluate the generic concept in genera of Selenastraceae such as Ankistrodesmus. this family. Phylogenetic trees inferred from the 18S Key index words: 18S rRNA, Ankistrodesmus, Chloro- rRNA gene sequences showed that the studied spe- phyta, Kirchneriella, Monoraphidium, molecular system- cies of autosporic Selenastraceae formed a well- atics, morphology, Podohedriella, pyrenoid, Quadrigula, resolved monophyletic clade within the DO group of Selenastraceae Chlorophyceae. Several morphological characteris- tics that are traditionally used as generic features Abbreviations: LM, light microscopy were investigated, especially the arrangement of au- tospores in the mother cells, colony formation, and pyrenoid structure. The parallel arrangement of au- The family Selenastraceae comprises coccoid green tospores was confirmed for the genera Ankistrodes- algae of more or less elongated shape. The solitary or mus, Podohedriella, and Quadrigula. In mother cells of colonial living cells are fusiform or cylindrical with Monoraphidium and Kirchneriella, the autospores were sharp or rounded ends; cell shape is straight or sickle- arranged serially. Colony formation was either stable like to spirally curved. The main criterion for defining (Quadrigula) or variable (Ankistrodesmus, Podohedriella) the Selenastraceae as a distinct family is their special within genera. All strains studied possessed naked or mode of cell division and autospore formation. Propa- starch-covered pyrenoids within the chloroplast. The gation begins with serial divisions of the protoplast pyrenoid matrix was homogenous or penetrated by perpendicular to the longer axis of the cell. After- thylakoids. In contrast to considerations of traditional ward, the daughter cells change their position in a systematics, the present study showed that the pres- longitudinal direction. Finally, the autospores are ar- ence and structure of pyrenoids are unsuitable for dif- ranged serially or parallel within the mother cell ferentiation of genera in Selenastraceae. Furthermore, (Düringer 1958, Pickett-Heaps 1970, Ettl and Ko- the molecular analyses showed that any morphological márek 1982). Whether this criterion is congruent with criterion considered so far is not significant for the sys- real phylogenetic relationships and whether the Sele- tematics of the Selenastraceae on the generic level. Spe- nastraceae is really a monophyletic group is unclear cies assigned to different genera such as Ankistrodesmus so far and needs to be investigated by DNA sequence and Monoraphidium were not monophyletic and there- analyses. fore not distinguishable as separate genera. Species Depending on the views of different authors, the of Monoraphidium appeared in four different lin- content of the family Selenastraceae changed in the eages of the Selenastraceae. Our phylogenetic analy- course of time. The family was established by Black- ses support earlier discussions to abandon the com- man and Tansley in 1903. However, it has been used mon practice of conceiving “small” genera (i.e. genera for some time synonymously with the Scenedes- that are differentiated from other genera by only a maceae Bohlin 1904, by including Selenastrum and Scenedesmus (Silva 1980). Elsewhere, the two families were clearly separated into Scenedesmaceae with 1Received 10 January 2001. Accepted 3 July 2001. Scenedesmus as type genus and Selenastraceae with Sele- 2Author for correspondence: e-mail [email protected]. nastrum as the type genus (West and Fritsch 1927). In 852 PHYLOGENY OF SELENASTRACEAE 853 this revised edition, the Selenastraceae included the rRNA gene sequence analyses (Huss and Sogin 1990). genera Actinastrum, Ankistrodesmus, Closteriopsis, Dacty- Therefore, the present study is the first attempt to lococcus, Kirchneriella, Quadrigula, and Selenastrum. Kor- evaluate the generic concept within the Selenastraceae, shikov (1953) established the family Ankistrodesmaceae, combining morphological, ultrastructural, and molec- a synonym of the Selenastraceae, and included nine ular data. genera. Komárek and Fott (1983) included 16 genera of selenastracean algae (within the subfamily Anki- materials and methods strodesmoideae) in their monograph of coccoid green Algal cultures and microscopy. Algal strains were obtained from algae. Marvan et al. (1984) investigated 18 genera of the SAG Culture Collection (Sammlung von Algenkulturen at /epsag/phykologiaف/the Selenastraceae by means of numerical evaluation Göttingen University, http://www.gwdg.de of morphological and ontogenetic characteristics. epsag.html; Table 1) and grown in a modified Waris solution The shape of the cells or colonies, the arrangement of (McFadden and Melkonian 1986) in aerated suspensions at room temperature under continuous cold fluorescent illumina- autospores inside the mother cell, the development tion of about 100 ␮mol photonsؒmϪ2ؒsϪ1. Cultures were aerated of mucilage and encrustations on the cell wall, and without CO2 enrichment to support the development of the presence, number, and type of pyrenoids were pyrenoids (Miyachi et al. 1986). used for differentiation of the genera. However, sev- The algae were investigated using a Diaplan (Leica, Wetzlar, eral genera differ from each other by only one of Germany) light microscope with differential interference con- trast. The algal strains were determined according to the keys the above characters. For example, Selenastrum dif- published by Komárková-Legnerová (1969), Komárek and Fott fers from Ankistrodesmus by the curvature of the cells (1983), and Hindák (1988). For TEM, the cultured cells were (Komárek and Comas 1982), Raphidocelis differs from prepared and investigated according to Krienitz et al. (1983) Kirchneriella by cell wall incrustations (Hindák 1977), (fixation in 1% KMnO4 and postfixation in 1% OsO4 using and Chlorolobion differs from Monoraphidium by the phosphate buffer) or Krienitz et al. (1996) (fixation in 2.5% glutaraldehyde and postfixation in 2% OsO4 using HEPES presence of a pyrenoid with starch envelope (Ko- buffer). Observations were made using a TESLA BS 500 or BS márek 1979). The use of only one diacritic character 540 (Brno, Czech Republic) electron microscope. for separation of genera has led to a confused situa- DNA extraction, PCR, and phylogenetic analyses. DNA was iso- tion in the taxonomy of the Selenastraceae. Often lated from cells as described previously (Huss et al. 1986). 18S rRNA genes were amplified by PCR and directly sequenced ei- these features are highly variable and difficult to dis- ther with a T7 Sequenase PCR Product Sequencing Kit (Amer- cern by light microscopy (LM) (Heynig and Krienitz sham Pharmacia Biotech, Piscataway, NJ) or in an ABI Prism 1982, Nygaard et al. 1986). Furthermore, it seems 310 Genetic Analyzer (Perkin-Elmer Cetus, Applied Biosystems to be questionable whether these “small” genera do Division, Foster City, CA). PCR conditions and sequencing primers were the same as described previously (Huss et al. reflect the real phylogenetic relationship within the 1999). All 18S rRNA gene sequences were deposited at the family. EMBL Nucleotide Sequence Database (Table 1). GenBank ac- To elucidate these questions, we studied an assort- cession numbers of reference 18S rRNA gene sequences are ment of strains of selenastracean algae that represent given in Table 1. The sequences were manually aligned with a broad range of diacritical characters. Strains repre- reference sequences as previously described (Huss et al. 1999). Highly variable regions that could not be aligned unambigu- senting the following morphological criteria were se- ously and regions of PCR primers were excluded from the anal- lected: (1) needle-like fusiform cells living in colonies yses (i.e. positions 1–34, 232, 233, 492–495, 665–667, 1362– (Ankistrodesmus, Quadrigula) versus solitary living fusi- 1371, 1703–1714, 1770–1796) compared with the Chlorella vul- form cells (Monoraphidium); (2) fusiform cells with het- garis sequence (X13688). The alignment used for phylogenetic analyses contained 1705 positions, 537 of which were variable eropolar differentiation (Podohedriella); (3) range of differ- and 392 informative for parsimony analyses. The alignment is ent morphotypes: fusiform cells (Monoraphidium braunii, available from T. Friedl ([email protected]) and from TreeBase M. neglectum, M. terrestre), cylindrical cells (Monoraphidium (http://herbaria.harvard.edu/treebase/) under the accession dybowskii), moon-like arcuated to bean-shaped cells (Anki- no. S664. To further examine phylogenetic relationships within strodesmus