Andrey A. Gontcharov 2 and Michael Melkonian - American Journal ...

American Journal of Botany 95(9): 1079–1095. 2008.
I N SEARCH OF MONOPHYLETIC TAXA IN THE FAMILY
DESMIDIACEAE (ZYGNEMATOPHYCEAE, VIRIDIPLANTAE): THE
GENUS COSMARIUM 1
Andrey A. Gontcharov
2
and Michael Melkonian
Botanisches Institut, Lehrstuhl I, Universit ä t zu K ö ln, Gyrhofstr. 15, D-50931 K ö ln, Germany
Nuclear-encoded small subunit (SSU) rDNA, 1506 group I introns, and chloroplast rbcL genes were sequenced from 97 strains
representing the largest desmid genus Cosmarium (45 spp.), its putative relatives Actinotaenium (5 spp.), Xanthidium (4 spp.),
Euastrum (9 spp.), Staurodesmus (13 spp.), and other Desmidiaceae (Zygnematophyceae, Streptophyta) and used to assess phylogenetic
relationships in the family. Analyses of single genes and of a concatenated data set (3260 nt) established 10 well-supported
clades in the family with Cosmarium species distributed in six clades and one nonsupported assemblage. Most of the clades contained
representatives of at least two genera highlighting the polyphyletic nature of the genera Cosmarium , Euastrum , Staurodesmus
, and Actinotaenium . To enhance resolution between clades, we extended the data set by sequencing the slowly evolving
chloroplast-encoded large subunit (LSU) rRNA gene from 40 taxa. Phylogenetic analyses of a concatenated data set (5509 nt)
suggested a sister relationship between two clades that consisted mainly of Cosmarium species and included C. undulatum , the
type species of the genus. We describe molecular signatures in the SSU rRNA for two clades and conclude that more studies involving
new isolates, additional molecular markers, and reanalyses of morphological traits are necessary before the taxonomic
revision of the genus Cosmarium can be attempted.
Key words: Actinotaenium ; clades; Cosmarium ; Desmidiaceae; Euastrum ; molecular phylogeny; molecular signatures; polyphyly;
taxonomy.
The conjugating green algae (Zygnematophyceae, Viridiplantae)
represent the most species-rich lineage in the Streptophyta
except for the embryophytic land plants ( Gerrath, 1993 ).
They share with most embryophytes not only a common ancestry
but also the absence of flagellate reproductive stages. A peculiar
mode of sexual reproduction (i.e., conjugation) sets the
class apart from other streptophytes and may have contributed
to their successful diversification (Brook, 1981). On the basis of
ultrastructural analyses of mitosis and cytokinesis, zygnematophycean
algae have been recognized as members of the streptophyte
algae known also as Charophyceae sensu Stewart and
Mattox ( Pickett-Heaps, 1975 ). Molecular phylogenetic analyses
of the Zygnematophyceae corroborated these results
( Chapman and Buchheim, 1992 ; Surek et al., 1994 ; McCourt
et al., 2000; Gontcharov et al., 2003 ), although the phylogenetic
position of the class among the streptophyte algae still remains
unresolved ( Chapman et al., 1998 ; Karol et al., 2001 ; Lewis and
McCourt, 2004 ; Turmel et al., 2007 ).
Phylogenetic analyses of the Zygnematophyceae using a
broad taxon sampling and multigene data sets have more recently
led to the conclusion that many traditional genera of the
class are polyphyletic, suggesting that the characters used to
delineate these taxa are either plesiomorphic, homoplasious or
unreliable ( Gontcharov et al., 2003 , 2004 ; Gontcharov and
Melkonian, 2005 ; Hall et al., 2008 ). This situation is well
1
Manuscript received 7 February 2008; revision accepted 11 June 2008.
The authors thank P. C. Silva and F. A. C. Kouwets for discussion on the
type species of Cosmarium . This work was supported by DFG grant
ME-658/26-1.
2
Author for correspondence (e-mail: gontcharov@biosoil.ru);
permanent address: Institute of Biology and Soil Science, 690022,
Vladivostok-22, Russia.
doi:10.3732/ajb.0800046
1079
known in microalgal systematics in which descriptions of genera
have often been based on single or very few morphological
characters visible in the light microscope without a careful investigation
of phylogenetic significance (one related example is
that of the coccoid green algal genus Chlorella and its relatives;
Krienitz et al., 2004 ; Luo et al., 2006 ). We have therefore started
to evaluate the genus concept in the most species-rich family
of the Zygnematophyceae, the Desmidiaceae (desmids), using
taxon-rich sampling and multigene phylogenetic analyses. Although
the traditional genus Staurastrum Meyen ex Ralfs was
shown to be polyphyletic, a monophyletic core of the genus
could be identified using such an approach ( Gontcharov and
Melkonian, 2005 ). It is anticipated that once a phylogenetic
framework of a desmid genus has been established, a reinvestigation
of its morphological traits should lead to the recognition
of hitherto overlooked, but more sound and reliable generic
morphological characters.
Here, we address the phylogenetic status of the genus Cosmarium
Corda ex Ralfs (Desmidiaceae, Zygnematophyceae),
the most species-rich desmid genus with more than 1000 species
described ( Gerrath, 1993 ). Together with Staurastrum
(~700 spp.) it constitutes about half of the total number of species
in the otherwise species-poor streptophyte green algae. It
should be mentioned that Cosmarium has always been regarded
as an artificial genus and thus taxonomically problematic ( West
and West, 1905 , 1908 ; Fritsch, 1953 , Hirano, 1959a ; Krieger
and Gerloff, 1962 , 1965, 1969; Prescott et al., 1981 , Croasdale
and Flint, 1988 ; Brook and Johnson, 2002 ; Gerrath, 2003 ). It
was poorly circumscribed by a vague diagnosis ( Ralfs, 1848 )
and linked morphologically to other genera such as Euastrum
Ehr. ex Ralfs and Xanthidium Ehr. ex Ralfs. Thus, although
over the 160 years since its description, numerous taxa have
been added to Cosmarium , the definition of the genus ( “ the
fronds are minute, simple, constricted in the middle; the segments
are generally broader than long and inflato-compressed,
but in some species orbicular or cylindrical; they are neither

1080 American Journal of Botany [Vol. 95
emarginate at the end nor lobed at the sides, and have no spines
or processes ” ; Ralfs, 1848 , p. 91) has not changed and its discriminatory
power diminished. Although widely acknowledged
to be polyphyletic, the genus is still adopted to date in its original
sense ( Lenzenweger, 1999 ; Brook and Johnson, 2002 ; Gerrath,
2003 ; Coesel and Meesters, 2007 ). Unfortunately, attempts
during the 19th century to resolve the taxonomic problems in
Cosmarium and establish more “ natural ” (morphologically uniform)
taxonomic entities were unsuccessful (e.g., N ä geli, 1849 ; de
Bary, 1858 ; Lundell, 1871 ; Kirchner, 1878 ; Gay, 1884 ; Hansgirg,
1888 ; de Toni, 1889 ; Raciborski, 1889 ; Turner, 1892 ). The
novel taxa were based on simple morphological features such
as cell and semicell shape, ornamentation of the cell surface,
degree of cell constriction, and chloroplast shape that occur in
any combination in the genus. In 1954, Teiling established a
new genus, Actinotaenium Teil ., for taxa with smooth-walled,
elongated cells, circular in apical view, and displaying a shallow
sinus. Although Actinotaenium is often regarded as a “ natural
group ” ( Prescott et al., 1981 , p. 1), its members are distinct
only in the combination of characters that individually occur in
many Cosmarium taxa. Another consequence of the unsatisfactory
taxonomic status of Cosmarium is the fact that only one
unfinished attempt of a monography of the genus dealing with
fewer than half of the described species exists ( Krieger and
Gerloff, 1962, 1965, 1969). The lack of type material and the
inaccessibility or vagueness of many original descriptions further
complicates a critical assessment of species that are distinguished
largely on the basis of the shapes of cells, semicells,
and chloroplasts; features of cell wall ornamentation; and zygospore
shape. The extent of the variability of these characters
is poorly known, and their taxonomic significance has never
been assessed within a cladistic framework.
The first tests of the genus concept of Cosmarium with molecular
tools confirmed the expected polyphyly of the genus,
but this result was based on a very limited taxon sampling, and
the phylogeny was affected by long-branched taxa and the limited
phylogenetic resolution of the marker used (nuclear-encoded
small subunit [SSU] rDNA). Six Cosmarium sequences
representing distinct morphotypes within the genus had no relationship
to each other; instead, some species formed clades with
members of other genera ( Gontcharov et al., 2003 ). Conversely,
phylogenetic analyses of two taxa of Cosmarium with the same
morphotype, revealed only a distant relationship between them,
questioning the validity of morphological characters traditionally
used in the taxonomy of the genus but confirming a previously
reported relationship between some smooth-celled species
of Cosmarium and spine-bearing Staurodesmus Teil. taxa
( Gontcharov et al., 2003 ; Gontcharov and Melkonian, 2005 ).
The primary goal of this study was to define major monophyletic
lineages within the traditional genus Cosmarium , related
genera, and the family Desmidiaceae and generate a
hypothesis of their phylogenetic relationship. To this end, we
sampled more than 120 species, representing major morphotypes
of Cosmarium , and its putative relatives, Staurodesmus ,
Euastrum , Xanthidium , and Actinotaenium . Nuclear-encoded
SSU rDNA, noncoding 1506 group I introns, chloroplast large
subunit (LSU) rDNA and protein-coding rbcL gene sequences
were obtained from these taxa and analyzed individually and in
concatenation using different phylogenetic methods. We identified
10 clades in the Desmidiaceae, with the genus Cosmarium
distributed over six of these. Furthermore, Cosmarium , Euastrum
, Staurodesmus , and Actinotaenium were shown to be
polyphyletic. Mapping traditional morphological traits that dis-
criminate these genera on the phylogenetic tree revealed extensive
homoplasy, calling into question the current genus concept
in the Desmidiaceae.
MATERIALS AND METHODS
Cultures — One hundred twenty-seven strains of Desmidiaceae and Peniaceae
used for this study were obtained from different sources (Appendix 1)
and grown in modified WARIS-H culture medium ( McFadden and Melkonian,
1986 ) at 15 ° C with a photon fluence rate of 40 µ mol ⋅ m − 2 ⋅ s
− 1
in a 14/10 h light/
dark cycle. The taxonomic designation of all strains was verified by light microscopy
prior to DNA extraction ( Krieger and Gerloff, 1962 , 1965 , 1969 ;
Prescott et al., 1981 ; Croasdale and Flint, 1988 ; Brook and Johnson, 2002 ;
Coesel and Meesters, 2007 ).
DNA extraction, amplification, and sequencing — After mild ultrasonication
to remove mucilage, total genomic DNA was extracted using the Qiagen
(Hilden, Germany) DNeasy Plant Mini Kit. Nuclear-encoded (nu) SSU rDNA
(including the 1506 group I intron) and chloroplast-encoded (cp) rbcL and LSU
rDNA were amplified by polymerase chain reactions (PCR) using published
protocols and 5 ′ -biotinylated PCR primers ( Marin et al., 1998, 2005 ;
Gontcharov, et al., 2004 ). PCR products were purified with the Dynabeads
M-280 system (Dynal Biotech, Oslo, Norway) and used for bidirectional sequencing
reactions (for protocols, see Hoef-Emden et al., 2002 ). Gels were run
on a Li-Cor IR 2 DNA sequencer (Li-Cor, Lincoln, Nebraska, USA).
Sequence alignments and tree reconstructions — Sequences were manually
aligned using the SeaView program ( Galtier et al., 1996 ). For coding regions of
the nu SSU rDNA, cp LSU rDNA, and noncoding 1506 group I introns, the
alignment was guided by primary and secondary structure conservation
( Bhattacharya et al., 1994 , 1996 ; Wuyts et al., 2000 , 2001 ; Gillespie et al.,
2006 ). All three codon positions of the rbcL gene were used for analyses. The
alignments are available at http://srs.ebi.ac.uk/, accessions ALIGN_001252,
ALIGN_001253 and ALIGN_001254.
The amount of phylogenetic signal vs. noise in our nu SSU rDNA, rbcL , and
cp LSU rDNA data were assessed by plotting the uncorrected against corrected
distances determined with the respective model of sequence evolution estimated
by the program MODELTEST version 3.06 ( Posada and Crandall, 1998 ).
The selected models and model parameters are summarized in Table 1 . Also,
the measure of skewness (g1-value calculated for 10c000 randomly selected
trees in the program PAUP* version 4.0b10; Swofford, 2002 ) was compared
with the empirical threshold values ( Hillis and Huelsenbeck, 1992 ) to verify the
nonrandom structuring of the data. To quantify the extent of substitution saturation
in data sets, we calculated the I ss statistic for the individual and combined
data sets with the program DAMBE ( Xia and Xie, 2001 ).
Phylogenetic trees were inferred with maximum likelihood (ML), neighborjoining
(NJ) distance, and maxiumum parsimony (MP) optimality criteria using
PAUP* 4.0b10 and Bayesian inference (BI) using the program MrBayes version
3.1.2 ( Huelsenbeck and Ronquist, 2001 ). Evolutionary models (for ML
and NJ analyses) were selected by the Akaike information criterion in
MODELTEST. ML and MP analyses used heuristic searches with a branchswapping
algorithm (tree bisection-reconnection); distances for NJ analyses
were calculated by ML. In BI, two parallel MCMC runs were carried out for
two million generations sampling every 100 generations for a total of 20 000
samples. The first 500 – 1500 samples were discarded as burn-in, and the remaining
samples were analyzed using the sumt command in MrBayes. The robustness
of the trees was estimated by bootstrap percentages (BP; Felsenstein,
1985 ) using 1000 (NJ and MP) or 100 (ML) replications and by posterior probabilities
(PP) for BI. BP < 50% and PP < 0.95 were not taken into account.
In MP, the stepwise addition option (10 heuristic searches with random taxon
input order) was used for each bootstrap replicate. The ML bootstrap used a
single heuristic search (starting tree via stepwise addition) per replicate.
Previous molecular phylogenetic studies resolved the families Peniaceae
(the genus Penium ) and Desmidiaceae as sisters and have shown that they
evolved with comparable evolutionary rates unlike the more distantly related,
fast-evolving desmid families Gonatozygaceae and Closteriaceae ( Besendahl
and Bhattacharya, 1999 ; McCourt et al., 2000; Denboh et al., 2001 ; Gontcharov
et al., 2003 ). Later Penium was found to not be monophyletic, and only one
sublineage was sister to the Desmidiaceae ( Gontcharov et al, 2004 ; Hall et al.,
2008 ). Therefore, only P. margaritaceum and P. spirostriolatum , comprising

September 2008]
Gontcharov and Melkonian — The genus COSMARIUM Corda ex Ralfs
1081
Table 1. Evolutionary models, log likelihood values ( – lnL) and model parameters identified by the program MODELTEST for different data sets used for Figs. 2 – 4 and for additional analyses.
127 taxa 97 taxa 40 taxa
Model and model parameter rbcL ( Fig. 2 )
nu SSU rDNA (with 1506
group I intron) rbcL
nu SSU rDNA (with intron)
+ rbcL ( Fig. 3 )
nu SSU rDNA (with intron)
+ rbcL cp LSU rDNA
cp LSU rDNA + nu SSU rDNA (with intron) +
rbcL ( Fig. 4 )
Model GTR+I+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G
– lnL 15157.271 12888.799 13512.904 27175.160 7373.987 17786.400 25536.258
I 0.5644 0.7145 0.5701 0.6583 0.7864 0.6779 0.7197
G 0.9369 0.4982 1.0241 0.6482 0.5697 0.6572 0.6007
Base frequencies
A 0.3002 0.2485 0.2975 0.2652 0.2783 0.2628 0.2694
C 0.1632 0.2108 0.1708 0.1937 0.2301 0.1981 0.2128
G 0.1857 0.2646 0.1884 0.2359 0.3100 0.2442 0.2710
T 0.3509 0.2512 0.3433 0.3052 0.1816 0.2949 0.2467
GC 0.3489 0.4754 0.3592 0.4296 0.5401 0.4423 0.4838
Rate matrix ([G ↔ T = 1.00])
[A ↔ C] 0.9147 1.5573 0.9356 1.2953 0.5478 1.2349 0.9605
[A ↔ G] 3. 6638 3.7675 3.9135 4.0542 1.1043 3.6121 2.5445
[A ↔ T] 1.2907 2.1920 1.4815 1.9766 1.6874 2.0898 2.0619
[C ↔ G] 1.0244 0.5683 0.9365 0.7169 0.1431 0.6480 0.4238
[C ↔ T] 7.1143 10.2938 7.5798 8.8722 2.9835 8.3522 6.8163
Aligned nt 1339 1921 1339 3260 2255 3254 5509
Constant nt 836 1524 846 2370 1960 2509 4469
MP-informative
a
416 281 402 683 155 514 669
MP-uninformative
a
86 119 90 209 140 231 371
Measure of skewness (g1) − 0324 − 0396 − 0451 − 0406 − 0564 − 0526 − 0655
0204/0766, < 0001 0185/0788, < 0001 0,21/0766, < 0001 0195/0806, < 0001 0222/0806, < 0001 0231/0793, < 0001 0163/0812, < 0001
I ss ( I ss / I ss + c, P -value of 32 taxon
data subsets)
a
Informative and uninformative (but not invariable) characters in MP

1082 American Journal of Botany [Vol. 95
this distinct clade, were used as an outgroup for the Desmidiaceae in our analyses,
and three more species of Penium were regarded as ingroup taxa (see
Results).
Combined analyses — For concatenated analyses, partitions were fused and
analyzed using a single “ concatenated model ” with averaged parameters. Before
that, models for individual partitions ( Table 1 ), ML topologies, and ML/
NJ(ML)/MP bootstrap support ( Table 2 ) were obtained and compared to reveal
significant discrepancies. We also assessed incongruence between the data sets
by the incongruence length difference (ILD) test ( Farris et al., 1994 ) in PAUP*
(partition homogeneity test with 1000 replicates).
The concatenated data set of nu SSU rDNA (including intron), rbcL , and cp
LSU rDNA was analyzed by BI using specific model parameters for each
partition.
RESULTS
Quality of the molecular data — Apart from the differences
in the alignment length, base and substitution frequencies, number
of parsimony-informative positions, pattern of substitution
distribution (G parameter), and proportion of invariable sites
between our data sets, the most complex GTR+I+G model of
sequence evolution was identified by the MODELTEST as the
best model fitting the data ( Table 1 ). A test of the data sets for
substitution saturation (distribution of the uncorrected vs. corrected
distances; Fig. 1 ) revealed a nearly linear correlation in
the SSU rDNA data indicating low saturation. The saturation
plot of rbcL was somewhat leveled off, suggesting the presence
of some saturation that would be expected from the third codon
position of this protein-coding gene ( Gontcharov et al., 2004 ).
However, according to the I ss statistics, neither of the data sets
was saturated ( P < 0,001; Table 2 ).
Comparison of the skewness of the tree length distribution
(g1 value) of random trees of all data sets with the empirical
threshold values ( Hillis and Huelsenbeck, 1992 ) showed that
the length distributions were considerably left-skewed, indicating
that the alignments were significantly more structured than
random data and likely contained a strong phylogenetic signal
( Table 1 ).
Noise assessment in the data sets without the outgroup (two
Penium species) yielded results identical to those obtained with
the complete data sets, suggesting that the outgroup did not interfere
with the phylogenetic signal (not shown).
rbcL data set (127 taxa) — ML analyses of 127 rbcL sequences
(1339 nt, GTR+I+G model; Table 1 ) yielded the phylogenetic
tree in Fig. 2 . Although resolution of internal branches
was mostly low, 11 terminal clades that included most of the
taxa studied were resolved and designated STD1, STD2, CO1,
CO2, CO3, CO4, ARTHR, Euastrum , Micrasterias , omniradiate,
and CAP ( Fig. 2 ). Most clades were well supported; only
STD2, Euastrum , omniradiate, and CAP attained weak to moderate
support ( Fig. 2 ; Table 2 ). In addition, two lineages containing
most of the multicellular (filamentous or colonial) taxa
analyzed and some Cosmarium species were recovered, the
larger multicellular 2 assemblage, and a long-branched Spondylosium
planum/S. secedens pair, multicellular 1. Species of
the genus Xanthidium (including Staurastrum tumidum ;
Gontcharov et al., 2003 ; Gontcharov and Melkonian, 2005 )
were also split between several individual branches and one
clade. Finally, Haplotaenium minutum formed an unresolved
assemblage with two Staurastrum species; however, bootstrap
analyses favored monophyly of Staurastrum with weak support
( Fig. 2 ; Table 2 ).
The majority of Cosmarium species were distributed among
seven clades (ARTHR, CO1, CO2, CO3, CO4, STD2, and omniradiate)
and one assemblage (multicellular 2). Most of these
clades (except for CO2 and CO 3) also included species of other
genera, namely Actinotaenium , Euastrum , Spondylosium , and
Staurodesmus ( Fig. 2 ). Two Cosmarium taxa, C. decedens and
C. ralfsii , were placed in the Euastrum and Micrasterias clades,
respectively. In both cases, affiliation of the Cosmarium species
to these lineages, however, was only weakly (60/55% BP) or
moderately (86/78% BP) supported. In addition, the longbranched
C. depressum diverged basally in the family together
with Actinotaenium cruciferum and three Penium species (CAP
clade; Fig. 2 ).
Cosmarium was most prominent (26 of 68 species analyzed)
in clade CO2. Strain SVCK482, identified as C. undulatum, the
type species of the genus ( Silva, 1952 ; Gerrath, 1993 ), was also
a member of this clade. Beside Cosmarium , this species-rich
clade accommodated three Actinotaenium and four Euastrum
species ( Fig. 2 ). CO2 was further divided into several subclades
and some single branches, but their relationships were largely
unresolved likely due to low sequence divergence in the clade.
Cosmarium species also formed the bulk of the taxa in clade
omniradiate, that also included Spondylosium panduriforme
and two additional Actinotaenium species ( Fig. 2 ). In the omniradiate
clade, the Cosmarium species formed four well-supported
subclades whose relationships, however, were unresolved
in the rbcL phylogeny ( Fig. 2 ).
Our analyses also revealed well-supported relationships between
some Cosmarium and Staurodesmus species. Members
of Staurodesmus were recovered in three distinct clades (STD1,
STD2, and ARTHR), and two of these clades also contained
species of Cosmarium . In both STD2 and ARTHR, Cosmarium
species diverged in paraphyletic succession before the Staurodesmus
taxa. Only in STD2 clade the derived position of the
Staurodesmus species was supported by high bootstrap values
( Fig. 2 ).
Two Cosmarium strains, M 2717 and SVCK 570, both identified
as C. punctulatum , were found in two distantly related
clades, CO2 and CO3, respectively. Light microscopic examination
revealed minor differences in cell dimensions and patterns
of the cell wall ornamentation between the strains, which,
nevertheless, fitted the rather broad species diagnosis (Prescott
et al., 1982). A similar discrepancy between taxonomic designation
and phylogenetic position in the tree was observed for
Staurodesmus extensus and S. extensus var. joshuae that showed
little affinity to each other in clade STD1 ( Fig. 2 ). It is likely
that the small difference in cell morphology between these conspecific
strains masked the conspicuous genetic distances between
the two taxa that should warrant the status of separate
species
Concatenated data set (SSU rDNA + rbcL) — To increase
phylogenetic resolution and probe the clades established in the
rbcL analyses with another marker, we combined SSU rDNA
(including 1506 group I introns) and rbcL sequences obtained
from the same strain. The taxon sampling was reduced to 97
species in this data set because in some taxa the SSU rRNA
gene could not be amplified using various primer combinations
(e.g., most members of the CO3 and CO4 clades) or their sequences
formed extremely long branches in the SSU rDNA
phylogeny (e.g., Cosmarium ovale and Euastrum moebii ). Concatenation
of SSU rDNA and rbc L sequences resulted in a data
set consisting of 3260 nt ( Table 1 ).

September 2008]
Gontcharov and Melkonian — The genus COSMARIUM Corda ex Ralfs
1083
Table 2. Significances (ML/NJ(ML)/MP/BI) for the clades and branches (encircled numbers in Figs. 2 – 4 ) in different analyses. If there is no number
before the slash, no ML bootstrapping was done for the data set.
127 taxa 97 taxa 40 taxa
Clade/branch rbcL ( Fig. 2 )
nu SSU rDNA
(with 1506 group I intron) rbcL
nu SSU rDNA
(with intron) + rbcL ( Fig. 3 )
cp LSU rDNA
nu SSU rDNA
(with intron) + rbcL
cp LSU rDNA + nu SSU rDNA
(with intron) + rbcL ( Fig. 4 )
ARTHR / 100/100/1.00 99/98/97/1.00 100 100 –/89/100/– 100 100
STD1 /100/100/1.00 100 100 100 96/100/100/1.00 100/96/100/1.00 98/100/100/1.00
CO1 /100/100/1.00 93/93/91/1.00 100 100 N/A N/A N/A
CO2 /96/78/0.99 86/79/73/1.00 83/99/80/1.00 100 97/99/100/1.00 99/100/99/1.00 100
CO3 /100/100/1.00 59/64/ − / − 100 100 N/A N/A N/A
CO4 /92/85/0.99 N/A N/A N/A N/A N/A N/A
Xanthidium / − / − / − -/60/-/1.00 56/63/68/- 78/96/85/1.00 -/58/100/- 95/100/100/1.00 98/100/100/1.00
STD2 /77/84/ − − / − / − / − 75/79/83/0.99 92/96/94/1.00 71/83/90/0.99 84/96/78/1.00 98/100/90/1.00
Multicellular 1+2 / − / − / − − / − / − / − − / − / − / − − / − / − / − − / − /67/- − / − /57/ − − / − /67–
Euastrum /60/55/0.99 − / − / − / − − / − / − / − − / − / − / − − / − / − / − 84/93/82/0.95 60/ − /51/0.99
Staurastrum /63/53/- 61/ − / − /- 57/60/ − / − 73/73/64/1.00 N/A N/A N/A
Micrasterias /86/78/1.00 80/93/69/1.00 93/91/93/1.00 99/100/93/1.00 − / − /97/0.99 99/100/100/1.00 91/98/97/1.00
Omniradiate /54/ − /0.99 68/71/ − /1.00 − / − / − /0.98 91/82/85/1.00 96/73/99/1.00 99/92/90/1.00 100/100/99/1.00
CO2 + CO3 / − / − / − − / − / − /1.00 − / − / − / − − / − / − / − − /62/67/ − − / − / − / − − /78/67/ −
CO2 + CO3 + / − / − / − − / − / − / − − / − / − / − − /63/ − / − − / − /68/ − − /70/67/ − 71/68/79/1.00
Xanthidium
1 /51/ − /0.95 − / − / − / − − / − / − 0.99 − / − / − / − − / − /52/ − 62/ − /56/0.97 78/ − /52/1.00
2 /56/56/0.97 − / − / − / − 63/53/55/0.99 56/51/ − / − 92/81/95/ − 87/81/88/1.00 98/97/95/1.00
Notes: N/A, not accessed; 100 =100/100/100/1.00
Comparison of the SSU rDNA- and rbcL -based topologies
for the 97 taxa data set and their bootstrap supports ( Table 2 )
showed general agreement between the markers (results not
shown). Only two clades attained somewhat weaker or no support
in the SSU rDNA phylogeny compared to the rbcL data set
(CO3 and STD2, respectively). Both partitions recovered two
nonsupported assemblages, one containing eight of nine Euastrum
species, the other the multicellular species and associated
Cosmarium taxa ( Table 2 ). Most members of these assemblages
had accelerated evolutionary rates in the SSU rRNA gene or in
both genes that likely affected bootstrap support for their grouping.
Different placement of these unresolved taxa/branches in
the SSU rDNA and rbcL trees was responsible for failure of the
combined data set to pass the ILD test ( P = 0.003). Only when
long branches such as C. depressum , Actinotaenium cruciferum ,
and members of the Euastrum and multicellular assemblages
were removed from the data set did it pass the test ( P > 0,005).
This result suggests that the nuclear and chloroplast data sets
are congruent for the data set as a whole. Problems with the ILD
test are well known (e.g., Dolphin et al., 2000 ; Yoder et al.,
2001 ; Darlu and Lecointre, 2002 ; Dowton and Austin, 2002 ;
Quicke et al., 2007 ), so failures due to localized cases of incongruence
are not surprising ( Thornton and DeSalle, 2000 ). Phylogenetic
analyses of the data set without the long-branched
taxa mentioned revealed that they have no effect on the support
levels of other terminal clades and the general tree topology.
Fig. 1. Analyses of saturation in the nu SSU rDNA, rbcL , and cp LSU rDNA data (uncorrected vs. corrected distances). Corrected distances
were calculated with the GTR+I+G model estimated by MODELTEST for each partition ( Table 1 ). (A) 97-taxa alignment ( Fig. 3 ), (B) 40-taxa alignment
( Fig. 4 ).

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In the combined analyses the clades ARTHR, STD1, CO1,
CO2, and CO3 attained 100%BP/1.00 PP in all analyses ( Fig. 3 ;
Table 2 ). The support increased also from weak or moderate to
high ( > 90% BP) for the omniradiate and STD2 clades. The earlier
unresolved Xanthidium lineage attained moderate bootstrap
values, whereas the two multicellular assemblages remained
without support. The more divergent SSU rDNA sequences
contributed also to the resolution of the terminal branches
within some clades, in particular CO2 ( Fig. 3 ).
Concatenated data set (nu SSU rDNA+rbcL+cp LSU
rDNA) — Although phylogenetic analyses using SSU rDNA
and rbcL led to the recognition of 10 well-resolved clades in the
Desmidiaceae, resolution of internal branches was low; thus relationships
among clades remained unclear ( Figs. 1, 2 ; Table
2 ). We therefore increased the data set by adding a more slowly
evolving marker, the chloroplast-encoded LSU rRNA gene
( Table 1 ). For these analyses, taxon sampling within the wellsupported
clades was reduced to 1 – 2 representatives per clade
(a total of 40 taxa).
The PCR products obtained by amplification of cp LSU
rDNA with primers that bound in the B19 and G20 domains of
the gene (nomenclature after De Rijk et al., 2000 ), varied significantly
in length between species. This inconsistency was
due to the presence of a single intron in 16 taxa that occurred at
four different insertion sites ( Fig. 4 ). BLAST searches indicated
that the introns in the cp LSU rDNA of desmids are similar to
group IA1, IA3, and IB4 introns from other green algal chloroplasts
and likely contain group I homing endonucleases
( Turmel et al., 1993 , 1995 ).
As expected, the phylogeny recovered with the concatenated
data set including the cp LSU rDNA (a total of 5509 nt) was
generally congruent with the phylogenies obtained in the previous
analyses ( Fig. 4 ). Compared to a concatenated data set of
the nu SSU rDNA and rbcL with a congruent taxon sampling
(40 taxa: Table 2 ), the cp LSU rDNA data set added phylogenetic
signal (and thus enhanced bootstrap values) to some internal
branches, in particular the Desmidiaceae (exclusive of the
Penium clades and Actinotaenium cruciferum ; 98% BP in ML;
Fig. 4 ), the omniradiate clade (100% BP in ML) and its basal
position in the Desmidiaceae (78% BP in ML for the Desmidiaceae
excluding omniradiate and the Penium clades + A. cruciferum
), and a putative novel clade consisting of CO2, CO3, and
Xanthidium (71% BP in ML). These results suggest that the cp
LSU rRNA gene could be a useful molecular marker for future
phylogenetic analyses in the Desmidiaceae, especially when the
focus is on resolving the deeper divergences in the family.
Mapping morphological characters that define genera on
the phylogenetic tree — Most of the 10 clades identified in the
Desmidiaceae by our molecular phylogenetic analyses, comprised
representatives of several desmid genera demonstrating
the artificial nature of these taxa and the inadequacy of the morphological
features used to define them. As a first step to characterize
the new clades, we mapped morphological features of
the cell and chloroplast of each species, on the rbcL topology
(NJ[ML] bootstrap consensus; Fig. 2 ) in all clades/assemblages
that contain Cosmarium species ( Fig. 5 ). The following morphological
characters were computed and mapped: (1) apical
view of the cell (circular [omniradiate], elliptical [biradiate] or
triangular), (2) ornamentation of the cell wall (smooth-walled,
ornamented [granular/spiny] or with [a few] stout spines), (3)
width of the isthmus (narrow [ < 1/2 of the cell width] or broad
[ > 1/2 of the cell width]), and (4) type of the chloroplast (axial
or parietal). In addition, distinct features of specific genera, e.g.,
a lobed cell outline (L), incision of the apical lobe (I), and presence
of filaments (F) or colonies (C) were also recorded ( Fig. 5 ).
Most members of clades that consist predominantly of Cosmarium
species (CO2, and omniradiate) display some sort of
cell wall ornamentation, i.e., granules or warts, arranged in
taxon-specific patterns. However, smooth-walled species (e.g.,
C. hammerii , C. cucumis , Actinotaenium spp.) were also members
of the same clades. None of the prominent Cosmarium lineages
was distinct in the degree of radiation. Biradiate cells are
generally more common in the genus and in our clades, but omniradiate
species also occur and often constitute distinct subclades
(e.g., Actinotaenium spp.; Fig. 5 ). Taxa with the same
chloroplast morphology, width of isthmus (which may influence
the pattern of cytokinesis; see Meindl (1986) , H ö ftberger
and Meindl (1993) ) or cell/semicell shape are also not confined
to single clades ( Fig. 5 ). Characters are mostly nonlinked and
occur together in various combinations. However, the 12 taxa
characterized by circular in vertical view cells (and distributed
over five clades/assemblages and five genera: Cosmarium , Actinotaenium
, Bambusina , Groenbladia , and Spondylosium ) all
had cells with a wide isthmus, whereas the opposite does not
apply ( Fig. 5 ).
Synapomorphies in the SSU rRNA — Mapping morphological
characters traditionally used to distinguish desmid genera
on the phylogenetic tree clearly demonstrated that these characters
cannot be used to circumscribe the clades identified in this
study ( Fig. 5 ). To initiate a molecular circumscription of the
clades, we searched for the presence of molecular signatures,
i.e., nonhomoplasious synapomorphies (NHS; according to
Marin et al., 2003 ) in the SSU rRNA. We restricted the analysis
to clades that contained a large number of Cosmarium species.
Screening of the secondary structure-based alignment revealed
several substitutions in the SSU rRNA that characterize the omniradiate
and CO2 clades ( Fig. 6 ).
All species comprising the omniradiate clade were distinct in
two transversions in an internal loop of Helix 25 (H673 after
Gillespie et al. (2006) ; nucleotides 30 (G → A) and 36 (A → U);
Fig. 5A ), whereas all other members of the Desmidiaceae (and
also the Desmidiales) reveal the plesiomorphic state (G, A) at
these positions. The two substitutions in the clade omniradiate
thus represent nonhomoplasious synapomorphies of this clade.
A compensatory base change (A-U → G-C) in base pair 38 of
Helix 49 (bp 47 of H1399; according to Gillespie et al. (2006) )
differentiated all members of clade CO2 from the rest of the
←
Fig. 2. The rbcL phylogeny of desmids (Desmidiaceae, Zygnematophyceae) based on 127 sequences (1339 nt, maximum likelihood [ML] topology,
for model and model parameters see Table 1 ; two Penium spp. as an outgroup). Nodes are characterized by bootstrap percentages (BP) ( ≥ 50%) and posterior
probabilities (PP) ( ≥ 0.95): neighbor joining (NJ[ML])/MP/Bayesian inference (BI). Branches with 100% BP in all methods and 1.00 PP are shown boldface.
For clade names see Results. Clades containing Cosmarium species are underlined. For species represented by more than one accession, strain data
are given. Cosmarium undulatum , the type species of the genus, is in bold.

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family ( Fig. 6B ). The A-U pair is conserved across the family
and the order Desmidiales making the CBC in clade CO2 an
NHS for this clade.
DISCUSSION
In this study, two ribosomal genes (nu SSU rDNA and cp
LSU rDNA), the protein-coding rbcL , and the noncoding 1506
group I intron of the SSU rDNA were used to assess phylogenetic
relationships among species of the Desmidiaceae (Zygnematophyceae,
Streptophyta). The phylogenetic structure of the
most species-rich genus in the family, Cosmarium , and its putative
relatives, Actinotaenium , Euastrum , Staurodesmus , and
Xanthidium was the major focus of our analyses. A large data
set and extensive taxon sampling revealed 10 distinct clades in
the Desmidiaceae.
Most of the clades established here were well supported
( Figs. 2, 3 ). Comparison of individual topologies and their
support values showed that the different markers used
contained a similar phylogenetic signal and yielded largely
congruent phylogenetic relationships between desmid taxa
( Table 2 ). In general, the rbcL gene provided better support
for most of the clades but was less informative in resolving
relationships among species within clades (e.g., CO2),
whereas divergence of the nu SSU rDNA and the 1506 group
I intron was sometimes too high to recover a clade (e.g., CO3,
STD2; Table 2 ). Differences in evolutionary rates between
markers were particularly pronounced in clade CO2, in which
divergence of rbcL at the species level was particularly low
compared to SSU rDNA (compare Figs. 2 and 3 ). The concatenated
analyses of SSU rDNA and rbcL resulted in increased
support for clades compared to phylogenetic analyses of individual
genes ( Table 2 ). Relationships among clades, however,
remained largely unresolved even when fast and slow evolving
sequences were combined ( Fig. 4 ). In the latter case,
though, taxon sampling was smaller, and phylogenetic resolution
may be much better, when taxon sampling is increased
to the same level as in the concatenated SSU rDNA and rbcL
analyses.
Phylogeny of Cosmarium — Not surprisingly, the results
presented here confirmed the long-anticipated artificial nature
of the genus (e.g., West and West, 1905 , 1908 ; Fritsch, 1953 ,
Krieger and Gerloff, 1962 ; Prescott et al., 1981 ). Early molecular
phylogenetic studies were indicative in this respect, but the
limited taxon sampling, presence of long-branched taxa, and
the relatively low phylogenetic signal of the markers made the
conclusions preliminary ( Lee, 2001 ; Nam and Lee, 2001 ;
Gontcharov et al., 2003 ; Moon and Lee, 2003 ; Gontcharov and
Melkonian, 2005 ; Hall et al., 2008 ). In the current study, 45 (68
in the rbcL analyses) species/strains of Cosmarium were distributed
over six clades, one nonsupported assemblage (multicellular),
and a long-branched basal lineage ( Fig. 3 ). Four of the
six clades also included species of other desmid genera. In the
rbcL analyses with 127 taxa, additional species of Cosmarium
associated with other clades ( Euastrum , Micrasterias ) or formed
a novel clade (CO4).
One reason for the rampant polyphyly of the genus Cosmarium
lies in its vague diagnosis that lacks any distinct synapomorphic
or even typological characters. When describing
Cosmarium , Ralfs (1848) stressed the lack of some characters
typical for other genera (e.g., incision at the apex, lateral lobes,
spines or processes), but he did not present a single character or
a combination of features that is unique to the genus. We have
shown that the morphology of the genus Cosmarium as defined
by Ralfs is one of the most common in the family and has likely
arisen independently in several lineages ( Fig. 5 ). Its evolutionary
status (ancestral vs. derived) could be also different. Noteworthy
in this respect is the close relationship between
smooth-walled Cosmarium and spine-bearing Staurodesmus
taxa in their common clades. It is very likely that in the STD2
clade, a triradiate cell with a stout spine at each angle, typical
for the genus Staurodesmus , was derived from a smooth-walled
biradiate “ Cosmarium ” taxon ( Figs. 2 – 4 ), whereas in the AR-
THR clade spines may have been lost in some members, in
which case the “ Cosmarium ” -type morphology may be the derived
state ( Fig. 2 ).
The presence of several morphotypes in most Cosmarium -
containing clades demonstrates homoplasy of morphological
features thought to be important for desmid taxonomy. Traditionally,
semicell shape, degree of radiation (biradiate vs. omniradiate),
cell wall features (smooth vs. ornamented), and
chloroplast morphology have been used to distinguish intrageneric
taxa in Cosmarium ( de Bary, 1858 ; Lundell, 1871; Hansgirg,
1888 ; De Toni, 1889 ; Raciborski, 1889 ; Turner, 1892 ;
West and West, 1905 ; Hirano, 1959a , b ; Bourrelly, 1966 ).
However, our study revealed that each of these morphological
features has a mosaic distribution in the tree and does not explicitly
characterize a specific clade ( Fig. 5 ). Our phylogenies
revealed a patchy distribution of the omniradiate morphotype
over our trees in several unrelated clades ( Figs. 1 – 4 ). Moreover,
we found that some species that differ in their degree of
radiation have nearly identical sequences (e.g., Cosmarium
portianum [biradiate]/ C. bisphaericum [omniradiate] in the
omniradiate clade; C. lundelli and C. pachydermum
[biradiate]/ Actinotaenium turgidum [omniradiate] in CO2 ( Fig.
2 ), and C. connatum [biradiate]/ C. pseudoconnatum [pseudoomniradiate]
in CO3, Fig. 2 ). The latter case is a good example
for the likely derived nature of the omniradiate morphotype at
least in some Cosmarium strains. In conclusion, the degree of
radiation is a homoplasious feature in the family Desmidiaceae
and its taxonomic significance has been largely overestimated.
Morphological heterogeneity of all clades containing Cosmarium
taxa ( Fig. 5 ) and lack of information on other phenotypical
features currently does not allow conclusions to be
drawn on the morphological characters that may unite their
members or, more importantly, that differentiate the clades.
The characters traditionally used to define taxa above the species
level are not suitable for this purpose, in particular features
of cell wall ornamentation and chloroplast morphology ( Fig. 5 ).
Many Cosmarium species have distinct patterns of granules,
←
Fig. 3. Phylogeny of Desmidiaceae (Zygnematophyceae) based on combined analyses of nu SSU rDNA, 1506 group I intron and rbcL sequences (97
taxa, 3260 nt, maximum likelihood [ML] topology, for model and model parameters see Table 1 ). The tree was rooted with two Penium spp. Nodes are
characterized by bootstrap percentages (BP ≥ 50%) and Bayesian posterior probabilities (PP ≥ 0.95): neighbor joining (NJ[ML])/MP/Bayesian inference
(BI). Branches with 100% BP in all methods and 1.00 PP are boldfaced. See Fig. 2 for further details.

1088 American Journal of Botany [Vol. 95

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Gontcharov and Melkonian — The genus COSMARIUM Corda ex Ralfs
1089
spines, or warts on the cell surface, and it is not clear whether
these types and patterns are homologous and what features
would represent character states here. Also very little is known
about such a distinct characteristic of the desmid cell wall as the
pores, their types, functions, significance of different distribution
patterns. Preliminary studies already revealed quite a diversity
of pore patterns ( Cout é and Tell, 1981 ; Neuhaus and
Kiermayer, 1981 ; Coesel, 1984 ; Gontcharov et al., 2002 ), however,
their possible suitability for desmid taxonomy above the
species level is virtually unknown.
Our knowledge about the diversity of chloroplast structures
in desmids is also very limited and has not been extended significantly
beyond the careful early studies of L ü tkem ü ller
(1893 , 1895 ) and Carter (1919a , b , 1920a , b ). Teiling (1952)
typified chloroplast shapes of desmids and presented a scenario
of their hypothetical evolution correlating it with cell morphology,
mostly radiation, but chloroplast morphology is still poorly
known in many Cosmarium species.
Type species of Cosmarium — Affiliation of a type species is
crucial for the identity of a genus. In Cosmarium this issue is
complicated by the uncertainty with the designation of the type
species. The Index Nominum Genericorum (ING; http://botany.
si.edu/ing/) recognizes C. margaritiferum as the type although
in the earlier version of ING C. undulatum had been suggested
( Silva, 1952 ). The designation of C. margaritiferum as the type
is credited to N ä geli (1849 , p. 114). However, N ä geli considered
Cosmarium as the subgenus of Euastrum and referred to E.
margaritiferum Ehr. Ehrenberg (1835) regarded his alga identical
with Ursinella margaritifera Turpin (1820) , so the correct
citation should be Euastrum margaritiferum (Turpin) Ehr. Because
the publications by Turpin and Ehrenberg were published
before the starting point of desmid taxonomy ( Ralfs, 1848 ),
N ä geli ’ s designation of the type species is invalid (ICBN, Art.
7.7). Obviously, N ä geli was not familiar with Ralfs ’ s publication
at that time, and his typification had no relation to the genus
Cosmarium Corda ex Ralfs.
Moreover, the alga described by Turpin is not identifiable
and obviously not identical to two or likely three species illustrated
by Ralfs under the name C. margaritiferum (1848, tables
XVI, XXXIII; figs. 2a – d , 3 a, b). In contrast, the choice of C.
undulatum as the type of Cosmarium was prompted by the fact
that it is the most clearly known of the species included in the
genus by Corda ( Silva, 1952 ). We agree with Silva that N ä geli ’ s
typification should be rejected because it is based on an invalidly
published name, and following Silva (1952), we regard C.
undulatum as the type species of the genus Cosmarium .
Our analyses placed C. undulatum in the CO2 clade ( Figs.
2 – 4 ) linking the genus name to this clade. The CO2 clade is a
member of a large, weakly supported assemblage that unites
two more clades, CO3 and the genus Xanthidium ( Fig. 4 ). The
species richness of the CO2 and CO3 clades and the diversity
of morphotypes in the clades suggest that these lineages
will accommodate the majority of the existing Cosmarium
species.
Cosmarium species from other clades that have no affinity to
CO2/CO3 should in the future either be classified together with
representatives of the genera in their clades or recognized as
new genera.
The omniradiate clade is phylogenetically most distant from
the rest of the genus Cosmarium and is likely one of the basal
branches in the Desmidiaceae ( Gontcharov et al., 2003 , 2004 ;
this study). Like other clades containing Cosmarium spp., this
clade includes a number of morphotypes distinct in cell/semicell
shape, chloroplast morphology, and cell wall ornamentation
( Fig. 5 ). Membership of two omniradiate Actinotaenium
(formerly Penium ) species with elongated, weakly constricted
cells and Spondylosium (formerly Cosmarium ) panduriforme
forming short filaments further complicates the circumscription
of the clade. However, we discovered several nonhomoplasious
molecular synapomorphies (NHS; for definition, see Marin et
al., 2003 ) in the SSU rDNA that discriminate members of the
omniradiate and CO2 clades from all other taxa in the family
Desmidiaceae and that might be useful in future taxonomic revisions
( Fig. 6 ).
The taxonomic affinity of Cosmarium species belonging to
the clades ARTHR and STD2, and the nonsupported assemblage
multicellular, is not yet clear. The distinctness of the
ARTHR and STD2 clades from STD1 that contains the type
species of the genus Staurodesmus, Std. triangularis ( Gontcharov
and Melkonian, 2005 ), received further support with the extended
taxon sampling and the larger data sets in this study. The
current lack of synapomorphic phenotypic characters for these
clades calls for further phenotypic study before taxonomic conclusions
should be made.
Within the assemblage of multicellular desmids, the Cosmarium
species either showed affinity to the colonial Heimansia
( C. sinostegos ), the filamentous Spondylosium pulchellum
( C. regnellii ) or formed an independent lineage ( C. diffi cile , C.
dilatatum ; Figs. 2, 3 ). Most taxa comprising this cluster are
characterized by fast evolutionary rates in all three genes and
were long-branched in the individual as well as the concatenated
analyses ( Figs. 2 – 4 ). One may hypothesize that the morphological
diversification of Desmidiaceae from a unicellular
to a multicellular life habit, a consequence of a peculiar cell
division mode ( Gerrath, 1970 , 1973 ; Krupp and Lang, 1985a ,
b ), may have been accompanied by accelerated rates of evolution
in several genes. These long branches likely affected topologies
and may have been responsible for lack of support for
this assemblage in almost all data sets ( Table 2 ).
Identification of monophyletic entities consisting of species
of the traditional genus Cosmarium should stimulate the search
for synapomorphic phenotypic characters distinguishing these
lineages.
Euastrum — The current study substantiated the polyphyletic
nature of the traditional genus Euastrum ( Hall et al., 2008 ) assigning
its species to several clades (see Results). Most Euastrum
taxa studied formed an assemblage that received low or no
support with the different data sets ( Figs. 2 – 4 ; Table 2 ). The
←
Fig. 4. Phylogenetic tree of 40 species representing major lineages (see Results) of desmids based on comparisons of concatenated nu SSU rDNA
(including 1506 group I intron), cp rbcL and LSU rDNA sequences (5509 aligned nt). The tree was constructed with maximum likelihood (ML) (GTR+I+G,
for model parameters, see Table 1 ). BP ≥ 50% for ML/neighbor joining (NJ)(GTR+I+G)/MP and PP ≥ 0.95 (Bayesian inference) values are given for the
nodes. Branches with bootstrap percentages (BP) 100% in all methods and 1.00 posterior probabilities (PP) are boldfaced. Presence of group I introns in
the cp LSU rDNA is indicated by asterisk and their positions relative to Escherichia coli 23S rDNA are given.

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Gontcharov and Melkonian — The genus COSMARIUM Corda ex Ralfs
1091
Fig. 6. Nonhomoplasious synapomorphies in the SSU rRNA molecule characterizing the “ Cosmarium ” clades (A) omniradiate and (B) CO2 of the
Desmidiaceae (see Results). Alignments contained only representative taxa for the clades. Taxa and nucleotides characterized by the synapomorphy are
boldfaced. SSU rRNA secondary structure after Wuyts et al. (2000) ; diagrams are based upon the last taxon in the alignment. The nomenclature of nucleotides
(nt, single stranded spacers and loops) and base pairs (bp, stem regions of Helices) depends on the polarity of the RNA: increasing numbers indicate
the 5 ′ → 3 ′ direction.
relatively high divergence of Euastrum sequences may have
been responsible for the uncertain status of this assemblage, so
that it requires further study with a more comprehensive taxon
sampling. It appears that the Euastrum assemblage is split into
two morphologically distinct lineages. One lineage comprises
large-celled taxa ( > 50 – 60 µ m long) with smooth cell walls and
a more or less regular porous cell surface often provided with
several large facial protrusions and excavations, e.g., E. oblongum
and E. affi ne . In these species, the polar lobe has a narrow
vertical incision with parallel margins. A similar
morphology is typical for a number of other Euastrum taxa, and
their affiliation with this lineage is anticipated ( Gontcharov
et al., 2003 ).
The second lineage contains relatively small-celled (typically
< 50 µ m long) and variously ornamented taxa. Their apical
incision is less pronounced and often appears as a V-shaped
invagination.
Euastrum taxa placed outside the Euastrum assemblage are
large-celled, with granules or spines arranged in specific patterns.
Their semicells are divided into one or two basal lobes
and a polar lobe, as is typical for the genus, but the polar lobe is
concave to nearly straight and lacks an apical incision. Species
with these characteristics were distributed between two putatively
related clades consisting mostly of Cosmarium taxa, i.e.,
CO2 and CO4. In most cases, their phylogenetic position could
be accessed with rbcL only; therefore, we consider their position
as tentative. However, high support values for the clades
that include Euastrum substellatum , E. verrucosum , E. germanicum
, E. spinulosum , E. moebii , and E. prowsei ( Fig. 2 )
suggest that these species are distinct from other Euastrum
taxa.
Actinotaenium — This study confirmed the polyphyletic status
of the genus Actinotaenium as suggested previously
( Gontcharov et al., 2003 ). In our analyses, six Actinotaenium
species were distributed among three only distantly related lineages
including the genus Penium (e.g., A. cruciferum ; Figs.
2 – 4 ). Their position in the tree highlights the homoplasious nature
of the characters used to define the genus (elongated, omniradiate
cells with two stellate [ A. cucurbita , A . cf. wollei ] or
numerous parietal [ A. turgidum ] chloroplasts and smooth cell
walls ( Teiling, 1954 ).
The status of phenotypic characters in A. phymatosporum
and A. silvae-nigrae , members of the omniradiate clade, and
particularly A. cruciferum , having a weak affinity to one of the
Penium clades ( Figs. 2 – 4 ), is unclear. The morphology of the
former species (elongate-elliptical, omniradiate cells with a
very weak median constriction and axial chloroplasts) is similar
←
Fig. 5. Bootstrap consensus topology of 10 major clades of desmids (Desmidiaceae) based on neighbor joining (NJ[ML]) analyses of rbcL sequence
data ( Fig. 2 ) with mapped features of cell and chloroplast morphology traditionally used to define genera.

1092 American Journal of Botany [Vol. 95
to that of the genus Penium in which they were placed until
relatively recently ( Kouwets and Coesel, 1984 ), and these characteristics
may be pesiomorphic.
The affiliation of the type species of the genus, A. curtum ,
remains currently unknown ( Gontcharov et al., 2003 ), and taxonomic
revision of Actinotaenium must await clarification of its
phylogenetic position.
Conclusions — Phylogenetic analyses based on several genes
from two genomes and extensive taxon sampling confirmed the
anticipated artificial nature of the desmid genera Cosmarium,
Euastrum, Staurodesmus , and Actinotaenium . Our results highlight
the inadequacy of morphological features such as cell and
semicell shape, degree of radiation, chloroplast morphology,
and cell wall ornamentation, traditionally used to discriminate
these genera and revealed numerous cases of homoplasy in distantly
related lineages. Many clades established during this
study deserve to be recognized as new genera but cannot be
formally described in the framework of the current morphology-based
taxonomic concept of desmids because either their
phenotypical features are insufficiently known or they do not
differ from those of other clades and genera. Molecular signatures
may be used to circumscribe the new taxa in the future,
but their utility is not straightforward in a group that accounts
for more than 2000 species. Although many desmid strains are
available in culture collections, the strains still represent only a
minute fraction of the taxa described, the unknown diversity of
desmids that may exist in the environment notwithstanding.
Many more isolates of desmids are needed as well as refined
analyses of morphological traits and additional molecular markers.
At the current state of knowledge, desmid systematics has
arrived at the beginning.
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A ppendix 1 . Strain information and EMBL/GenBank accession numbers for taxa used in this study. A dash ( — ) indicates the region was not sampled. New sequences
are boldfaced. ACOI = Coimbra Collection of Algae, University of Coimbra, Portugal (http://www1.ci.uc.pt/botanica/ACOI.htm); ASW = Sammlung von
Algen-Kulturen, University of Vienna, Austria ( Kusel-Fetzmann and Schagerl, 1992 ); CCAC = Culture Collection of Algae at the University of Cologne,
Germany (http://www.ccac.uni-koeln.de); M = Culture Collection Melkonian, Botanical Institute, University of Cologne, Germany (strains available upon
request); NIES = Microbial Culture Collection at National Institute for Environmental Studies, Tsukuba, Japan (http://www.nies.go.jp/biology/mcc/home.htm);
SAG = Sammlung von Algenkulturen, University of G ö ttingen, Germany (http://www.epsag.uni-goettingen.de/html/sag.html); SVCK = Sammlung von
Conjugaten-Kulturen, University of Hamburg, Germany (http://www.biologie.uni-hamburg.de/b-online/d44_1/44_1.htm). Taxon names in parentheses correspond
to those used in the culture collection catalogue.
Taxon ; strain; nu SSU rDNA+1506 group I intron (if a separate entry); rbcL ; cp LSU rDNA.
Actinotaenium cruciferum (de Bary) Teil.; M2025; AM920332 ; AM911235 ;
AM919466 . A. cucurbita (Ralfs) Teil.; M1199; AJ428099+ AM910431 ;
AM911236 ; AM919439 . A. phymatosporum (Nordst) Coes. et Kouwets;
M1368; AJ428088+ AM910432; AM911233; AM919434 . A . ( Penium )
silvae-nigrae (Raban.) Kouwets. et Coes. var. parallelum . (Krieg.)
Kouwets. et Coes.; SVCK295; AM920333; AM911234 ; —. A. turgidum
(Ralfs) Teil. ex Rouzicka et Pouzar; M1192; —; AM911238 ; —. A. cf.
wollei (W. et G. S. West) Teil. ex Rouzicka et Pouzar; M2945; AM920384;
AM911237 ; —. Bambusina borreri (brebissonii) (Ralfs) Cl.; CCAC
0045; AJ428118+ AM910433 ; AJ553935; AM919437 . Cosmarium
amoenum Br é b. in Ralfs ; M1172; —; AM911322 ; —. C. angulosum
Br é b. ; ACOI378; —; AM911328 ; —. C. binum Nordst. ; ACOI896; —;
AM911329 ; —. C. bioculatum Br é b. ex Ralfs; CCAP612/17; AM920354;
AM911265 ; —. C. biretum Br é b. in Ralfs; M2123; AM920339;
AM911267; AM919440 . C. bisphaericum Printz; SVCK436; AM920338;
AM911266 ; —. C. blyttii Wille; CCAP612/19; AM920374; AM911290 ;
—. C. botrytis Menegh. ex Ralfs; SVCK274; AM920378; AM911295;
AM919462 . C. broomei Thwaites ex Ralfs; M2075; AM920340;
AM911269 ; —. C. caelatum Ralfs; ACOI826; —; AM911319 ; —. C.
connatum Br é b. in Ralfs ; ACOI1152; —; AM911317 ; —. C. contractum
Kirchn.; SVCK396; AJ428112+AJ829661; AJ553937; —. C. cf.
contractum ( hians ) ; NIES452; AM920365; AM911283; AM919459 . C.
crenatum Ralfs ex Ralfs; M2164; AM920370; AM911268 ; —. C.
cucumis Corda ex Ralfs; M2715; AM920334; AM911270; AM919442 .
C. cyclicum Lund.; M1208; AM920388 ; AM911304 ; —. C. decedens
(Reinsch) Racib. ; ACOI794; —; AM911330 ; —. C. depressum (N ä g.)
Lund. ; ACOI1030; —; AM911325 ; —. C. depressum ; ; AM920367;
AM911285 ; —. C. difficile L ü tkem. ; ACOI403; —; AM911326 ; —. C.
dilatatum L ü tkem. et Gr ö nbl.; SVCK463; AJ829665; AM911274 ; —. C.
elegantissimum Lund.; M1887; AJ428115+ AM910434; AM911271;
AM919465 . C. granatum Br é b. in Ralfs; M2127; AM920364; AM911282;
AM919445 . C. hammeri Reinsch; ACOI349; AM920386; AM911302 ;
— . C. holmii Wille; M1211; AM920387; AM911303; AM919461 . C.
impressulum Elfv.; SVCK58; AM920362; AM911279 ; — . C. laeve
Rabenh.; SVCK35; AM920363; AM911280 ; — . C. lundellii Delp.;
SVCK357; AJ428113+ AM910446; AM911310 ; — . C. maculatum
Turn. ; SVCK422; — ; AM911315 ; — . C. margaritiferum Menegh. ex
Ralfs ; SVCK88; — ; AM911311 ; — . C. meneghinii Br é b. ex Ralfs;
SVCK59; AM920366; AM911284; AM919458 . C. notabile de Bary var.
medium (Gutw.) Krieg. et Gerloff; ACOI936; AM920369; AM911287 ;
— . C. obsoletum (Hantz.) Reinsch; M2303; — ; AM911277 ; — . C.
obtusatum Schidle; M2275; AM920389; AM911305 ; — . C. ochthodes
Nordst.; M1205; AM920376; AM911293 ; — . C. ornatum Ralfs;
SVCK569; AM920372; AM911288 ; — . C. ovale Ralfs; SVCK342;
AJ428114+ AM910447; AM911309 ; — . C. pachydermum Lund.;
SVCK24; — ; AM911321 ; — . C. perforatum Lund. ; SVCK109; — ;
AM911318 ; — . C. phaseolus Br é b. in Ralfs; M2302; AM920382;
AM911299 ; — . C. portianum Archer; M2560; AM920337; AM911273;
AM919450 . C. protractum (N ä g.) de Bary; SVCK460; AM920381;
AM911298 ; — . C. pseudoconnatum Nordst. ; M1272; — ; AM911316 ;
— . C. pseudonitidum Nordst.; ACOI1160; AM920377; AM911294 ; — .
C. punctulatum Br é b.; M2717; AM920383; AM911300; AM919464 . C.
punctulatum; SVCK570; AM920373; AM911289; AM919451 . C.
quadratum Ralfs ; SVCK484; — ; AM911323 ; — . C. quadratum; M2946;
— ; AM911313 ; — . C. quadrum Lund. var. sublatum (Nordst.) W. et G. S.
West f. dilatatum Scott et Gr ö nbl.; ACOI368; AM920335; AM911272 ;
— . C. ralfsii Br é b. in Ralfs ; SVCK300; — ; AM911324 ; — . C. regnellii
Wille; M2947; AM920350; AM911276 ; — . C. reniforme (Ralfs) Arch.;
SVCK34; AM920336 ; AY964179; AM919447 . C. sinostegos Schaarschm.
var. obtusius Gutw.; ACOI406; AM920349; AM911275; AM919435 . C.

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Gontcharov and Melkonian — The genus COSMARIUM Corda ex Ralfs
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sportella Br é b.; M2152; AM920390; AM911306 ; —. C. subcrenatum
Hantz.; M1200; AM920368; AM911286 ; —. C. subcucumis Schmidle ;
ACOI103; —; AM911312 ; —. C. subgranatum (Nordst.) L ü tkem. ;
M2629; —; AM911281 ; —. C. subochthodes Schmidle; ACOI377 ;
AM920379; AM911296 ; —. C. subprotumidum Nordst.; SVCK373;
AM920375; AM911292 ; —. C. tesselatum (Delp.) Nordst. ; SVCK381;
—; AM911320 ; —. C. tetraophthalmum K ü tz. ex Ralfs; SVCK220; —;
AM911314 ; —. C. tinctum Ralfs; M2301; AM920355; AM911278;
AM919455 . C. trachypleurum Lund.; ACOI935; AM920391; AM911307 ;
—. C. trilobulatum Reinsch; ACOI866; AM920385; AM911301 ; —. C.
undulatum Corda ex Ralfs; SVCK482; AM920380; AM911297;
AM919463 . C. vexatum West; M2119; AM920392; AM911308 ; —.
Cosmarium sp.; M2093; AM920394; FM163363; —. Cosmarium sp.;
M2856; —; AM911327 ; —. Cosmarium sp.; M2731; AM920395;
AM911291 ; —. Euastrum affi ne ( humerosum ) Ralfs; SVCK185;
AM920342; AM911240; AM919432 . E. bidentatum N ä g.; ACOI282;
AM920345; AM911243 ; —. E. binale Ralfs. var. gutwinskii (Schm.)
Homfeld; ACOI488; AM920347; AM911245 ; —. E. biverrucosum
Gontcharov et Watanabe; SVCK464; AM920346; AM911244 ; —. E.
divaricatum Lund.; SVCK 156; AM920344; AM911242; AM919444 . E.
germanicum (Schmidle) W. Krieg. ; SVCK461; —; AM911251 ; —. E.
moebii (Borge) Scott et Prescott; SVCK358; AM920393; AM911248 ; —.
E. oblongum (Grev.) Ralfs; ASW 07018; AJ428095+ AM910435;
AM911239 ; —. E. prowsei Scott et Prescott ; SVCK353; —; AM911249 ;
—. E. spinulosum Delp. var. henriquesii Sampaio ; ACOI1092; —;
AM911252 ; —. E. subalpinum Messik.; ACOI855; AM920348;
AM911246; AM919432 . E. subhexalobum W. et G. S. West ; ACOI1671;
—; AM911253 ; —. E. substellatum Nordst.; SVCK364; AM920371;
AM911247; AM919446 . E. trigiberrum W. et G. S. West; ACOI1174;
AM920343; AM911241 ; —. E. verrucosum Lund. ; SVCK798; —;
AM911250 ; —. Groenbladia neglecta (Racib.) Teil.; SVCK478;
AJ428119+ AM910436 ; AJ553943; —. Haplotaenium (Pleurotaenium)
minutum (Ralfs) Bando; SVCK302; AJ428090+ AM910437 ; AJ553947;
AM919438 . Heimansia (Cosmocladium) pusilla (Hilse) Coes.; SVCK428;
AJ428125+ AM910448 ; AJ553948; —. Micrasterias fi mbriata Ralfs;
M1188; AJ428098+ AM910450; AM911257 ; —. M. cf. thomasiana Arch.
var. notata (Nordst.) Gronbl.; M2253; AM920341; AM911256;
AM919430 . Penium cylindrus (Ehr.) Br é b. ex Ralfs; ACOI780;
AJ553930+ AM910438 ; AJ553959; AM919467 . P. exiguum W. West;
M2159 ; AJ553929+ AM910439 ; AJ553960; — . P. margaritaceum (Ehr.)
ex Br é b. in Ralfs; SAG 22.82; AF115440; AM911254; AM919469 . P.
polymorphum (Perty) Perty; M2335; AM920331; AM911255; AM919468 .
P. spirostriolatum Barker.; SVCK189; AJ553928+ AM910440 ; AJ553961;
— . Phymatodocis nordstedtiana Wolle; SVCK327; AJ428122+ AM910449 ;
AJ553962; — . Spondylosium panduriforme (Heimerl) Teil.; SAG 52.88;
AJ428124+ AM910441 ; AJ553969; AM919449 . S. planum (Wolle) W. et
G. S. West; SAG 41.81; AJ428123+ AM910442; AM911260; AM919441 .
S. pulchellum Arch.; SVCK365; AJ428130; AM911261 ; — . S. pulchrum
(Bail.) Arch.; SVCK331; AJ428129+ AM910443 ; AJ553970.1;
AM919436 . S. secedens (de Bary) Arch.; SVCK31; AJ428128+ AM910445;
AM911259 ; — . Staurastrum lunatum Ralfs; SVCK15;
AJ428106+AJ829640; AJ553971; AM919431 . S. orbiculare Ehr. ex
Ralfs; M2217; AJ829660; AM911331; AM919456 . S. sebaldi Reinsch;
M1133; AJ829630; AM911332 ; — . S. tumidum Br é b. ex Ralfs; SVCK85;
AJ428108+AJ829666; AJ553972; AM919452 . Staurodesmus bienianus
(Rabenh.) Florin; M1130; AJ829659; AM911341; AM919457 . S.
brevispina (Br é b. ex Ralfs) Croasd. var. obversus (West) Croasd.;
ACOI881; AM920361; AM911340 ; — . S. convergens (Ehr. ex Ralfs)
Lillier; M1886; AJ428102+AJ829651; AM911344 ; AM919460 . S.
extensus (Borge) Teil.; ACOI956; AM920358; AM911337 ; — . S. extensus
var. joshuae (Gutw.) Teil.; ACOI1000; AM920360; AM911339 ; — . S.
glaber (Ehr.) Teil.; ACOI954; AM920356; AM911334 ; — . S.
( Staurastrum ) isthmosus (Heimerl) Croasd.; SVCK466; AM920359;
AM911338 ; — . S. mucronatus (Ralfs ex Br é b.) Croasd.; M1394;
AJ428103+AJ829658; AM911342 ; — . S. omearii (Arch.) Thom.; M0751;
AJ829655; AM911333; AM919453 . S. spencerianus (Mask.) Teil.;
ACOI735; AM920357; AM911335 ; — . S. ( Arthrodesmus ) triangularis
(Lagerh.) Teil.; SVCK280; AJ829654; AM911336; AM919454 . S.
( Arthrodesmus ) validus (W. et G. S. West) Scott et Gr ö nbl.; SVCK457;
AJ829653; AM911343 ; — . Triploceras gracile Bail.; SVCK173;
AJ428089+ AM910444; AM911258; AM919448 . Xanthidium
antilopaeum var. canadense Josh.; SVCK147; AM920353; AM911264 ;
— . X. armatum (Br é b.) Ralfs; ASW 07059; AJ428094+AJ829667;
DQ026262; — . X. cristatum Br é b. var. uncinatum Ralfs f. ornatum
Jackson; SVCK426; AM920352; AM911263 ; — . X. subhastiferum W.
West; CCAP 690/1; AM920351; AM911262; AM919443 .