C S I R O  P U B L I S H I N G
Australian Journal 
of Botany
Volume 47, 1999
? CSIRO Australia 1999
An international journal for the publication of 
original research in plant science
w w w. p u b l i s h . c s i r o . a u / j o u r n a l s / a j b
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Published by CSIRO PUBLISHING
for CSIRO Australia and 
the Australian Academy of Science
Monophyly of Genera and Species of Characeae based on rbcL
Sequences, with Special Reference to Australian and European
Lychnothamnus barbatus (Characeae: Charophyceae)
Richard M. McCourt
AE
, Kenneth G. Karol
B
, Michelle T. Casanova
C
and Monique Feist
D
A
Department of Botany, Academy of Natural Sciences, Philadelphia, PA 19103, USA.
B
Laboratory of Molecular Systematics, National Museum of Natural History, MSC, 
MRC-534, Smithsonian Institution, Washington, DC 20560, USA.
C
Botany Department, University of New England, Armidale, NSW 2351, Australia.
D
Laboratoire de Pal?obotanique, Institut des Sciences de l?Evolution, 
Universit? des Sciences, Place Bataillon, 34095 Montpellier, France.
E
Corresponding author.
Abstract
Sequences for the chloroplast-encoded large subunit of the Rubisco gene (rbcL) were used to test the
monophyly of multiple isolates within species, and multiple species within genera, of green algae in the
Characeae (Class Charophyceae). Parsimony and maximum likelihood analyses supported the monophyly
of genera and most species, with the exception of a paraphyletic assemblage comprising isolates of two
?species?, dioecious Chara connivens Salzm. ex A.Br. and monoecious C. globularis Thuill., which
together constitute a monophyletic group. The rbcL data support the independent evolution of either
monoecious or dioecious sexual systems in the two connivens-globularis, clades. Comparisons of disjunct
isolates of the monotypic Lychnothamnus barbatus (Meyen) Leohn. revealed nearly identical rbcL
sequences in isolates from Croatia, Germany and Australia, although all three sequences were unique.
The variation exhibited by these isolates was similar to variation between isolates within species of Chara
and Lamprothamnium from different continents. The limited variation may be due to dispersal of thalli or
oospores between continents; however, the rarity of known intercontinental transfers of Characeae in the
last two centuries suggests that the Australian population is probably not an exotic from Europe.
Lychnothamnus barbatus populations in Australia and elsewhere thus merit continued protected status.
Introduction
The monospecific charalean genus Lychnothamnus occurs in widely separated freshwater
habitats on three continents (Wood and Imahori 1965). Although this alga sometimes forms
dense stands in shallow water, L. barbatus (Meyen) Leonh. is not abundant or common
throughout its range. Indeed, collecting this species is exceedingly difficult and some
molecular studies have had to employ herbarium specimens as sources of DNA (McCourt et al.
1996). Sequences for rbcL (the plastid-encoded large subunit of the enzyme ribulose-1,5-
bisphosphate carboxylase oxygenase [Rubisco]) (McCourt et al. 1996) and the small subunit
of ribosomal DNA (18S rDNA) (Meiers et al. 1999) have been published for European
specimens of L. barbatus. This report presents rbcL sequences for L. barbatus from a
restricted and possibly endangered population in Australia and for an additional European 
L. barbatus, and discusses the implications of these molecular data for phylogenetic
relationships of the three populations. By analysing the sequences along with new rbcL
sequences from other genera and conspecific isolates we also test the monophyly of genera
and selected species in the Characeae. 
Lychnothamnus barbatus has been collected from sites in Europe, India, China and Australia
(Jao 1947; Pal et al. 1962; Wood and Imahori 1965). The species has been recorded from two
sites in south-east Queensland, Australia. Specimens were collected more than 36 years ago
Aust. J. Bot., 1999, 47, 361?369
0067-1924/99/030361' CSIRO 1999
from a population in Warrill Creek by R. D. Wood in 1960 (Wood 1972), and no further
collections were made until specimens were collected from Warrill Creek in October 1996, and
from one other site in December 1996 in the same region (Casanova 1997). The herbarium
records and the specimens collected by Wood indicate that the species formed robust, perennial
stands in deep pools in at least two locations in Warrill Creek in 1960. The Warrill Creek
population in October 1996 consisted of a few weak specimens in a single location, and was
decimated 2 months later (December 1996) when the site was subjected to high, turbid flows.
Lychnothamnus barbatus is rare in Australia. Under the criteria of risk of extinction (Chalson
and Keith 1995) the species would be classified as critical (there are fewer than 2500 mature
individuals, the geographic range less than 40 km, and there is a pattern of decline).
The locality and catchments of habitats of L. barbatus in Australia are heavily utilised for
cattle grazing and irrigated horticulture (Thomas 1973). These processes represent threats to
the alga?s survival in Australia. The species has been listed under the Australian Endangered
Species Protection Act (1992). The act provides protection for native Australian species, and
determination of whether L. barbatus is native to Australia, or is an exotic introduced from
Europe, is important in deciding whether the species should remain protected under that act.
Analysis of the genetic similarity of European and Australian L. barbatus may help
determine whether the specimens collected in Australia are actually derived from European
L. barbatus. Given the rarity of specimens, extensive population genetic analysis was not
feasible for this study. Instead we chose to obtain sequence data from the rbcL gene and
compare specimens from three populations: one in Croatia, one in Germany and one in
Australia. Differences between these specimens from three populations of L. barbatus were
compared to differences among multiple isolates of species in other characean genera (Chara,
Nitella, Tolypella and Lamprothamnium). This taxon sample expands on the data set of
McCourt et al. (1996), who used a representative species of each genus and two of Tolypella.
The sequence data thus provide a test of the monophyly of some species and genera, as well
as a test of the original hypothesis of relationships proposed for genera and tribes (Wood and
Imahori 1965). The rbcL gene was chosen because of the availability of sequence data from
other extant Characeae, in particular for multiple representatives of several widespread
species of Chara, and the gene?s rate of change, which suggested that intraspecific
differences might be present but limited in number.
Materials and Methods
Species sampled are included in Table 1. Nomenclature follows the Microspecies Index in Wood and
Imahori (1965, pp. 763?786). Species identifications were based on morphological observations, not on
tests of potential interbreeding; the latter sometimes falsifies morphospecies identifications (Proctor
1970), and crossing experiments were beyond the scope of this study. Species chosen were widely
distributed so that comparisons of sequence differences between disparate populations could be compared.
New rbcL sequences for 18 species in five genera of Characeae are provided. Outgroup taxa were
chosen from other charophycean green algae and land plants, which are well supported as members of
the same clade containing the Characeae (Graham et al. 1991; Mishler et al. 1994; McCourt 1995). 
Thalli were cleaned of possibly contaminating epiphytes under a dissection microscope prior to DNA
extraction. Methods for DNA extraction and purification, rbcL amplification, and direct sequencing of
the double-stranded amplification product followed the techniques described in McCourt et al. (1996).
Sequences were determined visually from autoradiographs and entered directly into a Nexus file (see
description of Paup* below). Sequences were proofread against the original autoradiographs. Primer
sequences for amplification and sequencing were those used in McCourt et al. (1996) and are available
upon request. None of the sequences contained indels or introns, consistent with prior studies of rbcL in
the Characeae (Manhart 1994; McCourt et al. 1996), and sequences were easily aligned. Sequences were
usually 1354 nt in length, although a 134-nt fragment of the sequence for Nitella praelonga P/CR7 was
not determined.
Phylogenetic analysis was performed by using the test version of Paup* (4.0d64PPC version) with
permission of the author D. Swofford (Smithsonian Institution, Washington, DC). Phylogenetic relationships
362 R. M. McCourt et al.
were inferred by maximum parsimony (MP) and maximum likelihood (ML) (Swofford et al. 1996). For
parsimony analyses, heuristic searches were performed by the MULPARS, TBR and steepest descent
options. All sites were weighted equally and character states were unordered. The input order of taxa
was shuffled randomly in 100 replications. Bootstrap re-sampling methods (Felsenstein 1985) were used
(1000 replications) in parsimony analyses to assess robustness of inferred clades. To choose the
appropriate ML model, likelihood scores under the various models in Paup* were determined for the
363rbcL Phylogeny of Genera and Species in Characeae
Table 1. Species used in the analysis of rbcL sequences
Nomenclature follows the Microspecies Index of Wood and Imahori (1965)
Species Isolate Collection Genbank 
source accession
Chara connivens JRM
AB
Manhart (1994) 
X-774
B
Carmona, Spain AF097160 
F140
C
NE Spain AF097161
X-214
B
Israel AF097162
C. globularis F124C
C 
Eastern Germany AF097163
X-692
B 
Tas., Australia AF097164
X-751
B
Alberta, Canada AF097165
C. vulgaris X-152
B
Denmark AF097166 
F101
C
Southern France AF097167
C. rusbyana X-066
B 
Argentina AF097168 
LG
D
Unknown AF097169
Lamprothamnium papulosum F137
C 
Southern France AF097170 
X-695
B
Tas., Australia U27534
Nitellopsis obtusa F131B
C
Western Germany U27530
Lychnothamnus barbatus Croa
E
Croatia U27533 
Aus
F
Australia AF097171 
Ger
C
Berlin, Germany AF097172
Nitella praelonga P/CR7
B
Costa Rica AF097173
N. translucens F108
C 
Western France AF097745
Nitella sp. JRM
AB
Manhart (1994) L13482
N. opaca F146
C
Western Poland AF097174
Tolypella prolifera F142
C 
Netherlands U27532 
F150
C
Maine et Loire, France AF097175
T. nidifica F138
C
Southern France U27531
T. glomerata F131A
C
Western Germany AF097176
Coleochaete orbicularis JRM
A
Manhart (1994)
Marchantia polymorpha JRM
A
Manhart (1994)
Oryza sativa JRM
A
Manhart (1994)
Pseudotsuga menziesii JRM
A
Manhart (1994)
Ophioglossum engelmanii JRM
A
Manhart (1994)
Psilotum nudum JRM
A
Manhart (1994)
Spriogyra maxima JRM
A
Manhart (1994)
A
Sequence from Manhart (1994).
B
Thalli provided by Dr V. Proctor, Texas Tech University, Lubbock, TX, USA.
C 
Thalli provided by M. Feist, Universit? de Montpellier, Montpellier, France.
D 
Thalli provided by L. Graham, University of Wisconsin, Madison, WI, USA. This dioecious isolate
has also been referred to as C. zeylanica (L. Graham, pers. comm.), but V. Proctor has identified it as
C. rusbyana. 
E 
From herbarium specimen of W. Krause (see McCourt et al. 1996). 
F 
Thalli collected by M. Casanova.
trees resulting from the MP search. As described in Swofford et al. (1996), likelihood ratios of tree
scores under the various models were compared by a likelihood ratio test. This procedure yielded the
choice of the general time-reversible model incorporating site-specific rate heterogeneity (GTRss). 
Pairwise distance comparisons were calculated by the mean character distance formula of Paup*, in
which the absolute distance is divided by the total weight of the characters for which data are not missing. 
Results
A single most parsimonious tree was found in the MP search (Fig. 1). The three C. connivens
isolates at the top of Fig. 1. possessed identical sequences and their branches are collapsed in
the tree shown. The ML analysis found three equally optimal trees, and the only differences
were in the ordering of the three C. connivens isolates with identical sequences (JRM, X774,
and F140) at the top of the tree in Fig. 1. 
The topology of the tree in Fig. 1 in terms of relationships of genera is identical to that for the
equal-weights parsimony analysis of McCourt et al. (1996). Monophyly of the family Characeae
was strongly supported, as was the topology of internal branches, with two exceptions. Bootstrap
support for the sister relationship of Nitella and genera of the tribe Chareae was weak (60%).
Therefore, basal relationships within the family were less clearly resolved. 
Strong bootstrap support (> 90%) was found for the monophyly of all the minor genera,
including L. barbatus; monophyly of the more species-rich genera Nitella and Chara was less
strongly supported (57 and 65%, respectively). Monophyly of isolates within species was
strongly supported, with the exception of a paraphyletic group containing two well-supported
clades, each with isolates of C. connivens and C. globularis. 
The three isolates of Lychnothamnus barbatus form a monophyletic group with 100%
bootstrap support. Each species isolate has a unique sequence, with few interspecific
differences. The Croatian Lychnothamnus exhibits one autapomorphy, while the Australian
and German isolates share one synapomorphy. Sequences of the latter pair of species differ
by one base change, an autapomorphy for the Australian Lychnothamnus; the German isolate
exhibits no autapomorphies. Sequence variation among isolates of Lychnothamnus was
comparable to that for conspecific isolates of other genera, for example multiple isolates of
Chara and Lamprothamnium show two to nine base pair differences for isolates from
different continents (Table 2). Pairwise distance measurements for the taxa (Table 2) show
that differences among isolates within species of Chara, Lamprothamnium and
Lychnothamnus (tribe Chareae) were never more than 1%, and differences between species
were only slightly higher. Differences between species within Nitella and Tolypella (tribe
Nitelleae) ranged from 4 to 8.6%.
Discussion
The phylogeny inferred from this enlarged sample of taxa is congruent with that in
McCourt et al. (1996) based on rbcL sequences from a representative species from each
genus and two of Tolypella. The tribe Chareae is monophyletic, while the relationships of the
three basal clades (Tolypella, Nitella and tribe Chareae) are not clearly resolved. Monophyly
of the genera is generally well supported, although Chara and Nitella, the two most species-
rich genera, were less strongly supported. Lamprothamnium isolates are monophyletic and
constitute the sister taxon to a monophyletic Chara. The latter finding conflicts with the
phylogenetic reconstruction based on sequence data from the small subunit of rDNA, a
nuclear-encoded gene (Meiers et al. 1997, 1999). The support for the internal branch leading
to Chara and Lamprothamnium is not strong when sequences from additional species of
Chara are included in the analysis (McCourt and Karol, unpubl. data), but the monophyly of
both Chara and Lamprothamnium are still supported. The conflict between 18S rDNA and
rbcL sequence analyses regarding the status of these two genera remains a mystery.
Monophyly of the species isolates was supported, except for C. connivens and C. globularis
which formed a paraphyletic cluster. For these two species the sequences differences among
364 R. M. McCourt et al.
isolates were comparable to the small differences (< 1%) found for conspecifics of other
species in the Chareae. The paraphyly of several isolates of the dioecious C. connivens and
the generally monoecious C. globularis is intriguing for several reasons. Wood and Imahori
(1965) combined this monoecious and dioecious species pair (plus other microspecies) into one
species, C. globularis Thuill., em. R.D.W. These authors classified these two microspecies as
365rbcL Phylogeny of Genera and Species in Characeae
Fig. 1. Single most parsimonious tree found in the MP search. Branch lengths shown above and
bootstrap support (1000 replications) below each branch. Tribe designations Chareae and Nitelleae are
based on Wood and Imahori (1965). Tree length = 1626 steps; CI = 0.5185 (0.4815 excluding
uninformative characters). The tree was obtained by using the heuristic search option of Paup* (4.0d64
PPC version), TBR, MULPARS, collapse zero length branches, and steepest descent options in effect. A
total of 100 random taxon additions were conducted. The tree is based on 473 parsimony informative
characters. Maximum likelihood analysis using GTRss model in Paup* found this tree and two other
equally optimal topologies that differed only in the placement of the three C. connivens species sharing
an identical sequence (JRM, X774 and F140). 
146
29
76
19
26
24
4
3
4
3
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0
0
0
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Chara. connivens JRM
C. connivens  X774
C. connivens F140
C. globularis F124C
C. globularis X751
C. connivens X214
C. globularis  X692
C. vulgaris X152
C. vulgaris   F101
C. rusbyana  X066 
C. rusbyana  LG
Lamprothamnium F137
Lamprothamnium X695
Nitellopsis obtusa F131B
Lychnothamnus  Croa
Lychnothamnus  Aust
Lychnothamnus  Ger.
Nitella praelonga
Nitella  sp. JRM
N. translucens  F108
N. opaca  F146
Tolypella prolifera  F142
T. prolifera F150
T. nidifica  F138
T. glomerata  F131A
Coleochaete
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366 R. M. McCourt et al.
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closely related forms within the variety ?globularis? of a much more broadly defined species of
C. globularis. These authors noted that the two forms are very similar except for a minor
difference in branchlet morphology. Proctor (1980) disagreed strongly with this combining
practice, which Wood and Imahori (1965) applied to many other monoecious?dioecious species
pairs, although in the case of globularis and connivens, Proctor (1975) agreed there was some
?slight? support for combining the two on the basis of limited success in interbreeding the two
species under laboratory conditions. But whereas Wood and Imahori (1965) combined these
microspecies (and numerous others) as part of a broad, morphologically defined species,
Proctor?s (1975) breeding experiments suggested the two may constitute a single biological
species separate from other taxa. Proctor (1975) postulated that C. connivens or closely related
dioecious forms may have given rise independently to several monoecious lines of ?C.
globularis? via relatively minor changes in the life cycle, although he stated that there is no
experimental evidence for this (Proctor 1980, p. 231). The rbcL data provide just such evidence
and strongly support Proctor?s hypothesis. Successful interbreeding between isolates of these
two species would corroborate the species boundary implied by the rbcL data. Sampling of
more variable genes, such as the internally transcribed (ITS-1 and ITS-2) regions of the nuclear
ribosomal RNA genes, might provide further evidence regarding the relationships of isolates
within species (Meiers 1997) and the ability of separate populations to interbreed (Coleman and
Mai 1997). These further studies may warrant combining of the two described species into one,
or a redefinition of each species based on molecular and breeding data. 
It is noteworthy that sequence differences between species of tribe Nitelleae sensu Wood
& Imahori (Nitella and Tolypella) were much greater than sequence differences between
species in the Chareae (i.e. the latter show much shorter branch lengths). One might attribute
this difference to the smaller number of Nitella and Tolypella species sampled, i.e. the
branches involved are missing many taxa that would shorten the internodes. However,
although additional unsampled species of Nitella and Tolypella might break up their long
branches, the total number changes in Nitella and Tolypella exceeds by far the number within
the Chareae as a whole. Unless there are some highly divergent and unsampled species of
Chara, we hypothesise that the divergence of Chara species may represent a more recent
radiation (descent from a more recent common ancestor); alternatively, Chara may have
experienced a different rate of sequence evolution in this clade. 
Comparison of Australian and European Isolates of Lychnothamnus
There are several possible explanations for the disjunct distributions of European and
Australian specimens of L. barbatus, which are mirrored by that of other monoecious species
(e.g. C. braunii, Lamprothamnium papulosum and Nitellopsis obtusa (Proctor 1970; Grant
and Proctor 1972)). One explanation for the disjunction is that L. barbatus was recently
introduced by human transport into Australia from Europe. Another one is that the widely
separate populations of L. barbatus are remnants of a formerly widespread species distributed
throughout several continents, including Eurasia, South-east Asia and Africa (Souli?-M?rsche
1981). There is no published record of fossil Lychnothamnus (nor of any fossil charophyte) in
Australia, but research underway at the University of New South Wales may provide more
data on the charophyte fossil record on this continent (A. Garcia, pers. comm.). This broad
distribution of L. barbatus may be the result of dispersal of a monoecious species via non-
human means (i.e. water birds (Proctor 1962; Proctor et al. 1967)). Alternatively, the disjunct
distributions may represent residual populations following break-up of Gondwana some 150
million years ago near the end of the Jurassic Period (Wilford and Brown 1994).
The nearly identical rbcL sequences in the three L. barbatus isolates suggest a close
relationship between the populations. Evidence suggesting that L. barbatus is an exotic
European species in Australia includes the morphological similarity among the Australian
specimens and those illustrated from other continents (Pal et al. 1962; Wood 1972), along with
367rbcL Phylogeny of Genera and Species in Characeae
the colonisation of the Fassifern region by German immigrants in the 1860s (McHugh 1978).
However, several lines of evidence argue against the hypothesis that the Australian population
is a product of recent European invasion. First, the specimens from the different continents are
not morphologically identical (they differ in the length of stipulodes and bract cells, the
number of branchlets in a whorl and the degree of cortication (Garcia and Casanova, unpubl.
data)). Second, the environments in which the populations occur on both continents are
different (Europe has cold, deep, clear lakes; south-east Queensland has subtropical, warm,
ephemeral streams). Third, disjunct distributions such as that of L. barbatus are well doc-
umented among aquatic plants in general (Sculthorpe 1967) and especially among charophytes
(Proctor 1970). Finally, circumstantial evidence suggests that the likelihood of transporting
viable charophyte thalli or oospores between continents is low. Vernon Proctor of Texas Tech
University (pers. comm.) has noted that there is evidence of but one probable introduction of a
European species of Chara into North America. Furthermore, no European Lychnothamnus
has been known to establish a viable population in North America. Further sampling of 
L. barbatus and other Characeae from Asia and South-east Asia would test hypotheses of
dispersal between areas where this species has historically occurred. 
If not an exotic, what is the explanation for the current distribution of L. barbatus? The
possibility exists that L. barbatus is capable of intercontinental dispersal. This species and
Lamprothamnium papulosum and Chara muelleri exhibit similar broad distributions (Wood
and Imahori 1965; V. Proctor, pers. comm.). All three species are monoecious and thus more
likely to be capable of successful dispersal to new areas (Proctor 1980). Gondwanan L.
barbatus may have dispersed to Europe via Africa after the continental break-up (Wilford
and Brown 1994). Australian L. barbatus would then be a descendant of a broadly distributed
species in Gondwana, where lacustrine and fluvial habitats were common in the region that
would become Queensland. In any case, the populations of L. barbatus in Australia and
elsewhere represent ?natural? populations worthy of conservation efforts. 
In conclusion, rbcL sequence data suggest that the three L. barbatus isolates are genetically
distinct, and the sequence divergence levels are comparable to those for isolates of species of
Chara and other genera sampled from different continents. Further studies with more variable
genes (e.g. ITS-1 and ITS-2) are planned. The data are consistent with what one would expect
from a native Australian population of a cosmopolitan species of the Characeae. The
responsible action, given the declining status of L. barbatus worldwide, is to assume that the
species is native to Australia and to continue to list it under the Endangered Species legislation.
Acknowledgments
The authors thank V. Proctor (Texas Tech University, Lubbock, Texas) for providing samples
of Characeae from his culture collection, for many stimulating discussions on the evolution of
these algae, and for a critical review of this manuscript. The authors thank Linda Graham
(University of Wisconsin, Madison) for the sample of C. rusbyana. Dave Swofford of the
Laboratory of Molecular Systematics, Smithsonian Institution, generously allowed us permission
to use Paup* for our analyses. The research was supported by NSF grant DEB 9407606 to RMM,
the Sloan Foundation, and the University Research Council of DePaul University, Chicago. 
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Manuscript received 23 September 1997, accepted 26 November 1998
369rbcL Phylogeny of Genera and Species in Characeae
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