bashan foundation - traditional generic …...852 j. phycol. 37, 852–865 (2001) traditional...

852

J. Phycol.

37,

852–865 (2001)

TRADITIONAL GENERIC CONCEPTS VERSUS 18S rRNA GENE PHYLOGENY IN THE GREEN ALGAL FAMILY SELENASTRACEAE (CHLOROPHYCEAE, CHLOROPHYTA)

1

Lothar Krienitz

2

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-vestigated by means of light microscopy, TEM, and18S rRNA analyses to evaluate the generic concept inthis family. Phylogenetic trees inferred from the 18SrRNA gene sequences showed that the studied spe-

cies of autosporic Selenastraceae formed a well-resolved monophyletic clade within the DO group ofChlorophyceae. Several morphological characteris-tics that are traditionally used as generic featureswere investigated, especially the arrangement of au-tospores in the mother cells, colony formation, andpyrenoid structure. The parallel arrangement of au-tospores was confirmed for the genera

Ankistrodes-mus

,

Podohedriella

, and

Quadrigula.

In mother cells of

Monoraphidium

and

Kirchneriella

, the autospores werearranged serially. Colony formation was either stable(

Quadrigula

) or variable (

Ankistrodesmus

,

Podohedriella

)within genera. All strains studied possessed naked orstarch-covered pyrenoids within the chloroplast. Thepyrenoid matrix was homogenous or penetrated bythylakoids. In contrast to considerations of traditionalsystematics, the present study showed that the pres-ence and structure of pyrenoids are unsuitable for dif-ferentiation of genera in Selenastraceae. Furthermore,the molecular analyses showed that any morphologicalcriterion considered so far is not significant for the sys-tematics of the Selenastraceae on the generic level. Spe-cies assigned to different genera such as

Ankistrodesmus

and

Monoraphidium

were not monophyletic and there-fore not distinguishable as separate genera. Speciesof

Monoraphidium

appeared in four different lin-eages of the Selenastraceae. Our phylogenetic analy-ses support earlier discussions to abandon the com-mon practice of conceiving “small” genera (i.e. generathat are differentiated from other genera by only a

few diacritic characteristics and that contain only asmall number of species) and to reestablish “large”genera of Selenastraceae such as

Ankistrodesmus.

Key index words:

18S rRNA,

Ankistrodesmus

, Chloro-phyta,

Kirchneriella

,

Monoraphidium

, molecular system-atics, morphology,

Podohedriella

, pyrenoid,

Quadrigula

,Selenastraceae

Abbreviations:

LM, light microscopy

The family Selenastraceae comprises coccoid greenalgae of more or less elongated shape. The solitary orcolonial living cells are fusiform or cylindrical withsharp or rounded ends; cell shape is straight or sickle-like to spirally curved. The main criterion for definingthe Selenastraceae as a distinct family is their specialmode of cell division and autospore formation. Propa-gation begins with serial divisions of the protoplastperpendicular to the longer axis of the cell. After-ward, the daughter cells change their position in alongitudinal direction. Finally, the autospores are ar-ranged serially or parallel within the mother cell(Düringer 1958, Pickett-Heaps 1970, Ettl and Ko-márek 1982). Whether this criterion is congruent withreal phylogenetic relationships and whether the Sele-nastraceae is really a monophyletic group is unclearso far and needs to be investigated by DNA sequenceanalyses.

Depending on the views of different authors, thecontent of the family Selenastraceae changed in thecourse of time. The family was established by Black-man and Tansley in 1903. However, it has been usedfor some time synonymously with the Scenedes-maceae Bohlin 1904, by including Selenastrum andScenedesmus (Silva 1980). Elsewhere, the two familieswere clearly separated into Scenedesmaceae with

Scenedesmus

as type genus and Selenastraceae with

Sele-nastrum

as the type genus (West and Fritsch 1927). In

1

Received 10 January 2001. Accepted 3 July 2001.

2

Author for correspondence: e-mail [email protected].

PHYLOGENY OF SELENASTRACEAE

853

this revised edition, the Selenastraceae included thegenera

Actinastrum

,

Ankistrodesmus

,

Closteriopsis

,

Dacty-lococcus

,

Kirchneriella

,

Quadrigula

, and

Selenastrum.

Kor-shikov (1953) established the family Ankistrodesmaceae,a synonym of the Selenastraceae, and included ninegenera. Komárek and Fott (1983) included 16 generaof selenastracean algae (within the subfamily Anki-strodesmoideae) in their monograph of coccoid greenalgae. Marvan et al. (1984) investigated 18 genera ofthe Selenastraceae by means of numerical evaluationof morphological and ontogenetic characteristics.The shape of the cells or colonies, the arrangement ofautospores inside the mother cell, the developmentof mucilage and encrustations on the cell wall, andthe presence, number, and type of pyrenoids wereused for differentiation of the genera. However, sev-eral genera differ from each other by only one ofthe above characters. For example,

Selenastrum

dif-fers from

Ankistrodesmus

by the curvature of the cells(Komárek and Comas 1982),

Raphidocelis

differs from

Kirchneriella

by cell wall incrustations (Hindák 1977),and

Chlorolobion

differs from

Monoraphidium

by thepresence of a pyrenoid with starch envelope (Ko-márek 1979). The use of only one diacritic characterfor separation of genera has led to a confused situa-tion in the taxonomy of the Selenastraceae. Oftenthese features are highly variable and difficult to dis-cern by light microscopy (LM) (Heynig and Krienitz1982, Nygaard et al. 1986). Furthermore, it seemsto be questionable whether these “small” genera doreflect the real phylogenetic relationship within thefamily.

To elucidate these questions, we studied an assort-ment of strains of selenastracean algae that representa broad range of diacritical characters. Strains repre-senting the following morphological criteria were se-lected: (1) needle-like fusiform cells living in colonies(

Ankistrodesmus

,

Quadrigula

) versus solitary living fusi-form cells (

Monoraphidium

); (2) fusiform cells with het-eropolar differentiation (

Podohedriella

); (3) range of differ-ent morphotypes: fusiform cells (

Monoraphidium braunii

,

M. neglectum

,

M. terrestre

), cylindrical cells (

Monoraphidiumdybowskii

), moon-like arcuated to bean-shaped cells (

Anki-strodesmus bibraianus

,

Kirchneriella aperta

); and (4) range ofdifferent pyrenoid types: naked (

M. neglectum

) versussheathed pyrenoids (

M. braunii

,

M. terrestre

); central lo-cated spherical one-pieced

pyrenoids (A. gracilis, M. dy-bowskii

) versus multiformed pyrenoids (

Podohedriella falcata

,

Quadrigula closterioides

); pyrenoids with (e.g.

Ankistrodesmusfusiformis

) or without (e.g.

A. gracilis)

traversing thylakoids.This selection includes the most common genera of thefamily.

The aim of our study was to investigate whether ornot the classification of genera according to the tradi-tional morphological approach is congruent with re-sults from 18S rRNA gene sequence analyses andwhether the Selenastraceae is a natural monophyleticfamily of chlorophycean algae. So far, only one spe-cies of the Selenastraceae,

Ankistrodesmus stipitatus

(Chodat) Komárková-Legnerová, was included in 18S

rRNA gene sequence analyses (Huss and Sogin 1990).Therefore, the present study is the first attempt toevaluate the generic concept within the Selenastraceae,combining morphological, ultrastructural, and molec-ular data.

materials and methods

Algal cultures and microscopy.

Algal strains were obtained fromthe SAG Culture Collection (Sammlung von Algenkulturen atGöttingen University, http://www.gwdg.de/

�

epsag/phykologia/epsag.html; Table 1) and grown in a modified Waris solution(McFadden and Melkonian 1986) in aerated suspensions atroom temperature under continuous cold fluorescent illumina-tion of about 100

�

mol photons

�

m

�

2

�

s

�

1

. Cultures were aeratedwithout CO

2

enrichment to support the development ofpyrenoids (Miyachi et al. 1986).

The algae were investigated using a Diaplan (Leica, Wetzlar,Germany) light microscope with differential interference con-trast. The algal strains were determined according to the keyspublished by Komárková-Legnerová (1969), Komárek and Fott(1983), and Hindák (1988). For TEM, the cultured cells wereprepared and investigated according to Krienitz et al. (1983)(fixation in 1% KMnO

4

and postfixation in 1% OsO

4

usingphosphate buffer) or Krienitz et al. (1996) (fixation in 2.5%glutaraldehyde and postfixation in 2% OsO

4

using HEPESbuffer). Observations were made using a TESLA BS 500 or BS540 (Brno, Czech Republic) electron microscope.

DNA extraction, PCR, and phylogenetic analyses.

DNA was iso-lated from cells as described previously (Huss et al. 1986). 18SrRNA genes were amplified by PCR and directly sequenced ei-ther with a T7 Sequenase PCR Product Sequencing Kit (Amer-sham Pharmacia Biotech, Piscataway, NJ) or in an ABI Prism310 Genetic Analyzer (Perkin-Elmer Cetus, Applied BiosystemsDivision, Foster City, CA). PCR conditions and sequencingprimers were the same as described previously (Huss et al.1999). All 18S rRNA gene sequences were deposited at theEMBL Nucleotide Sequence Database (Table 1). GenBank ac-cession numbers of reference 18S rRNA gene sequences aregiven in Table 1. The sequences were manually aligned withreference sequences as previously described (Huss et al. 1999).Highly variable regions that could not be aligned unambigu-ously and regions of PCR primers were excluded from the anal-yses (i.e. positions 1–34, 232, 233, 492–495, 665–667, 1362–1371, 1703–1714, 1770–1796) compared with the

Chlorella vul-garis

sequence (X13688). The alignment used for phylogeneticanalyses contained 1705 positions, 537 of which were variableand 392 informative for parsimony analyses. The alignment isavailable from T. Friedl ([email protected]) and from TreeBase(http://herbaria.harvard.edu/treebase/) under the accessionno. S664. To further examine phylogenetic relationships withinthe Selenastraceae, a second reduced data set was constructedthat contained only selenastracean sequences as ingroup, andno sequence positions were excluded. This data set contained1741 positions, 206 of which were variable and 93 were parsi-mony informative.

For the phylogenetic analyses of the first data set, the rDNAsequences of

Nephroselmis olivacea

and

Pseudoscourfieldia marina

were used as outgroup taxa (Steinkötter et al. 1994). Three in-dependent types of data analyses were used to assess the evolu-tionary relationships resolved in the rDNA phylogenies andwere done using PAUP* V4.0b8 (Swofford 2001). In the maxi-mum parsimony analyses, the sites were weighted (rescaledconsistency index over an interval of 1–1000; Bhattacharya1996) and then used as input for bootstrap analyses (2000 repli-cations). Gaps were treated as missing data. Heuristic searchconditions were with starting trees built stepwise with 10 ran-dom additions of taxa, using the tree bisection-reconnectionbranch-swapping algorithm to find the best tree. Best scoringtrees were held at each step. For distance and maximum likeli-hood (ML) analyses, a model for the process of DNA substitu-tion (Felsenstein 1988) that fits the data best was chosen using

854

LOTHAR KRIENITZ ET AL.

the program MODELTEST 3.04 (Posada and Crandall 1998).This program calculates the likelihood test statistic from theevaluation of a null hypothesis tree under 56 different modelsof nucleotide evolution (Huelsenbeck and Crandall 1997,Posada and Crandall 1998). For MODELTEST, the null hy-pothesis tree is calculated from the data set under the assump-

tion of equal base frequencies, transition rate equals tranver-sion rate, rates equal among sites, and no invariable sites (JC69model; Jukes and Cantor 1969). The program uses a matrix oflog likelihood scores resulting from the execution of a block ofPAUP* (Swofford 2001) commands, which is available from thedocumentation of MODELTEST (Posada and Crandall 1998).

Table

1. List of algal strains used in this study, their origin, and GenBank accession numbers for 18S rRNA gene sequences.

Class GenBank

Family/group/order accession

Species Origin number

ChlorophyceaeSelenastraceae

a

Ankistrodesmus bibraianus

(Reinsch) Korshikov SAG

b

12.94 Y17924

Ankistrodesmus fusiformis

Corda SAG 2005 X97352

Ankistrodesmus gracilis

(Reinsch) Korshikov SAG 278-2 Y16937

Ankistrodesmus stipitatus

(Chodat) Kom.-Legn. SAG 202-5 X56100

Kirchneriella aperta

Teiling SAG 2004 AJ271859

Monoraphidium braunii

(Näg. in Kütz.) Kom.-Legn. SAG 2006 AJ300527

Monoraphidium dybowskii

(Wolosz.) Hindák et Kom.-Legn. SAG 202-7e Y16939

Monoraphidium neglectum

Heynig et Krienitz SAG 48.87 AJ300526

Monoraphidium terrestre

(Bristol) Krienitz et Klein SAG 49.87 Y17817

Podohedriella falcata

(Düringer) Hindák SAG 202-2 X91263

Quadrigula closterioides

(Bohlin) Printz SAG 12.94 Y17924DO group

Botryococcus braunii

Kütz. Berkeley strain X78276

Bracteacoccus aerius

Bisch. et Bold UTEX

c

1250

3

U63101

Bracteacoccus grandis

Bisch. et Bold UTEX 1246 U63100

Bracteacoccus minor

(Chodát) Petrová UTEX 66 U63097

Desmodesmus communis

(Hegew.) Hegew. UTEX 76 X73994

Hydrodictyon reticulatum

(L.) Lagerh. Carolina Biol. Supply M74497

Neochloris aquatica

Starr UTEX 138 M62861

Scenedesmus obliquus

(Turp.) Kütz. SAG 276-3a X56103CW group

Chlamydopodium vacuolatum

(Lee et Bold) Floyd et Watanabe UTEX 2111 M63001

Dunaliella salina

Teodorescu O. Parra strain M84320

Haematococcus pluvialis

Flotow em. Wille SAG 34-1b AF159369

Protosiphon botryoides

(Kützing) Klebs UTEX 99 U41177Chaetopeltidales

Chaetopeltis orbicularis

Berthold UTEX 422 U83125

Hormotilopsis tetravacuolaris

Arce et Bold UTEX 946 U83126

Planophila terrestris

Groover et Hofstetter UTEX 1709 U83127Trebouxiophyceae

Chlorella vulgaris

Beijerinck SAG 211-11b X13688

Choricystis minor

(Skuja) Fott SAG 251-1 X89012

Dictyochloropsis reticulata

(Tschermak-Woess) Reisigl CCHU

d

5616 Z47207

Fusochloris perforata

(Lee & Bold) Floyd et Watanabe UTEX 2104 M62999

Microthamnion kuetzingianum

Näg. UTEX 1914 Z28974Myrmecia astigmatica Vinatzer ASIBe T76 Z47208Nanochlorum eucaryotum Wilhelm et al. SAG 55.87 X06425Trebouxia impressa Ahmadjian UTEX 892 Z21551Watanabea reniformis Hanagata et al. SAG 211-9b X73991

UlvophyceaeAcrosiphonia sp. Aghard SAG 127.80 UO3757Enteromorpha intestinalis (L.) Link AF189077Monostroma grevillei (Thurét) Wittrock AF015279Ulothrix zonata Kütz. SAG 38.86 Z47999Ulva curvata (Kütz.) De Toni AF189078

PrasinophyceaeNephroselmis olivacea Stein SAG 40.89 X74754Pseudoscourfieldia marina (Throndsen) Manton CCMPf 717 X75565Scherffelia dubia (Scherffel) Pascher SAG 17.89 X68484Tetraselmis striata Butcher CCMP 443 X70802

a 18S rRNA gene sequences for this group were all determined in this work except for Ankistrodesmus stipitatus (Huss and Sogin1990).

b SAG-Culture Collection of Algae at University of Göttingen, Germany.c UTEX-Culture Collection of Algae at University of Texas, Austin, USA.d CCHU-Culture Collection of Algae at University of Hiroshima, Japan.e ASIB-Culture Collection of Algae at University of Innsbruck, Austria.f CCMP-Culture Collection of Marine Phytoplankton, West Boothbay Harbor, Maine, USA.

PHYLOGENY OF SELENASTRACEAE 855

Based on the likelihood statistic, base frequencies and a ratematrix of base substitutions were chosen as calculated underthe GTR model (Rodrìguez et al. 1990) with the proportion ofinvariable sites of 0.5578 and a gamma distribution with a shapeparameter � � 0.8244. Distance analyses were then done in twoways. In the first approach, the neighbor-joining method (Sai-tou and Nei 1987) was used to build a tree from a distance ma-trix calculated using the above settings for the GTR nucleotideevolution model. In the second approach, the minimum evolu-tion criterion (ME, Rzhetsky and Nei 1992) was used in con-junction with the same model and the same heuristic searchprocedure as in the maximum parsimony analyses was selected.Bootstrap analyses with 2000 replications were done with thetwo distance methods. As a third independent type of analysis,ML searches were done under the GTR model and values forthe proportion of invariable sites and gamma distribution asused in the distance analyses. The tree topology obtained fromthe first ML run was used as input for a second run to obtain atree with a better –ln likelihood. Due to computational limits,no bootstrap tests were performed using the ML method. Forthe second (reduced) data set, ME distance analyses, weightedmaximum parsimony, and ML analyses were applied as for thefirst data set. In addition, ML bootstrap analysis was done with200 replications. Desmodesmus communis, Scenedesmus obliquus,and Neochloris aquatica were chosen as outgroup taxa becausethese species appeared to be next to the Selenastraceae in anal-yses of the first (complete) data set.

resultsMicroscopy. The cells were investigated for their

shape, dimensions, autospore arrangement, colonyformation, and pyrenoid structure (Table 2). A moredetailed documentation of the diacritical charactersof the strains including description and micrographsare available from L.K. Here we focus on newly foundmorphological facets, especially overlapping charac-ters in colony formation (Fig. 1, a–d, h) and criteriathat led to new designations of strains (Fig. 1, b–d).Mostly, the cells of Ankistrodesmus stipitatus were ar-ranged in parallel in free floating fascicular colonies.However, numerous cells, especially such that were at-tached to the walls of the glass tubes, produced muci-lage on their ends (Fig. 1a). The basal mucilage andcone-shaped fragments of the mother cell wall rem-nants joined different ontogenetic stages of cells.These affixed colonies showed transitions to the ge-nus Podohedriella. The colonies of Ankistrodesmus fusi-formis often were unstable and disintegrated into soli-

tary cells or cruciform colonies that were joined by acentral mucilaginous area; sometimes they did not ex-hibit any mucus (Fig. 1b). The cells of Podohedriella fal-cata showed heteropolar morphology. The apical endswere sharply pointed, whereas the basal ends were bluntlypointed. After negative staining with India ink, the basalend exhibited a mucilaginous lump (Fig. 1c). The cellswere solitary or formed colonies of four or eight cells at-tached by the basal ends in a common mucilaginouslump (Fig. 1d). Whereas the cells of Monoraphidium brau-nii and Monoraphidium dybowskii were mostly solitary (Fig.1, e and f), an increasing tendency of production of mu-cilage on the apical ends in Monoraphidium neglectum wasobserved (Fig. 1g). Especially in Monoraphidium terrestrethe apices were covered by remarkable amounts of muci-lage. Therefore, the cells were often attached to eachother with one pole (Fig. 1h).

In all strains that were studied we found pyrenoids,even visible under LM (Fig. 1, e–g, i). The ultrastruc-ture of pyrenoids showed a wide range of features re-garding the formation and distribution of the pyrenoidmatrix within the chloroplasts and the development ofstarch envelopes and penetrating thylakoids. In Anki-strodesmus bibraianus a spherical pyrenoid was situatedin the central region of the chloroplast (Fig. 2a). Thyla-koids did not penetrate the pyrenoid matrix but wereloosely arranged within its periphery. The pyrenoidwas naked (i.e. not covered by a starch envelope; Fig.2a) or some small starch grains (not shown) coveredit. The naked ellipsoidal pyrenoid of A. fusiformis waspenetrated by numerous thylakoids (Fig. 2b). Ankistrodes-mus gracilis formed spherical pyrenoids, surrounded by asometimes reduced envelope of small starch grains, andpenetrated only in the peripheral part by thylakoids(Fig. 2, c and d). In A. stipitatus the naked ellipsoidalpyrenoid was penetrated by thylakoids. (Fig. 2e). Thepyrenoid in Kirchneriella aperta was spherical and starchcovered (Fig. 3a). The pyrenoid matrix was penetratedby only few thylakoids (Fig. 3b). In Monoraphidium brau-nii the spherical or ellipsoidal pyrenoid was covered bystarch grains and penetrated by thylakoids (Fig. 4a). Asection of the pyrenoid showed thylakoids in longitu-dinal and crosswise direction. Thus, the thylakoids canpenetrate even the central region of the pyrenoid. Se-

Table 2. Morphological characteristics of algal strains used in this study.

Species Cell size

Cell shape(length � breadth)

(�m)Arrangementof autospores

Colonyformation

Starchenvelope

Pyrenoidpenetratingthylakoids

Ankistrodesmus bibraianus Thick fusiform, crescent-shaped 15–36 � 2–4.5 Parallel � � �Ankistrodesmus fusiformis Fusiform 30–45 � 3–6 Parallel � � �Ankistrodesmus gracilis Fusiform, sickle-like bent 20–35 � 2–4 Parallel � � �Ankistrodesmus stipitatus Fusiform, spindle-shaped 45–78 � 2.5–6 Parallel � � �Kirchneriella aperta Bean-shaped 6–11 � 5–8 Serial � � �Monoraphidium braunii Spindle-shaped, cylindrical 15–45 � 2–8 Serial � � �Monoraphidium dybowskii Cylindrical 6–17 � 3–6 Serial � � �Monoraphidium neglectum Spindle-shaped 15–37 � 2–6.5 Serial � � �Monoraphidium terrestre Spindle-shaped 14–30 � 2–8 Serial � � �Podohedriella falcata Needle-shaped, heteropolar 28–45 � 1.5–3.5 Parallel � � �Quadrigula closterioides Cylindrical 15–36 � 2–4.5 Parallel � � �

856 LOTHAR KRIENITZ ET AL.

FIG. 1. Light micrographs of Selenastraceae in culture. Scale bar, 10 �m. Arrowheads indicate the pyrenoid region in the chloro-plast. (a) Ankistrodesmus stipitatus SAG 202-5; different stages of mother cells are attached at their basal region, a cone-shaped mothercell wall remnant (empty star) is covering the bases of two mother cells; mucilage is excreted by one cell (arrow). (b) Ankistrodesmus fusi-formis SAG 2005; young cruciforme colony. (c) Podohedriella falcata SAG 202-2; young solitary mother cell with mucilaginous basal end(arrow). (d) Podohedriella falcata SAG 202-2; autospores that are fixed with their basal ends within a mucilaginous lump (arrow); nega-tive staining. (e) Monoraphidium braunii SAG 2006; autospores and vegetative cells. (f) Monoraphidium dybowskii SAG 202-7e; vegetativecells. (g) Monoraphidium neglectum SAG 48.87; vegetative cells. (h) Monoraphidium terrestre SAG 49.87; young vegetative cells connectedby a mucilaginous lump (arrow) in groups of two or four cells. (i) Quadrigula closterioides SAG 12.94; colony with four vegetative cells.


rial cuts of the pyrenoid region of M. dybowskii showedthat the pyrenoid was covered but not penetrated by thy-lakoids. The chloroplasts contained numerous starchgrains sometimes covering the pyrenoid region (Fig. 4,

b–e). The ellipsoidal pyrenoid in M. neglectum was na-ked and the pyrenoid matrix was not penetrated by thy-lakoids (Fig. 4f). In Monoraphidium terrestre we foundnaked pyrenoids situated near the inner edge of the

FIG. 2. Transmission electron micrographs of members of the genus Ankistrodesmus in culture. Scale bar, 1 �m. (a) Ankistrodesmus bi-braianus SAG 278-1; the chloroplast contains a large central pyrenoid; the protoplasm is filled with numerous vacuoles. (b) Ankistrodesmusfusiformis SAG 2005; the pyrenoid is penetrated by numerous thylakoids; fixation with KMnO4. (Note the difference to fixation with glu-taraldehyde, e.g. in e. Fixation with KMnO4 leads to a darker contrast of thylakoids.) (c) Ankistrodesmus gracilis SAG 278-2; cross-section ofchloroplast with a centrally situated pyrenoid. (d) Ankistrodesmus gracilis SAG 278-2; diagonal section. (e). Ankistrodesmus stipitatus SAG202-5; the naked ellipsoidal pyrenoid is penetrated by thylakoids. The pyrenoid and the nucleus are separated only by the double mem-branes of the chloroplast and the nucleus with a narrow zone of protoplasm in between. C, chloroplast; D, dictyosome; M, mitochon-drion; N, nucleus; V, vacuole; PY, pyrenoid; S, starch grain; arrowheads indicate thylakoid membranes penetrating the pyrenoid matrix.


chloroplast. The pyrenoid matrix was penetrated by onlyfew thylakoids (Fig. 4g). The chloroplasts of Podohedriellafalcata contained multiform compound pyrenoids thatwere sometimes distributed as subunits in different partsof the chloroplast. These subunits were always in closevicinity to the nucleus and mitochondrion. Starch en-velopes (Fig. 5a) did not surround pyrenoid subunits,although the chloroplast contained numerous starchgrains between the thylakoids. The pyrenoid matrixwas not penetrated by thylakoids. The basal end of thecell exhibited no distinct substructure and was filledwith protoplasm (Fig. 5b). The parietal chloroplast ofQuadrigula closterioides contained an elongated pyrenoid in

opposite position to the nucleus. With LM this pyrenoidcould be distinguished as an elongated homogeneousarea (Fig. 1i). With TEM it became evident that the elon-gated pyrenoid occupied a relative large area of thechloroplast. The pyrenoid was penetrated by thyla-koids (Fig. 5c) and occurred with or without coveringstarch grains.

Phylogenetic analysis. The Selenastraceae formed anindependent lineage whose monophyletic origin withinthe DO group of the Chlorophyceae was well supportedin bootstrap tests (Fig. 6). The DO group comprises coc-coid green algae that form biflagellate zoospores withdirectly opposed basal bodies (Bracteacoccus, Neochloris,Hydrodictyon) or are autosporic (Selenastraceae, Desmodes-mus, Scenedesmus; without the ability to form zoospores).Monophyly of the DO group, including the Selena-straceae, within the Chlorophyceae was also well sup-ported in bootstrap analyses (Fig. 6). Taxa with clock-wise oriented basal bodies in motile cells (CW group)formed another well-supported lineage within the Chlor-ophyceae (Fig. 6). CW and DO groups had a sister–group relationship in our analyses (Fig. 6), but it was onlyweakly supported in bootstrap analyses using distancemethods. The Chaetopeltidales, which form quadriflagel-late motile stages that exhibit directly opposed (DO)basal bodies, did not cluster with DO group membersbut formed an independent lineage that was basal tothe radiation of CW and DO groups (Fig. 6). Mono-phyly of the Chlorophyceae was well supported in boot-strap tests (Fig. 6). The sister–group relationship of thisclass with the Trebouxiophyceae and the monophyleticorigin of the Trebouxiophyceae received only low ormoderate bootstrap support (Fig. 6).

Absolute pairwise distances among rDNA sequencesfrom the Selenastraceae ranged from 7 (between A.stipitatus and A. fusiformis) to 58 (between Q. closterioidesand Kirchneriella aperta). Within the Selenastraceae onlyvery few relationships were unambiguously resolved.There was good bootstrap support, using both distanceand parsimony methods, for the monophyletic originof Monoraphidium dybowskii and Ankistrodesmus gracilisand a sister–group relationship of Quadrigula closteri-oides with the M. dybowskii/A. gracilis lineage (Fig. 6).Two pairs of taxa with almost identical sequences re-ceived high bootstrap support. Between Podohedriellafalcata and M. neglectum the absolute pairwise differ-ences were seven, and between A. stipitatus and A. fusi-formis, there were eight sequence positions different.The clustering of Ankistrodesmus bibraianus with Kirch-neriella aperta was only supported in bootstrap tests us-ing distance methods but not with maximum parsi-mony. The weighted maximum parsimony analysesrevealed moderate bootstrap support for some addi-tional relationships within the Selenastraceae, butthey were not supported using distance methods (Fig.6). Distance, maximum parsimony, and ML tree to-pologies were identical within the Selenastraceae. Tofurther examine phylogenetic relationships within thegroup, a second (reduced) data set with only selena-stracean sequences was analyzed. However, no better

FIG. 3. Transmission electron micrographs of Kirchneriellaaperta in culture. Scale bar, 1 �m. (a) Kirchneriella aperta SAG2004; the longitudinal section shows the parietal chloroplastwith a central pyrenoid and numerous vacuoles in the proto-plasm. (b) Kirchneriella aperta SAG 2004; transversal section ofthe convex part of the cell with a pyrenoid surrounded bystarch grains and penetrated by some smooth thylakoids (ar-rowhead). Abbreviations are as in Figure 2.


FIG. 4. Transmission electron micrographs of members of the genus Monoraphidium in culture. Scale bar, 1 �m. (a) Monoraphid-ium braunii SAG 2006; the starch-sheathed pyrenoid is penetrated by numerous thylakoids that are sectioned longitudinal (arrow-heads) or transversal (arrow). (b–e) Serial sections of Monoraphidium dybowskii SAG 202-7e; the longitudinal section are arrangedfrom the outer to the inner region of the cell. (b) The pyrenoid is sectioned only at its surface. (c) The pyrenoid matrix becomes vis-ible but covering thylakoids are also sectioned. (d) The pyrenoid matrix is free of thylakoids. (e) Section through the central regionof the pyrenoid. (f) Monoraphidium neglectum SAG 48.87; the naked ellipsoidal pyrenoid filling the central region of the chloroplast issituated close to the nucleus that is divided into two daughter nuclei. (g) Monoraphidium terrestre SAG 49.87; the naked pyrenoids arepenetrated by few thylakoids (arrowheads); starch grains are distributed irregularly in the chloroplast but do not surround thepyrenoid matrix. Abbreviations are as in Figure 2.


resolution was obtained with this second data set.Bootstrap support for some nodes was even lower thanwith the first data set (Fig. 7). The monophyletic ori-gin of A. gracilis with M. dybowskii received only littlebootstrap support in ME and ML bootstrap analyses,and also the sister–group relationship of the A. graci-lis/M. dybowskii/Q. closterioides lineage with M. neglec-tum and Podohedriella falcata was only poorly supportedin ME and ML bootstrap analyses of the second dataset (Fig. 7). As with the first data set, other relationshipswithin the Selenastraceae were ambiguous except forthose pairs of taxa whose 18S rRNA gene sequences werealmost identical (Fig. 7). Absolute pairwise distancesamong rDNA sequences from the Selenastraceae rangedfrom 7 (between A. stipitatus and A. fusiformis) to 58 (be-tween Q. closterioides and Kirchneriella aperta).

discussionMonophyly of the family Selenastraceae and within-group

relationships. All 11 species studied formed a mono-phyletic clade within the Chlorophyceae. The Selena-straceae were included in the DO group. Chapman etal. (1998) already showed that some autosporic chlo-rococcalean taxa (i.e. Scenedesmus, Ankistrodesmus) wereincluded in this lineage. The family Selenastraceae inthe broad traditional sense (summarized by Komárekand Fott 1983) contained genera that, based on mo-lecular phylogenetic analyses, should be transferredinto other families and classes: Hyaloraphidium curvatumKorshikov SAG 235-1 was shown to belong to lowerfungi, close to Chytridiomycetes (Ustinova et al. 2000),Closteriopsis acicularis (G.M. Smith) Belcher et Swale SAG11.86 was closely related to the genus Chlorella (Ustinovaet al. 2001), and Keratococcus bicaudatus (A. Braun)Boye-Petersen SAG 202-11 and Pseudococcomyxa simplex(Mainx) Fott KR 1986/1, which also belonged to theTrebouxiophyceae (unpublished data). Our findingssupport the view of Kessler (1982), who summarizedhis chemotaxonomical studies on the Selenastraceaeby emphasizing the high uniformity in biochemicalcharacters within this family. Resolving relationshipswithin the Selenastraceae using 18S rRNA genes se-quences is, at present, still rather limited despite theconsiderable number of variable sequence positionsamong Selenastracean taxa (7–58). There was a con-flict in bootstrap support for some nodes between thelarge data set (Fig. 6) and the second data set that wasreduced in the number of ingroup taxa to only Sel-enastracean taxa (Fig. 7) between maximum parsi-mony and distance analyses (e.g., for the placement

FIG. 5. Transmission electron micrographs of Podohedriellaand Quadrigula in culture. Scale bar, 1 �m. (a) Podohedriella falcataSAG 202-2; two regions of the multiformed pyrenoid near the up-per and lower part of the nucleus. (b) Podohedriella falcata SAG202-2; basal cell end without any substructure. (c) Quadrigula clos-terioides SAG 12.94; longitudinal section of two young vegetativecells; the chloroplast is filled with pyrenoid matrix penetrated bynumerous thylakoids. Abbreviations are as in Figure 2.


of Monoraphidium terrestre ; 95 vs. 52/55; Fig. 6). Theweighted maximum parsimony analysis may generallyoverestimate the significance of relationships amongthe Selenastraceae. Because of these discrepancies,such relationships cannot be regarded as sufficiently

resolved for taxonomic conclusion. Although no se-quence positions were excluded in the second dataset, it comprised only slightly more variable/parsi-mony informative sites (�14/6). The addition of in-group taxa outside of the Selenastraceae clade may

FIG. 6. Phylogenetic analyses of 18S rRNA gene sequences from members of the Selenastraceae and other green algal lineages.Phylogeny inferred with the maximum likelihood method. The tree is rooted on the branch leading to the prasinophytes Nephroselmisolivacea and Pseudoscourfieldia marina. Numbers shown at the branches are bootstrap percentages from 2000 replicates using neighbor-joining distance (above lines, left), minimum-evolution distance (above line, right), and weighted maximum parsimony (below lines)methods. Bootstrap values are shown only for groupings within the Selenastraceae, for the relationships of that group within the Chlo-rophyceae, and for deep branches of the green algal phylogeny. Arrows are used to indicate bootstrap values at nodes where thesenumbers do not fit on the branches. Thick lines mark internal nodes that are defined by bootstrap support above 70% and shared bythe distance, weighted parsimony, and maximum likelihood trees. Underlined taxa names mark autosporic coccoid green algae.


account for this discrepancy. These relationships, al-though well supported in weighted maximum parsi-mony analyses of the full data set (79/95/80 in Fig.6), therefore cannot be regarded as sufficiently re-solved for taxonomic conclusion. However, it may beanticipated from the inclusion of additional selena-stracean taxa to better resolve the within-group rela-tionships.

Arrangement of autospores and colony formation. These cri-teria are of high taxonomic value on the genus levelin traditional systematics of the Selenastraceae (Ko-márek and Fott 1983, Marvan et al. 1984). In our in-vestigations, a parallel arrangement of autospores wasconfirmed for the genera Ankistrodesmus, Podohedriella,and Quadrigula, whereas in mother cells of Monoraphid-ium and Kirchneriella the autospores were arranged seri-ally. Although the presence of colonies or solitary cellshave traditionally been used to delimit genera, we foundsome intermediates in the formation of colonies. In fastgrowing cultures, colonies of Ankistrodesmus often disin-tegrate into solitary cells and can hardly be differenti-ated from Monoraphidium species. The results of our 18SrDNA analyses indicated that these important traditionalcharacteristics do not reflect natural phylogenetic rela-tionships. Our phylogenetic analyses (Fig. 6) do not re-solve lineages comprised exclusively taxa with seriallyarranged autospores (e.g. Monoraphidium) from thosewith only parallel oriented autospores (e.g. Ankistrodes-mus). Solitary cells versus colony formation are also notconsistent characters within the Selenastraceae.

Characteristics of pyrenoids. One of the most confus-ing criteria in the generic conception of Selena-straceae is the presence or absence of pyrenoids. Theproblem of generic relevance of pyrenoids particularlyconcerns the genera studied in this work. The investi-gation of pyrenoids by LM is influenced by method-ological problems. Pyrenoids without starch envelopes(so-called naked pyrenoids) are almost impossible todiscern using conventional LM except after stainingwith Acetocarmin G (1% in acetic acid) according toEttl (1965). Often, Selenastraceae with naked pyrenoidswere considered to be without pyrenoids (Korshikov1953, Komárek and Fott 1983). In contrast, pyrenoidscovered with a starch envelope are clearly visualizedwith a conventional microscope, for example, the py-renoids in the genus Chlorolobion (Hindák 1970). How-ever, even naked pyrenoids can be discerned usingthe differential interference contrast technique in LM.Nevertheless, TEM is necessary for the study of detailsof the pyrenoid structure. Pyrenoids also develop differ-ently within the chloroplast depending on the culturetechnique. Especially in aerated cultures, the concen-tration of CO2 has an influence on the developmentof pyrenoids (Miyachi et al. 1986). Under normal airconditions, the pyrenoids are well developed, whereasthe pyrenoids can disappear under CO2 enrichment(Krienitz and Klein 1988). According to Eloranta(1979), pyrenoid fine structure in Monoraphidium grif-fithii is very changeable depending on the physiologi-cal stage of the algae.

FIG. 7. Maximum-likelihood tree for the Selenastraceae on which diacritical morphological features for the group have beenmapped. Numbers shown at the branches are bootstrap percentages from 2000 replicates using ME distance (above lines, left),weighted maximum parsimony (below lines), and from 200 replicates using ML (above line, right) methods. Only bootstrap valuesequal or greater than 50% were recorded. Drawings right to the species names show features of cell shape, arrangements of au-tospores, and colony formation (see text).


Members of the genus Ankistrodesmus and Monoraphid-ium were considered to be pyrenoidless (Komárková-Leg-nerová 1969, Komárek and Fott 1983). In the presentstudy, naked ellipsoidal or spherical pyrenoids were foundin all strains of Ankistrodesmus and Monoraphidium investi-gated both by LM and TEM. The pyrenoid matrix waspenetrated by thylakoid membranes in Ankistrodesmusfusiformis and Ankistrodesmus stipitatus but not in Anki-strodesmus gracilis SAG 278-2. However, the pyrenoid ispenetrated by thylakoids in another strain of A. gracilis(KR 1981/231, Krienitz et al. 1985). Obviously, thischaracter is highly variable in the genus Ankistrodes-mus. For Monoraphidium similar varied pyrenoid struc-tures were observed.

Comments on genera: Ankistrodesmus. Traditionally,Ankistrodesmus is distinguished by the colonial habitusand by the parallel arrangement of autospores withinthe mother cell wall, whereas Monoraphidium is charac-terized by solitary cells. However, in our phylogeneticanalyses, different species of Ankistrodesmus and Monora-phidium were found in different clades supporting the ap-proach to combine both genera in one.

The designation of strain SAG 2005 (formerly KR1988/9, Krienitz and Scheffler 1994) as Ankistrodesmusfalcatus has to be revised to Ankistrodesmus fusiformisbecause of its tendency to develop cruciform colonies.These colonies produce only small amounts of muci-lage concentrated in the center of the colonies andtherefore were relatively unstable with a tendency todisintegrate into solitary cells. In contrast, A. falcatusproduces large amounts of mucilage that covers theentire colony, resulting in a stable colony formation(Komárková-Legnerová 1969, Komárek and Fott 1983).

Comments on genera: Selenastrum. Whereas Komárekand Comas (1982) made a distinction between the colo-nial genera Ankistrodesmus and Selenastrum by means ofthe higher curvature of the cells of Selenastrum, our datado not support such a distinction. We investigated twotaxa, which were placed in the genus Selenastrum byKomárek and Comas (1982): Ankistrodesmus gracilis (syn.Selenastrum gracilis) and Ankistrodesmus bibraianus (syn.Selenastrum bibraianum). In our phylogenetic analyses, A.gracilis clustered close to straight needle-shaped speciesof the genus Monoraphidium and A. bibraianus was closelyrelated to Kirchneriella aperta.

Comments on genera: Monoraphidium. We investigatedthree morphologically very similar taxa from the solitaryneedle-shaped representatives of the genus Monoraphid-ium: M. braunii, M. neglectum, and M. terrestre. These spe-cies were almost identical in size, in cell shape, and in themorphology of the empty mother cell wall remnants afterliberation of autospores. Nevertheless, we found somedifferences in the pyrenoid structure: the pyrenoid inM. braunii is surrounded by starch grains and pene-trated by thylakoids. It is visible even under a conven-tional LM. Therefore, formerly it was included into thegenus Chlorolobion, which is characterized by starch-sheathed pyrenoids (Komárek 1979). According to tra-ditional systematics, Chlorolobion is closely related toKeratococcus Pascher. Hindák (1970) combined both

genera, but Marvan et al. (1984) contradicted this viewand established the additional genus DrepanochlorisMarvan, Komárek et Comas as “an intermediate taxonbetween Chlorolobion and Monoraphidium.” Our studies onMonoraphidium braunii (syn. Chlorolobion braunii [Nägeli]Komárek) and the other strains of Monoraphidium showthat Chlorolobion and Drepanochloris are doubtful genera.Furthermore, molecular analyses demonstrated thatKeratococcus bicaudatus has to be excluded from the Sele-nastraceae and to be placed to the Trebouxiophyceae(unpublished data). We suggest that M. braunii shouldbe kept within the genus Monoraphidium. According toHeynig and Krienitz (1982), M. neglectum differs fromM. braunii by the absence of a pyrenoid. This study showsthat both species have a pyrenoid, but the pyrenoid inM. neglectum is naked and not penetrated by thyla-koids. The edaphic species M. terrestre exhibits transi-tional traits of morphology and pyrenoid substructurebetween M. neglectum and M. braunii. The weightingof these structural characters is very difficult. Accord-ing to the 18S rRNA analyses, the three species appearon three different branches within the Selenastraceae:M. neglectum clusters with Podohedriella falcata, while M.terrestre and M. braunii constitute independent lineages.

Monoraphidium dybowskii represents another mor-photype within the genus Monoraphidium. This speciesis a member of a group of small cylindrical taxa thatare straight or more or less curved like M. convolutumand M. minutum. These species are characterized by aspherical pyrenoid that is facultatively covered with astarch envelope and not penetrated by thylakoids.Hindák (1988) transferred M. dybowskii and M. minu-tum (Nägeli) Komárková-Legnerová into the genusChoricystis and established the new combinations Chori-cystis dybowskii (Woloszynska) Hindák and Choricystisminuta (Nägeli) Hindák. However, Choricystis is a tre-bouxiophycean alga (Krienitz et al. 1996, 1999). Ourresults demonstrate that M. dybowskii belongs to theSelenastraceae and is grouped in one cluster with Ank-istrodesmus gracilis. Therefore, M. dybowskii is a memberof the class Chlorophyceae and not a member of theTrebouxiophyceae, and the new combinations sug-gested by Hindák (1988) do not reflect the real phylo-genetic position of these algae.

Comments on genera: Podohedriella. Strain SAG 202-2,formerly designated as Ankistrodesmus falcatus, was des-ignated as Podohedriella falcata based on its heteropolar-ity and cell morphology. This assignment remains pro-visional until further investigations of the systematicrelationships of Ankistrodesmus, Podohedra, and Podohed-riella can be performed.

In A. stipitatus, cell aggregations that are fixed attheir basal region are common. This indicates a tran-sition state to the genus Podohedriella. Hindák (1988)separated Podohedriella from Podohedra by the absenceof a pyrenoid in Podohedriella. In the light of this study,this is a questionable criterion. Nevertheless, thereare other diacritic characters that separate these gen-era: the autospores are arranged in parallel in Podohed-riella and serially in Podohedra. Furthermore, Podohedra


is an aerophytic alga, whereas Podohedriella is a com-ponent of the freshwater periphyton (Düringer 1958,Hindák 1988). On the other hand, Podohedriella has ahigh morphological and ecological similarity to Anki-strodesmus. According to Hindák (1988), the most im-portant criterion is the heteropolarity of the cells.However, the present study gives no support to thisview because the apical end of Podohedriella does notpossess special structures, like, for example, the an-chors in the genus Ankyra (Reymond and Druart1980). Furthermore, some species of Ankistrodesmus,such as A. stipitatus, can also be attached by their basalends to the substrate. Taken together, the separationof Ankistrodesmus and Podohedriella on one hand andPodohedra Düringer and Podohedriella on the other re-mains unclear. It would also be necessary to studythe type species of the genus Podohedriella, P. longipesDüringer. However, this taxon is not available fromculture collections. No clear distinction among Podo-hedriella, Monoraphidium, and Ankistrodesmus was possiblein the 18S rDNA analyses presented here, which revealsthat the differentiation among these genera is veryquestionable. The 18S rDNA sequences from Podohedri-ella falcata and Monoraphidium neglectum were almostidentical and both taxa had a sister–group relationshipwith the lineage comprising Ankistrodesmus gracilis, M.dybowski, and Quadrigula closterioides (Fig. 6). Therefore,the differentiation between Podohedriella, Monoraphid-ium, and Ankistrodesmus seems to be questionable.

Comments on genera: Quadrigula. Members of the ge-nus Quadrigula are discerned from Ankistrodesmus by thedistant position of adult cells within colonies (Komárekand Fott 1983). Quadrigula was never found to disinte-grate in solitary cells like Ankistrodesmus. Even in quicklygrowing cultures, Q. closterioides produces prominent mu-cilaginous sheaths. The large elongated naked pyrenoidsof Quadrigula were penetrated by thylakoids and weresimilar to those of strains of Ankistrodesmus. Nevertheless,the 18S rRNA analyses do not provide molecular supportfor combining Quadrigula and Ankistrodesmus into a sin-gle genus, unless the “large genus” Ankistrodesmus in itsclassical sense is reestablished to include Monoraphidiumand Quadrigula (cf. Korshikov 1953).

Comments on genera: Ankistrodesmus bibraianus/Kirchneriella. Morphological and molecular criteriadid not place Ankistrodesmus bibraianus into the groupof needle-shaped Selenastraceae as proposed previously.Rather, A. bibraianus belongs to the section of semilu-nate or crescent-shaped morphotypes of Kirchneriella.The presence of starch-covered peripheral pyrenoidstraversed by only few thylakoids supports, from the ultra-structural point of view, the clustering of A. bibraianusand K. aperta in a joint clade.

Conclusions to the generic concept in Selenastraceae. In aprevious study on the morphologically uniform andtaxonomically difficult genus Chlorella, phylogeneti-cally significant biochemical and ultrastructural datawere found that were supported by the 18S rRNA phy-logeny (Huss et al. 1999). Similar morphological datathat could be used as phylogenetic relevant diacritical

features for the Selenastraceae could not be identi-fied in our study. In contrast, the molecular analysesshowed that the following criteria are not significantfor the systematics of the Selenastraceae on the genuslevel: cell shape and colony formation, arrangementof autospores in the mother cell wall, and shape andultrastructure of pyrenoids. These criteria can only beused for a differentiation at the species level. The 18SrDNA phylogeny of the Selenastraceae (Fig. 6) is fur-cated in three separate clusters. The first two clusters(one includes eight strains of Ankistrodesmus, Monoraphid-ium, Podohedriella, and Quadrigula and the second joins astrain of Kirchneriella aperta and Ankistrodesmus bibraianus)seem to support the conception of reestablishing two“large” genera in their classical circumscription (usede.g. by Korshikov 1953 and Belcher and Swale 1962),namely (1) Ankistrodesmus, including Monoraphidium,Podohedriella, and Quadrigula, and (2) Kirchneriella, in-cluding related semilunate or crescent-shaped genera.However, the independent position of Monoraphidiumbraunii apart from the two other lineages demonstratesthat it is too early to decide on these two large genera.Also, there was no such distinction in three clades/lin-eages with the second (reduced) data set (Fig. 7). Ob-viously, there might be other lineages within the Sele-nastraceae that are not covered by our selection ofstrains of the most common genera. The following raregenera of the Selenastraceae, and preferentially theirtype species, should be investigated in future studies:Coenolamellus Proskina-Lavrenko, Diplochloris Korshikov,Podohedra, Pseudoquadrigula Lacoste de Diaz, and Seleno-derma Bohlin. Unfortunately, no cultures are availablefor these genera at present.

The discussion of the taxonomical value of differ-ent generic features based on morphology or ultra-structure demonstrates that applying the concept of“small genera” that include only a few species (cf. Ko-márek and Fott 1983) for the Selenastraceae is prob-lematic because of many existing transitional forms.To define genera within the Selenastraceae, a differ-entiation based on only one single criterion (e.g. thepresence of pyrenoids) cannot be accepted. At the pres-ent time, we do not want to draw taxonomical conclu-sions. Nomenclatural changes can only be approachedafter more data from more taxa are available.

We are grateful to M. Papke, V. Sasse, and G. Steingräber for ex-cellent technical assistance and to M. Garcìa Carles for sequenc-ing work. This work was supported by the Deutsche Forschungs-gemeinschaft (Fr 905/6-1, 7-1, Hu 410/6-4, Kr 1262/6-1). Wolf-Henning Kusber provided some classical references. Dr. Mar-vin Fawley has kindly read and commented on the manuscript.We thank Dr. Wytze T. Stam and two unknown reviewers forsuggestions to improve the manuscript.

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