Chapter 1: The Characeae Plant

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Chapter 1: The Characeae Plant
1.1 General Morphology
1.2 Morphology of individual species
1.2.1 The Chara corallina/australis group of species
1.2.2 The Lamprothamnium papulosum/macropogon group of species
1.2.3 The Lamprothamnium succinctum group of species
1.2.4 Lamprothamnium inflatum
1.2.5 Species of Tolypella
1.2.6 Species of Nitellopsis
1.2.7 Species of Nitella suitable for physiological studies
1.3 Cellular structures and processes
1.3.1 Cells
1.3.2 Gametangia
1.3.3 The oospore
1.3.4 The germinating oospore
1.4 Characeae names
1.4.1 Systematics and the concept of species
1.4.2 Individual species’ stories
1.4.2.1 Chara australis R. Br.
1.4.2.2 Lamprothamnium inflatum (Fil. & G.O. Allen ex Fil.) A. García & K.G.
Karol
1.4.2.3 Chara longifolia C.B. Robinson
1.4.3 Lessons to be learned
1.5 Ecology
1.5.1 Distribution and abundance
1.5.2 Processes controlling characeae distribution
1.5.3 Ecological role of characeae
1.5.4 Abiotic environmental factors
1.5.4.1 Light
1.5.4.2 Water chemistry and physical properties
1.5.4.3 Nutrients
1.5.4.4 Temperature

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1.5.4.6 Depth
1.5.5 Culturing characeae
1.5.6 Life History
1.5.7 Population ecology
1.5.8 Application of ecological knowledge
Abstract
The aim of this chapter is to give physiologists a thorough grounding in the morphology,
taxonomy and ecology of the characeae plant. The morphology of characeae is depicted and
explained, with specific examples of the morphological characteristics of different species or
species groups that are used in physiological studies. The details of characeae cellular
structure in growing plants and in the reproductive organs are reviewed. The history of
characeae taxonomy and nomenclature is outlined, along with the most recent approaches to
charophyte systematics (and what name to use for characeae e plants in physiological
studies), and finally the patterns of characeae distribution and requirements for characeae
growth in natural situations are explained, and related to the culture and growth of
characeae for physiological studies.
Michelle T. Casanova
Centre for Environmental Management, University of Ballarat, Mt Helen, Vic. 3350, Australia
and Royal Botanic Gardens, Melbourne. Birdwood Ave, South Yarra, Victoria 3141,
Australia
Current Address: 273 Casanova Rd, Westmere Vic 3351 Australia
Email: amcnova@netconnect.com.au

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1.1 General Morphology
The characeae thallus (or plant body) is similar in appearance and size to the plant body of
other submerged plants such as Ceratophyllum or Myriophyllum. Charophytes consist of long
photosynthetic stem-like structures (axes) anchored in the soil, with whorls of leaf-like
organs (branchlets) along the stem (Fig. 1.1a). Close examination reveals that the structure of
characeae is very different to that of flowering plants. Instead of roots they have colourless
rhizoids, instead of leaves they have whorls of branchlets of limited growth, instead of stems
they have an axis of giant cells joined end-on-end, and instead of flowers and fruit they have
relatively simple reproductive structures (the oogonium and antheridium, Fig. 1.1b) that
produce gametes. The product of fertilisation of the gametes is an oospore (Fig. 1.1c) rather
than a seed.
The thallus of characeae is essentially filamentous. The axes (stems) are made up of long,
multinucleate, single cells interrupted by multicellular nodes (Fig. 1.2). There is no
development of tissues such as parenchyma in characeae, although the axial nodes approach
such an arrangement. Several organs of limited growth (branchlets, stipulodes, cortical
filaments) arise in whorls at the nodes (Fig. 1.2). Branchlets are the ‘leaf-like’ organs that
occur in spreading whorls, and below these there are often whorls of smaller cells called
stipulodes. In many species of Chara the stipulodes occur in two whorls, the upper whorl
pointing upwards, the lower whorl pointing downwards (Fig. 1.2a, b). In the genus
Lamprothamnium and some species of Chara the axial node can also be the site of
gametangial development (Fig. 1.2c).
Branchlet arrangement (Fig. 1.3) and morphology (Fig. 1.4) varies among the genera, but is
characterised by elongate multinucleate cells interrupted by multicelluar branchlet nodes.
Other cellular structures can be produced at the branchlet nodes, namely bract cells (Fig. 1.3a,
b, Fig. 1.4a, b), secondary and tertiary (et seq.) branchlet segments or rays (Fig. 1.3c, d, Fig.
1.4c, d), cortical filaments (Fig. 1.4a) and gametangial initials (Fig. 1.4). Some species of
Chara have elongate bract cells in whorls (verticillate) at the branchlet nodes, as well as at
the apicies of the branchlets (Fig. 1.3a). Other species produce unilateral bract cells (Fig.
1.4a). Lamprothamnium exhibits the same overall branchlet morphology as Chara but the
bract cells are generally inserted at angles of 45° to 90° to the branchlet, forming a ‘cage-like’
structure around the nodal complexes (Fig. 1.3b, Fig. 1.4b). Nitella species have branchlets
that are divided or forked (furcate) into separate rays or segments (Fig. 1.4c), which can be

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very evenly arranged (Fig. 1.3d) or messy (Fig. 1.3c). Some species of Nitella can produce
more than one whorl of branchlets at the nodal complexes, a condition referred to as
‘heteroclemous’ (Fig. 1.3d). Tolypella branchlets are different from those in other genera,
usually with a central pluricellulate ray, secondary rays and clusters of gametangia (Fig.
1.4d).
The gametangial initial cell produces the gametangia (oogonia and/or antheridia), bracteoles
and sometimes a bractlet (Fig. 1.5). Oogonia have a striped appearance with a small but
distinctive crown of cells (coronula) at their apex. Oogonia vary in colour from green to
bright orange, and in older parts of the thallus the fertilised oospore within the oogonium
becomes darker as it matures. The antheridia can also be green, but are often orange to red in
colour, and in dioecious species can be so large (approaching 1 mm in diameter) as to be
mistaken for ‘berries’ on the branchlets. The gametangia (oogonia and antheridia
collectively) are often surrounded by bracteoles (that arise from the base of the gametangia)
and bract cells (that arise from the branchlet node). These can be longer (Fig. 1.5a) or shorter
than the oogonium (Fig. 1.5b). A bractlet can occur at the base of the oogonium in female
dioecious plants instead of an antheridium.
The distinctive characteristic of the characeae thallus is that all of the parts consist (with the
exception of the gametangia and nodes) of single cells or uniseriate filaments of cells. A
notable characteristic of some species of characeae is the development of a coating of
calcium carbonate, ‘marl’ or ‘lime’, when growing in hard-water lakes or streams. In general,
species of Lamprothamnium, Tolypella, a few species of Nitella and corticated species of
Chara can develop a calcareous layer on the thallus and/or a calcified coating on the oospore
(called a lime-shell or gyrogonite). However, the species that do so are not used generally in
physiological studies. Calcium carbonate can be precipitated in bands on the axes and
branchlets of many species of characeae as a consequence of their photosynthesis and
metabolism, but this is generally distinct from the thick encrustation that occurs in calcareous
habitats (see section 2.4).
The growing thallus of characeae plants varies in complexity and size depending on the
species. Most of the species of Chara that occur in the Northern Hemisphere (Krause 1997;
Han et al 1986; Scribalo 2010) have internodes covered by a cortex of smaller, linearly
aligned cells (Fig. 1.2a, b). This layer of cortical cells can prevent physical access to the

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largest cells, and for this reason corticated species of Chara are rarely used in physiological
studies (Winter et al. 1987). The rows of cortical cells are often interrupted by spine cells,
which can be globular (Fig. 1.2a) or long and acute (Fig. 1.2b). They can be sufficiently
abundant in some species as to make the entire thallus appear prickly. In contrast to corticated
species of Chara, all members of the genera Nitella, Tolypella, Lamprothamnium and
Nitellopsis and species of Chara in subgenus Charopsis are without a cortex (ecorticate), at
least in part of the thallus (Fig. 1.2c). The species most commonly used in physiological
studies are Chara australis, C. corallina, C. longifolia, and species of Lamprothamnium
(Table 1).
There are additional species that could be used in physiological studies: Chara braunii is a
widespread ecorticate species (Proctor 1980), but has an annual life history, so is not
commonly maintained in laboratory culture. There are also several short-range endemic
species that have ecorticate stems (C. simplicissima, C. lucida in Australia, C. walichii in
India), or ecorticate branchlets (C. baueri in Europe, C. curtissii in North America, C.
gymnopitys and C. muelleri in Australia). A large number of Nitella species produce robust
thalli with long internodes, but this genus is less commonly used in physiological studies.
1.2 Morphology of individual species
There are six recognised extant (as opposed to fossil) genera in family Characeae: Chara and
Nitella contain the majority of species in the family, Lamprothamnium, Nitellopsis and
Tolypella have several species each, and Lychnothamnus has a single species, Ly. barbatus
which is rare world-wide (Sugier et al. 2009). Many species could be useful for physiological
studies, but the majority of studies are done on those species of Chara or Lamprothamnium
that have already been cultured, or are common and easily introduced into culture in the
laboratory (Table 1).
The following list of species have been used in physiological studies and are characterises by
their large cell size, lack of cortication on the axis or branchlets, relatively simple
morphology and ease of collection or culture. As such, they do not represent the entire range
of variation that exists in characeae plants, and have some very specialised characteristics.

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1.2.1 The Chara corallina/australis group of species
The species in this group have a relatively simple morphology compared to most other
species of Chara (which are illustrated in Figs 1-5). The internodes and branchlets are
without a cortex (ecorticate or naked), which allows direct manipulation of the giant cells that
make up the internodes (Fig. 1.6a). The gametangia occur either together (oogonium above
the antheridium in Chara corallina Kl. ex Willd.) or on separate plants (in the dioecious
Chara australis R. Br.), so sometimes two plants (male and female) are needed for sexual
reproduction to take place (Fig. 1.6b, c). The absence of a cortex coincides with a lack of
spine cells. There is one row of stipulodes beneath the branchlets (Fig. 1.6f), and this is
usually poorly developed (Fig. 1.6g). Bract cells and bracteoles are reduced in comparison
with many species (Fig. 1.6e). Chara corallina and C. australis were amalgamated in the past
(Wood 1962; 1965; 1972), and the two species are distinguished primarily on the basis of
sexuality (i.e. C. corallina is monoecious (both male and female organs occur on the one
plant) and C. australis is dioecious (separate male and female plants)). Oogonia and
antheridia occur on the branchlets as well as inside the base of the branchlet whorl (Fig. 1.6a,
b, c).
The taxonomy of this group has been problematic and over time (see section Charophyte
names) and members of this group have been variously placed in the genera Tolypellopsis,
Protochara, Chara and Nitella (Table 1. Wood 1972). Recent morphological (e.g. Casanova
2005) and genetic analysis indicates that Chara australis from Australia and New Zealand,
and Chara corallina from south-east Asia, should be retained as separate species, and that
Chara lucida A. Braun (a very thin species with short internodes from tropical northern
Australia) and Chara stuartiana Kütz. (a very robust species from Tasmania) can also be
distinguished. The Asian species Chara wallichii A. Braun (India) and C. fulgens Filarszky
(Indonesia) have also been described and these differ from C. australis and C. corallina in
the arrangement of the reproductive structures and expression of bract cells (Zaneveld 1940).
1.2.2 The Lamprothamnium papulosum/macropogon group of species
Lamprothamium papulosum (Wallr.) J. Groves was the first species to be described in this
group. It was originally placed in the genus Chara and later transferred to the genus
Lychnothamnus. Later, when it was found to be different from the other species of
Lychnothamnus it was placed in a genus on its own (Lamprothamnus), which was renamed

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Lamprothamnium in 1916 to avoid a nomenclatural conflict with a flowering plant genus
(Groves 1916). Other species of Lamprothamnium were added over time, so that in 2010 the
genus had approximately seven recognised species. All members of the genus
Lamprothamnium have ecorticate internodes and branchlets (similar to C. australis), but
differ in the presence of downward pointing stipulodes and angular whorls of bract cells at
the branchlet nodes (spreading, verticillate bract cells) (Fig. 1.7a). Many specimens have
distinctive, spherical white bulbils at the rhizoid nodes (McNicol 1907; Ophel 1947). These
bulbils are full of starch grains, and they allow the plant to persist when it is unable to
photosynthesise, and to regenerate when the vegetative axis is uprooted or destroyed. There is
a high degree of morphological similarity among species of Lamprothamnium (García and
Casanova 2004), and the most recent taxonomic treatments use the arrangement of the
reproductive organs as a guide for distinguishing different species (Casanova et al. 2011).
Lamprothamnium papulosum has antheridia and oogonia together on the branchlet nodes,
with the antheridium above the oogonium (Fig. 1.7e). There are a number of species of
Lamprothamnium in Australia, many of which were amalgamated with European L.
papulosum in the past (Wood 1972), however none of them exhibit the particular
arrangement of reproductive organs found in that species. The most commonly mentioned
species is L. macropogon (A. Braun) I.L. Ophel. In non-reproductive plants of L.
macropogon there can be a whorl of upward-pointing stipulodes within the branchlet whorl
(Ophel 1947). The oogonia are clustered inside the base of the branchlet whorl, and one or
two can be found on the first branchlet node (Fig. 1.7b, c). The antheridia are rarely present
singly inside the branchlet whorls (jammed in with the oogonia), they are mostly confined to
the first and second branchlet nodes (Fig. 1.7d). Male and female gametangia occur together
(side by side) at the same branchlet node rarely. The shoots of species of Lamprothamnium
often have a dense ‘fox-tail’ appearance (‘alopecuroid’) because the upper internodes are
short and the branchlets, stipulodes and bract cells overlap. There are several additional taxa
of Lamprothamnium similar to L. papulosum and L. macropogon, including L. hansenii in
which the branchlet segments are rather stout and constricted at the nodes (Wood 1965), L.
heraldii which is dioecious (García and Casanova 2004), L. papulosum vars toletanus,
carrissoi and aragonense from Spain (Cirujano et al. 2008) and L. sonderi in Germany
(Schubert and Blindow 2003), but these are less frequently used for physiological studies.
Additional, undescribed species of Lamprothamnium exist in Australia.

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1.2.3 The Lamprothamnium succinctum group of species
The original collection of Lamprothamnium succinctum was made in Libya, North Africa,
and was described as a species of Chara (A. Braun in Ascherson 1876). It was recognised as
a species of Lamprothamnium by Wood (1962). The species was confusing because of the
poor expression of the vegetative characters that characterise the genus i.e. downward
pointing stipulodes and verticillate bract cells, and the presence of reproductive organs at the
base of the whorl which indicated a relationship with Chara corallina and C. australis (Braun
and Nordstedt 1882). Wood (1965) amalgamated specimens from Mauritius and New
Caledonia with the Libyan specimens, because those specimens also had reduced stipulodes
and bract cells, and gametangia at the base of the whorl. Later he placed similarly
depauperate species of Lamprothamnium in Australia in L. papuluosum, describing a new
form, f. succintoideum (Wood 1972) for them (Fig. 1.8). However, despite records of the
species in Australia (García et al. 2002) there is no evidence that L. succinctum occurs over
such a wide area, from Northern Africa to islands in the Indian and the Pacific Oceans, and in
southern Australia. It is more likely that there are additional species with similar
characteristics. The illustrations provided of the Mauritian species in Imahori and Wood
(1965) display a different arrangement of reproductive organs from the type material, so that
specimen, at least, is unlikely to be conspecific with L. succinctum. In the absence of an
updated taxonomy specimens of L. succinctum that have been collected from places other
than the Libyan desert (Braun and Nordstedt 1882) and used in physiological studies, can be
called L. sp aff. succinctum. Australian specimens in this group are generally long and
slender, with few or short stipulodes below the branchlet whorls (Fig. 1.8a), and few or short
bract cells (Fig. 1.8b). The fertile whorls can be contracted into the typical ‘fox-tails’ of the
genus, but the antheridia occur on short stalks outside the base of the whorls, and on the
branchlets (Fig. 1.8c, d), and the oogonia are clustered within the whorl of branchlets as well
as on the branchlets (Fig. 1.8c, e). This taxon is particularly useful in physiological studies
because of its elongated internodes.
1.2.4 Lamprothamnium inflatum
The ‘inflated’ charophyte Lamprothamnium inflatum (Fil. & G. O. Allen ex Fil) A. García &
K.G. Karol has had a long and confused taxonomic history, largely because of its unusual
morphology (Fig. 1.9). When growing in the field the young thallus appears as a (dense or
sparse) mat of green bubbles erupting from the surface of the mud, the bubbles being the
swollen tips of the branchlets. As plants mature the narrow axis elongates and dense whorls

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of inflated branchlets develop, clustered towards the top of the axis. The inflated parts of the
plant are the branchlet cells, and often only the second (or penultimate) cell of the branchlet
(Fig. 1.9b). In comparison the axis cells are narrow and thick-walled. The plants are
monoecious and the oogonia and antheridia are clustered at the bases of whorls and at the
branchlet nodes (Fig. 1.9c, d). Many specimens of Lamprothamnium have inflated sterile
branchlets, but L. inflatum has also inflated fertile branchlets. In common with other species
of Lamprothamnium it tolerates fluctuations in the salinity of its medium, and probably
doesn’t thrive in fresh water. Its natural distribution is in brackish to saline temporary
wetlands in the south of Western Australia, South Australia and Kangaroo Island.
1.2.5 Species of Tolypella
The genus Tolypella (Fig. 1.10) appears to be restricted to latitudes greater than c. 40° in both
the Northern and Southern Hemispheres (Wood 1965). The genus is characterised by its
relatively disorganised thallus, with large numbers of sterile and fertile branchlets, branches
and reproductive organs arising from the nodal complexes. Plants usually consist of a long
protonema or primary axis cell from which a series of complex nodes arise, each with many
simple sterile branchlets, branches and reproductive organs arranged in a disorganised
fashion (Fig. 1.10a). The structure of the branchlets is different from that of members of
Chara, Lamprothamnium or Nitella, consisting of a primary branchlet cell, a nodal complex
that produces the continuation of the primary ray, a number of secondary rays and
reproductive organs (Fig. 1.10b, c). The antheridia and oogonia are often stalked (stipitate)
and the oogonia have 10 coronula cells (Fig. 1.10d). In some of the species of Tolypella the
coronula is deciduous (i.e. falls off at maturity) and the spiral cells become swollen (Fig.
1.10e). Some species produce gyrogonites (lime-shells) around the oospores. The oospores
are terete in cross-section (c.f. Nitella in which the oospores are flattened in cross-section),
and often flanged on the striae.
The common name for some species of Tolypella is the ‘birds nest’ or ‘tassel’ stonewort,
eflecting the highly divided and disorganised nature of the thallus. Many species can tolerate
salinity to varying degrees (Winter et al. 1996), but they do not grow in strictly marine
systems. Although species of Tolypella occur in permanent lakes in North America, there
appears to be a higher diversity of species in temporary systems, requiring disturbance or
drying of their habitat for the maintenance of populations (Stewart and Church 1992).

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1.2.6 Species of Nitellopsis
Nitellopsis (Fig. 1.11) is a Northern Hemisphere genus, so far absent from Africa, Australia
and South America. The genus occurs in relatively deep water in European lakes (Shubert
and Blindow 2003), as well as in North America. Nitellopsis obtusa (Desv. in Lois.) J.
Groves can be dominant in lakes and frequently co-occurs with the rare charophyte
Lychnothamnus barbatus (Pełechaty et al. 2010). It reproduces sexually as usual, but also
asexually via distinctive star-shaped bulbils (Fig. 1.11d) sometimes called starch-stars
(Fritsch 1948), attached to the lower stems and rhizoids. The capacity for asexual
reproduction could improve the success of long-term culturing in the laboratory. The long,
ecorticate internodes and branchlets can be large enough for physiological studies (Winter
and Kirst 1990; Winter et al. 1999), and it is tolerant of low levels of salinity.
The plant body is relatively simple (Fig. 1.11a) with whorls of elongate branchlets and simple
bract cells. Stipulodes are absent or rare at the base of the whorl, there is no cortication and
few accessory structures (Fig. 1.11c). The oogonia and antheridia are on separate plants
(dioecious), and the oogonia can be calcified, with the oospore surrounded by a gyrogonite
(Fig. 1.11b). Oospores and gyrogonites of Nitellopsis can be among the largest of extant
charophyte reproductive organs.
1.2.7 Species of Nitella suitable for physiological studies
Nitella species are identified on the basis of a coronula of 10 cells on the oogonium (Fig.
1.12b) and furcate or forked branchlets, at least in the fertile parts (Fig. 1.12a, c). The
terminal branchlet segments or rays are called ‘dactyls’ and the arrangement of cells in the
dactyls is used to determine the subspecies and species of Nitella (Fig. 1.4c). The species of
Nitella that have been used for physiological studies in the past are generally characterised by
long internodes, interrupted by few, sparsely forked whorls of branchlets. Species with short
internodes and dense whorls (e.g. Fig. 1.12a) are unlikely to be useful, simply because access
to their large cells would be difficult. In general species of Nitella have branchlets in whorls
of 6 to 9 (Fig. 1.3c, d, Fig. 1.12c), except where there is more than one whorl of branchlets at
the node (a condition termed ‘heteroclemous’ Fig. 1.3d). Branchlets can be once, twice or
more times furcate (Fig. 1.4c). Fertile branchlets can be similar to the sterile ones, or
contracted into ‘heads’ or ‘spikes’. They can sometimes be covered with a jelly-like
substance called ‘mucus’. The reproductive structures occur at the branchlet nodes (forks),

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where the antheridia are terminal or central in the fork (Fig. 1.12d), and the oogonia are
lateral. The taxonomy of Nitella species is gradually being sorted out by various authors, and
the overall morphology as well as the characteristics of the oospore are useful clues to
identity (Sakayama et al. 2002; Casanova 2009). Species with single-celled dactyls (terminal
branchlet segments) (e.g. Fig. 1.12d) are in two groups; Palia and Nitella, those with
pluricellulate (multicelluar) dactyls are in subgenus Hyella, and those with two-celled
dactyls, of which the terminal one is small, conical and acute (bicellulate e.g. Fig. 1.12c), are
in subgenus Tieffallenia. The validity of these higher taxonomic groupings is under
investigation, as is the membership of those groups.
1.3 Cellular structures and processes
1.3.1 Cells
Characeae organs (internodes, branchlet cells, stipulodes, bract cells etc.) are single cells or
groups of single cells. In general they have a typical plant-cell structure with a cellulose cell
wall, plasma membrane enclosing a thin layer of cytoplasm containing chloroplasts,
mitochondria, nuclei, protein bodies and statoliths, surrounding a large tonoplast-bound
vacuole (Fig. 1.13). At the ultrastructural level charophyte cells have characteristics in
common with the cells of other algae (Zygnematophytes), mosses, liverworts and vascular
plants (Casanova 2007). The plant axis is produced via sequential divisions of a dome-shaped
apical cell, the products of which differentiate into discoid nodes and elongate internodes
(Fritsch 1948). Axis nodal initials divide into central cells and a series of peripheral cells that
differentiate into branchlet, stipulode, cortical and gametangial initials (Bharathan and
Sundralingham 1984). Branchlet initials divide further into branchlet internode cells and
nodal complexes (Fig. 1.13). The cortical filaments, bract cells and gametangial initials
develop from the branchlet nodes.
The cell wall construction in charophytes is basically fibrillar, crystaline cellulose (Casanova
2007), sometimes with a superficial layer of mucilage (check Mary). The chloroplasts are
lodged in the peripheral layer of the cytoplasm, close to the cell wall, and are arranged in
longitudinal lines. Internode cells contain multiple copies of the nucleus formed via amitosis
(Fritsch 1948). The axial node complex consists of different numbers and arrangements of
cells and can be used to distinguish among genera: in Chara there are two central cells in the
nodal complex, and in Nitella, Tolypella and Lamprothamnium one of the central cells
undergoes further division into daughter cells (Frame and Sawa 1975).

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The contents of the cell are in continual movement in healthy internodal cells, with active
streaming clearly visible under relatively low magnification (10-20 x magnification). The
lines of chloroplasts in the peripheral cytoplasm are interrupted by a less-dense part of the
cytoplasm, around which the cytoplasm rotates (Fritsch 1948). The vacuole that takes up the
majority of the cell volume in the centre of elongate characeae cells is responsible for
generating and maintaining cell turgor (the internal pressure that maintains the rigidity and
shape of the cell) (Pickett-Heaps 1975).
1.3.2 Gametangia
The charophyte plant body is haploid (i.e. n chromosomes) and gametes (also n
chromosomes) are produced via mitoitic division in the reproductive organs. Characeae male
gametes consist of motile sperm cells (n chromosomes) (Fig. 1.14a) that swim through the
water to fertilise the female egg cell (n chromosomes). Specialised, diploid (2n), seed-like
oospores (Fig. 1.1c) are the product of fertilisation of the egg cell by a sperm cell. Characeae
gametangia, or reproductive organs, have a distinctive morphology (Fig. 1.5). Unlike other
algae, the gamete-producing cells (spermatogenous threads (Fig. 1.14b) and egg cell) are
surrounded by sterile cells that protect them. These organs are variously called (for male
organs) the globule or antheridium; and (for female organs) the nuclule, oogonium or
oosporangium. Development of the oogonium progresses from a single peripheral cell on the
branchlet node, producing the five spiral cells and an oosphere mother cell. The spiral cells
elongate around the mother cell which divides to produce the oosphere or egg cell (Leitch et
al. 1990). The oogonium has one or more basal cells. The spiral cells divide to produce a
crown-like structure (coronula) of either five cells (in Chara, Lamprothamnium, Nitellopsis
and Lychnothamnus) or ten cells (in Nitella and Tolypella). Antheridial development
similarly proceeds from a peripheral cell (Pickett-Heaps 1968), and the mature antheridium
consists of a mass of spermatogenous filaments or threads attached to manubria cells via
capitula cells, surrounded by (usually) eight shield cells (Fig. 1.14b). When the male gametes
(spermatids (Pickett-Heaps 1968b): Fig. 1.14a) are mature they exit the spermatogenous
threads via pores (Fritsch 1948), and the shield cells separate slightly to release them,
eventually falling apart altogether (Fig. 1.14c). When the egg cell is ready to be fertilised the
cells below the coronula become swollen and gaps are formed between the spiral cells
allowing entry of the sperm cells. After fertilisation the egg cell becomes the diploid zygote
(i.e. 2n chromosomes) and a complex, multilayered wall is formed by both the developing

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zygote and the maternal spiral cells (Leitch et al. 1990). This specialised zygote is called the
oospore and its multilayered wall allows it to resist desiccation and destruction during
dispersal and dormancy.
1.3.3 The oospore
Characeae oospores are distinctive and easy to recognise (Fig. 1.1c). In all the genera except
Nitella there is sometimes the development of a gyrogonite, or calcified ‘shell’ around the
oospore, deposited during the last stages of development on the parent plant (Fig. 1.15a).
Gyrogonites can gradually decompose over time, or break open when the oospore germinates.
The oospore wall, within the gyrogonite, is formed by both the egg cell (internal layers) and
the oogonium cells (external layers), and is characterised by spiral sutures or lines on its
surface (Leitch 1989). The flat part of the oospore wall is called the fossa, and the sutures are
called the striae. Sometimes the striae have flanges (where the oospore wall is formed on the
side walls of the spiral cells; Fig. 1.15b), and sometimes a thinner structure called a ‘ribbon’
(John et al. 1989) or thicker structures (pachygyra; Fig. 1.15c) are formed. The fossa can be
variously ornamented with grains (typically so for Lamprothamnium) or fibres. The
ornamentation becomes amazingly diverse in the genus Nitella, with verrucae, tuberculae,
reticulae and every combination of wall construction imaginable (Fig. 1.15d, e, f) (John and
Moore 1987). The base of the oospore is impressed with the shape and arrangement of the
basal cell complex. In Chara there is a single basal cell (Fig. 1.15g), in other genera there can
be more (Fig. 1.15h). The apex of the oospore exhibits a ‘w-shaped’ suture where the five
spiral cells join up (Fig. 1.15i). Oospores can be different colours, ranging from pale yellow
to black. In some Nitella species oospore colour is an indication of the maturity of the
oospore, and degree of development of the oospore wall (Casanova 1991). Sometimes
oospore colour is useful for distinguishing species of Chara (Zaneveld 1940). Oospores are
densely packed with starch grains (amyoplasts), the primary reserve for the germinating
plant. Healthy oospores are turgid and resistant to external forces. They can be handled easily
with fine forceps. The distinctive differences among charophyte oospores allows them to be
used in identification of different charophyte species (Haas 1994, Sakayama et al. 2002,
Casanova 2005, Casanova 2009). Cultures of charophytes for physiological studies can be
started by collecting mature oospores, sterilising their surface and germinating them under
controlled or known conditions (see section 1.5.5).

14
1.3.4 The germinating oospore
At some time between fertilisation of the egg cell on the parent plant, and germination of the
oospore, reduction division (meiosis) occurs, resulting in the regeneration of haploid (n)
thallus cells (Fritsch 1948). Despite some thorough studies (e.g. Leach 1989) the exact timing
of meiosis has not yet been discovered. The start of the germination process sees the oospore
wall split along the ‘w-shaped’ suture at the apex. The first two cells of the germinating
charophyte develop into the protonema (first thread) and the rhizoid initial (Ross 1959). Axes
and whorls of branchlets develop from the first node of the protonema (Fritsch 1948; Ross
1959). The rhizoids are positively geotropic (Kiss and Staehelin 1993) and grow into the
substrate. The protonema is negatively geotropic and positively phototropic (Fritsch 1948)
and grows up through the soil or substrate, often from depths of 25 mm or more,
exceptionally from depths of at least 100 mm (Dugdale et al. 2001). Germination rates can be
enhanced by light of certain wavelengths (Sokol and Stross 1992), although germination is
possible in the absence of exposure to light (de Winton et al. 2004). However, successful
establishment of the young plant appears to be light dependent when the oospore starch
reserves are exhausted (de Winton et al. 2004).
1.4 Characeae names
Characeae are commonly called ‘stoneworts’ because of the deposition of calcium carbonate
on the axes and branchlets, especially in lime-rich habitats. However, many species,
particularly in the genus Nitella, never accumulate calcium carbonate, and the name is quite
unsuitable for these often delicate, mucus-covered, flexible species. Occasionally characeae
are referred to as ‘musk-grass’, due to the strong, pungent, sometimes rank smell associated
with certain corticated species (Anthoni et al. 1980). The smell can be so strong and
distinctive that a single shoot can be detected in a handful of other vegetation. However, not
all species produce the odour, so the term ‘musk-grass’ is inappropriate in many cases. For
this reason, in this text, we use the term ‘characeae’ as a name for the group. This, of course,
does not replace the need for recognised scientific binomial names. Characeae taxonomy and
the application of binomials has been difficult for non-specialists, and only slightly easier for
specialists. The application of genetic analytical techniques has helped to solve many
problems.

15
1.4.1 Systematics and the concept of species
The superficial resemblance of characeae to flowering plants has led, in the past, to confusion
about their place in botanical systematics (Fritsch 1948). Early botanists discussed whether
they were the simplest of flowering plants or the most complicated of algae. Recent
phylogenetic studies (Karol et al. 2000) have shown that they are the closest living relatives
of the ancestors of land plants. They have evolved an analogous morphology to aquatic
angiosperms, in the same environment. The common and ancient ancestry of characeae and
land plants means that many of their cellular processes and mechanisms are the same as those
of land plants, a fact that makes the study of characeae physiology directly relevant to the
physiology of land plants, including plants and processes of economic interest.
Characeae have been recognised as fossils in sediments dating as far back as the Silurian
(Grambast 1974) and have been through various changes in diversity and abundance over the
millennia. Living characeae were among the species first described in Linnaeus’ Species
Plantarum in which he sought to develop a consistent taxonomy. Characeae were not
recognised as being substantially different from other plants, and most workers at that time
saw them as a watery Equisetum or horsetail. It was later that their unique differences were
recognised, and they were set apart in Division Charophyta.
Although a number of characeae species are regularly used in physiological studies,
physiologists seeking to have their plant identified to species can obtain erroneous or
conflicting answers. There are a number of reasons for this. Firstly characeae morphology is
affected by the environment for growth, and there is variation in plant size and appearance
depending on the light availability and water chemistry of the habitat. Secondly, although
characeae have been well known for a long time, the taxonomy of characeae is undergoing a
revolution, based on recent advances in the understanding of variation and speciation in the
group and genetic data about relationships among species and species groups.
The taxon level called ‘species’ is not the same in all groups of organisms. In birds and
mammals relatively small genetic differences are present between ‘species’. In plants there
are often larger genetic differences. In algae the genetic distances among ‘species’ can be
larger still. The detection of characteristics that allow us to reliably discriminate among
different taxa are often based on historical precedent or subjective assessment (i.e. what the
first taxonomist used) rather than objectivity or experimentation (Proctor 1980), and linking

16
what has been described as a species with a genetically discrete ‘clade’ or group of
individuals is one of the frontiers of taxonomy today. Traditionally characeae species have
been organised into groups within a genus (subgenera, sections, subsections) and ‘complexes’
of closely related species have been recognised. A charophyte ‘species complex’ is a group of
morphologically similar, closely related taxa that have, historically, had the morphological
extremes described as different species by some authors (e.g. Braun and Nordstedt 1882), and
described as different varieties or forms by other authors (Wood 1965; Boegle et al. 2007;
Blume et al. 2009). Examples of these are the Nitella hookeri complex of Australia and New
Zealand (Casanova et al. 2007) and the Chara baltica complex of central Europe and the
Baltic Sea (Boegle et al. 2007; Blume et al. 2009).
Historically the first comprehensive taxonomic treatment on characeae was by Braun and
Nordstedt (1882), and was based on the specimens from around the world available to those
experts at the time. They distinguished between the genera Chara and Nitella, but the other
genera were not delineated and named until later in the 19 th (Tolypella, Lychnothamnus,
Nitellopsis) or 20 th centuries (Lamprothamnium). Researchers in the past often saw few
specimens of each species, so the conclusion that many characeae ‘species’ had a broad range
of characteristics was a natural conclusion. Subsequent taxonomists used Braun and
Nordstedt (1882) as a basis for their taxonomy (Zaneveld 1940, Wood 1962), but it was not
until 1965 that a world-wide treatment of family Characeae was attempted. Wood (1965)
tried to rationalise the species concept in Characeae, amalgamating monoecious and
dioecious species and grouping morphologically similar specimens from around the world.
His work resulted in a reduction of the 350 recognised species to just 18 species of Chara,
three of Lamprothamnium, 90 of Nitella, three of Nitellopsis and two of Tolypella.
Subsequent research has shown Wood’s species concept to be erroneous, and rather than
there being c. 180 species in family Characeae, the older treatments (c. 380 species) give a
better representation of the true diversity. This has consequences for physiologists seeking to
identify charophytes. Firstly Wood’s work resulted in confusion rather than consensus among
taxonomists. Long-held species concepts had been demolished and in many places in the
English-speaking world Wood’s treatment was accepted without challenge. Few people using
Wood’s taxonomy were able to reliably identify characeae to species. This was especially
true for some of the species most frequently used by physiologists: Chara
australis/corallina/inflata, Lamprothamnium macropogon/paulosum/succinctum. With the
use of molecular taxonomy (e.g. McCourt et al. 1996) and an experimental approach to the

17
definition of species (Proctor 1975), taxonomists in the 21 st century can provide a better
taxonomy.
The use and citation of designated clones in physiological studies is to be encouraged, given
the past taxonomic difficulties with the group. Specimens used for physiological studies
should also be vouchered (preserved by pressing or in 70% alcohol, and lodged in an
internationally recognised herbarium) as a standard practice. Misidentification of taxa in the
past (at both species and genus level) could go some way towards explaining difficulties in
generalisation about characeae physiological processes.
1.4.2 Individual species’ stories
1.4.2.1 Chara australis R. Br.
One of the most popular species used in physiological studies is the dioecious species Chara
australis. The species was collected first from the vicinity of Sydney in New South Wales,
Australia, by Robert Brown, a botanist on Matthew Flinders’ exploratory expedition to
circumnavigate Australia in 1802-03. It was one of only two species in family Characeae
described for Australia in the early 19 th century (Brown 1810). At least two collections in the
1800s were named Chara australis (which means ‘southern Chara’), but the one named by
Brown has priority, so it is the one that retains the name. Later, Alexander Braun (1843)
described a similar specimen as Chara plebeja, which he later amalgamated with C. australis
as a subspecies (Braun and Nordstedt 1882). In the same publication (Braun and Nordstedt
1882) Braun was inclined to distinguish more varieties and forms of Chara australis, so he
presented a descriptive list including C. australis var. nobilis form stuatiana, var. lucida form
tenerior, var. vieillardii and forms vitiensis and simplicissima as well as subspecies plebeja.
The group was reviewed by Zaneveld (1940) who had the advantage of seeing more material
from a diversity of places, but he followed Braun’s taxonomy closely. In the 1960s R.D.
Wood amalgamated the entire complex with monoecious Chara corallina Kl. ex Willd. from
south-east Asia (Wood 1962), on the basis of its morphological similarities. Wood (1965)
retained many of the subdivisions recognised by Braun and Zaneveld, but this time within the
species Chara corallina. Thus since the 1960s we have had Chara corallina with three
varieties and eight forms, at least half of which were dioecious, the other half monoecious.
Proctors work (summarised in Proctor 1975) established that monoecious Chara corallina
and dioecious Chara australis were different species, but the status of many of the other

18
entities within the complex has not been fully examined. We can be sure, at least, that
dioecious specimens that originated in eastern Australia and have the morphology illustrated
in Fig. 1.5, can be called Chara australis, and that monoecious specimens that have a similar
morphology are likely to be Chara corallina.
1.4.2.2 Lamprothamnium inflatum (Fil. & G.O. Allen ex Fil.) A. García & K.G. Karol
This curious species (Fig. 1.8) has been well known, and misnamed for a long time. The first
collection of this species was made by Nancy Burbidge, the eminent Australian taxonomist,
in 1933, while swimming in Lake Parkeyerring near Wagin in Western Australia. It is an
attractive plant when actively growing, looking like small bunches of glistening green grapes.
The preserved specimens were sent to G.O. Allen (in England) who was the world expert on
charophytes in the English-speaking world at the time. He sent a subsample to F. Filarszky in
Hungary, who described the species as Nitellopsis inflata. In recognition of the role that Allen
had in the discovery, Filarszky included Allen in the authorship of the species, although Allen
was not consulted about the determination (Wood 1964). Next, this odd species was included
in the genus Protochara by South Australian phycologists Womersley and Ophel (1940)
although they had not examined any specimens (Womersley and Ophel 1940). When
Macdonald and Hotchkiss (1956) investigated the genus Protochara (specifically Protochara
australis) their results indicated that the genus should be amalgamated with Chara, so
Protochara inflata became Chara inflata (Fil. & G. O. Allen ex Fil) Macdon. & Hotch.,
again, without a thorough examination of any specimens of the species. Then, in a revision of
the Australian charophyte flora Wood (1962, 1965, 1972) reduced the species to a form of
Chara corallina (C. corallina var. nobilis f. inflata) despite the fact that it was monoecious
and all other Australian specimens in that group were dioecious. Thus the species was
allocated to three different genera. However, recent evidence places the species firmly within
the genus Lamprothamnium (García and Karol 2004). This placement is supported by
morphological characteristics such as the presence of gyrogonites (calcium carbonate ‘shells’
around the oospore), contracted apical whorls and occasional downward-pointing stipulodes
(Fig. 1.8).
1.4.2.3 Chara longifolia C.B. Robinson
This enigmatic species has been described as three different species, in two genera in the
past: Chara longifolia (which is the currently accepted name), C. buckellii and Nitellopsis

19
bulbilifera. The natural range of the species is in North, South and Central America in
brackish habitats (Mann et al. 1999). Chara longifolia was originally collected from Kansas,
USA by M.A. Carleton in 1891 (Robinson 1906). The species was dioecious, had an
irregularly corticated axis, poor development of stipulodes (absent in older nodes) with c. 8
ecorticate branchlets in each whorl with distinctive decumbent bract cells. Robinson
commented on the similarity of this species with Nitellopsis, but described it as a new species
of Chara (Robinson 1906). The species was known only from its type collection (Wood
1965). In 1928 Dr Edward Buckell, a friend of the British charophyte specialist James
Groves, collected another specimen from British Colombia, Canada which was described by
Allen (1951) as Chara buckellii. Again, the specimens had irregularly or incompletely
corticated axes, with ecorticate branchlets and posterior bract cells distinctively deflected
downwards. Again, this species was known only from the type locality (Allen 1951; Wood
1965). In 1959 a specimen was cultured from soil collected from Laguna La Brava near
Buenos Aires, Argentina, by Carlotta Carl de Donterberg, who described it as Nitellopsis
bulbilifera (Carl de Donterberg 1960). Tindall et al. (1965) reported N. bulbilifera (cited as N.
bulbillifera) from New Mexico, and illustrated the morphology of the thallus. Although
Tindall et al. (1965) received Chara buckellii from V.W. Proctor in Texas for examination at
the time of their study of N. bulbilifera, they concluded that despite the common
characteristics with C. buckellii (dioecious nature, and similarity of morphology of the
branchlets and bract cells), the presence of a corticated stem and stipulodes in C. buckellii
was sufficient to distinguish the two taxa. Daily (1967) also examined the taxon, and
transferred all the previously described species to the genus Lamprothamnium, but this
taxonomic change was very short-lived, as in the next article in the same issue of J. Phycol.
Proctor et al. (1967) presented evidence that all of the species (Chara longifolia, C. buckellii
and N. bulbilifera) were conspecific. The evidence consisted of 1) similar morphology, 2) the
same number of chromosomes, and 3) the production of viable oospores when crossed. This
finding, and the designation to the genus Chara, has been further supported by genetic studies
(Meiers et al. 1999). Since all these taxa are the same species of Chara, the oldest name has
priority, thus the species is Chara longifolia C. B. Robinson.
1.4.3 Lessons to be learned
The morphology of ecorticate charophytes is relatively simple, and there are few outstanding
characters with which to distinguish species. Those that are available (arrangement and
angles of stipulodes and bract cells, arrangement of gametangia) are not always obvious or

20
expressed on plants in culture. Added to this is the fact that there is more species-level
diversity among ecorticate characeae than has been recognised in the past. The only way to
be sure that the entity used in experimentation is the same as that used by someone else is to
use the same culture. However, approaches to taxonomy are improving all the time, and
chances of correct identification are increasing with every new taxonomic work undertaken,
especially with the establishment of gene sequencing approaches, DNA fingerprinting and
DNA bar-coding. Isolation of new culture material from wild populations is likely to have
significant benefits for physiological studies, and could produce novel insights in relation to
old problems. In all cases preservation of reference voucher material in a recognised
herbarium is vital so that the species used can be reliably identified in the future, even if the
taxonomy changes. It should be standard protocol for the herbarium material to be referred to
in the description of methods for physiological studies. This can contribute to making the
research timeless and applicable into the future.
1.5 Ecology
1.5.1 Distribution and abundance
Characeae occur in non-marine water bodies of all kinds, on every continent except
Antarctica. The only occurrence of characeae in marine waters is in estuaries and saltmarshes
associated with marine influence, and in the Baltic Sea, where they are restricted to
areas of relatively low salinity, or sheltered bays and inlets (Blindow 2000; Appelgren and
Matilla 2005). They can grow in water as shallow as a few centimetres deep, or down to c. 40
m at the limits of plant growth under water (Casanova et al. 2007). They grow in both
temporary and permanent water bodies (Casanova 1993), lakes, rivers (Bornette and Arens
2002), swamps, seeps, bogs, fens, marshes, creeks, lagoons, clay-pans, wadis, water-holes
and billabongs (Casanova and Brock 1999b). Most species prefer water with little current or
wave action, but some can grow in relatively fast-flowing water, water that experiences
waves, as well as in the backwaters and shallows of rivers and streams (Bornette and Arens
2002). Characeae populations can occur as dense monospecific ‘beds’ across the bottom of
lakes and wetlands, or as isolated single plants, restricted to a particular depth or soil type.
Early studies into characeae distribution distinguished Chara-lakes (shallow, calciumcarbonate-rich
lakes) from other kinds of lakes (e.g. John et al. 1982) and there was
recognition of the value of temporary wetlands as characeae habitat (e.g. Corillion 1957).
Other historical and current studies of characeae ecology have focused on their distribution in
the landscape (García and Chivas 2007). The two questions ‘what species occur?’ and ‘where

21
do they occur?’ are central to such studies and have been vital in the formulation of
hypotheses concerning characeae distribution and abundance. In other studies ecological
experiments have been undertaken, in order to understand the mechanisms (i.e. ‘how?’) and
reasons (i.e. ‘why?’) for the diversity and distribution of characeae that we see (Blindow et al.
2002).
1.5.2 Processes controlling characeae distribution
Many characeae species develop a long-lived bank of oospores in the soil of permanent and
temporary wetlands and riparian zones (Casanova and Brock 1990). The density of oospores
in this ‘seed’ bank is dependent on 1) the longevity of oospores; 2) the rate of oospore
addition to the bank; 3) the loss from the seed bank via germination of new plants, death or
dispersal (via invertebrate ingestion, or dispersal of the substrate (mud) attached to animals)
(Fig. 1.16). Oospore density can vary from just a few oospores/m 2 (Casanova and Brock
1990) to hundreds of thousands of oospores/m 2 or per litre of surface soil (van den Berg
1999; Porter 2007). The presence and density of oospores in the seed bank depends on
oospore production by adult plants, and in most systems there is likely to be a mass
deposition of oospores of a single age in the late summer and autumn, or as temporary water
bodies dry up, as this is the time when the oospores of most species mature (Casanova and
Brock 1990; Casanova 1994; Casanova and Brock 1999a). In permanent water bodies there
can be immediate germination of fresh oospores, although many oospores are dormant upon
release (Casanova and Brock 1997). Oospores can remain viable in seed banks for years
(Casanova and Brock 1997) and even decades (Simons et al. 1994). There is one record of
oospores germinating from sediment estimated to be at least 45 years old (Rodrigo et al.
2010). Oospore viability and subsequent germination generally decreases over time
(Casanova and Brock 1994), as even very low respiration rates can exhaust the oospore starch
reserves over time, and there is likely to be loss to bacterial infection of oospores as well as
direct herbivory. Germination followed by failure to establish is also a mechanism by which
oospore banks can be depleted (de Winton et al. 2004). As a result of deposition of oospores
over many seasons and the longevity of oospores, oospore banks can contain oospores of
various ages, accumulated over time. Germination from the seed bank, and the differential
germination requirements of characeae species can partly explain characeae distribution in
the landscape (Casanova and Brock 1996).

22
Dispersal of characeae into new habitats occurs most commonly via water-fowl (Proctor
1962; Bonis and Grillas 2002; Casanova and Nicol 2009), and in riparian systems oospores
are probably transported downstream with sediment by flow (Bornette and Arens 2002).
Although characeae occur in a diversity of systems, they are not always a dominant or
permanent component of the submerged vegetation. Classical succession theory suggests that
when characeae are early or ‘pioneer’ components of aquatic vegetation (Stewart and Church
1992), they are replaced by angiosperms and phytoplankton as nutrient concentrations and
turbidity increase (Crawford 1981; Blindow 1992). Thus characeae can be rare in eutrophic
or highly modified systems (Casanova and Brock 1999b), despite the fact that they are
capable of tolerating very low light conditions (de Winton et al. 2004). The alternative stable
states hypothesis (Sheffer et al. 1993 van den Burg et al. 2001) postulates that characeae
(along with other submerged vegetation) produce a stable, macrophyte-dominated state at a
variety of nutrient levels, a condition that can be restored via turbidity reduction. An
appropriate disturbance regime is likely to be vital in maintaining characeae populations and
diversity in ecosystems. Such disturbance can be imposed by water-bird herbivory
(Noordhuis et al. 2001), or by drawdown and drying (Casanova and Brock 1991). Too much
disturbance can reduce or prevent characeae establishment (de Winton et al. 2002). In general
we find that characeae can be permanent components of the vegetation in a number of
situations, and can dominate the submerged flora of calcareous lakes (Forsberg 1960), saline
lakes (Porter 2007) and temporary wetlands (Casanova 2003). They are found more rarely in
farm ponds, although oospores can be present in a majority of farm pond seed banks
(Casanova and Brock 1999b).
1.5.3 Ecological role of characeae
Characeae undertake many ecosystem services including provision of resources to other
organisms and maintenance of processes that enhance biodiversity. Charophytes provide
habitat and shelter for algal epiphytes, invertebrates and fish. The density and diversity of
invertebrates associated with characeae populations can be greater than those associated with
other macrophytes (Kingsford and Porter 1994, Kuczyńska-Kippen 2007). In Great Lake,
Tasmania, characeae are thought to provide resources for a number of rare and endangered
invertebrate species (Davies 2001) and most wild-collected characeae thalli have a diversity

23
of animal and algal epiphytes (Kairesalo et al. 1987). In general, characeae are thought to be
indicators of healthy, clear-water ecosystems (Krause 1981).
Characeae provide food, both directly for invertebrates (Proctor 1999) and vertebrates
(Noordhuis et al. 2002), and as particulate organic matter (Pereya-Ramos 1981). They
remove nutrients from the water column and stabilise sediment (Kufel and Kufel 2002),
reducing turbidity and nutrient release from the sediment. The presence of characeae is
correlated with high species diversity in phytoplankton communities (Casanova and Brock
1999b) and when characeae are removed experimentally phytoplankton (particularly bluegreen
algae) concentrations have been shown to increase (Villena and Romo 2007),
coincident with increased nutrient concentrations and sediment resuspension. Where dense
characeae beds exist they can be responsible for patches of clear water through their capacity
to dampen water movement and allow sediment to settle (Scheffer et al. 1994). There is
thought to be a positive feed-back mechanism operating between the development of
characeae populations in shallow lakes and the maintenance of the clear water conditions
they need to thrive (Fig. 1.17). The thalli also provide habitat and refuge for cladocerans
(water-fleas) that consume phytoplankton, thus reducing the biological turbidity. Both these
processes maintain conditions conducive to charophyte growth (clear water and low watercolumn
nutrient concentrations). When charophyte establishment is inhibited the opposite
occurs, leading to high turbidity, high water-column nutrient concentrations and a stable,
phytoplankton-dominated state. Studies have shown a statistically significant relationship
between charophyte abundance and a reduction in wind-driven resuspension (Hamilton and
Mitchell 1996), and a high correlation between characeae abundance and water clarity
(Blindow et al. 2002), but there is still discussion about the stability and mechanisms by
which the different states are achieved (Blindow et al. 2002). The capacity of charophytes to
modify community dynamics through allelopathy (production of substances that adversely
affect the growth of other organisms) has also been under investigation (Wium-Andersen et
al. 1982). There is general consensus, however, that characeae populations are important in a
variety of ecosystem processes in shallow lakes.
1.5.4 Abiotic environmental factors
1.5.4.1 Light
Light intensity affects the morphology of the characeae thallus and the distribution of
characeae species within lakes (Schwarz et al. 1996). Different species exhibit different

24
degrees of acclimation to low or high light intensities (Wang et al. 2008). In general, high
light intensities (that can be experienced in very shallow water, or very clear water) lead to a
contracted thallus with short internodes (Wang et al. 2008), whereas low light intensities (in
deep water or shaded situations) produce a more diffuse plant (Wang et al. 2008). Characeae
also respond to light intensity by modifying their pigment ratios (Wang et al. 2008) and in the
timing of reproduction (Wang et al. 2008). Exposure to light, and certain wavelengths of light
can also be important for stimulating germination of oospores (Sokol and Stross 1992),
although there is experimental evidence that although germination can take place in darkness,
light is required for establishment of the young thallus (de Winton et al. 2004).
1.5.4.2 Water chemistry and physical properties
The physical properties pH and conductivity (as an analogue for salinity or total dissolved
solids) are relatively easy to measure and have frequently been reported for charophyte
habitats, and it is common for tables of species to be listed in relation to these factors
(Corillion 1957, García and Chivas 2007). In general the most common species of
charophytes tolerate a range of pH values, with species of Chara preferring slightly alkaline
pH and species of Nitella preferring slightly acid to circum-neutral pH. There are a few
species that are found only in water with specific pH, but this is the exception rather than the
rule. To date there are no reports of a mechanism by which pH might select for, or influence
the distribution of characeae species. Salinity, on the other hand, imposes specific constraints
on the physiology and osmotic processes in charophyte cells. Several species of Chara and
Nitella can be found in, and appear to prefer slightly saline conditions. However, species in
the genera Lamprothamnium and Tolypella are frequently found in water of varying salinity,
and Lamprothamnium species can be the only plants occurring in waters with high salinity
(Brock and Lane 1980).
1.5.4.3 Nutrients
Characeae can take up nutrients through the above-ground thallus as well as through the
rhizoids (Littlefield and Forsberg 1965; Andrews 1987; Vermeer et al. 2003). Nitrogen is
preferentially accumulated as ammonium, and it can be circulated throughout the plant body
via cytoplasmic streaming (Vermeer et al 2003). In field studies characeae can have a major
impact on the nitrogen budget of lakes (Rodrigo and Alonso-Guillén 2008), responsible for
12 to 95 % of the total nitrogen content of the system. Characeae can also accumulate
phosphorus (Kufel and Ozimek 1991). Mean daily rates of phosphate uptake can exceed 27
nmol g -1 fresh weight h -1 (Andrews 1987).

25
1.5.4.4 Temperature
Storage temperature can affect germination of characeae (Casanova and Brock 1996), so that
winter germinating species will only germinate after they have been exposed to cues to the
onset of winter. The rate at which characeae germinate can be related to the ‘day-degrees’
they have experienced (Casanova 1994). Some species occur only in tropical regions, others
have a temperate distribution (Casanova 1993) and many populations experience seasonal
changes in biomass (Fernández-Aláez et al.2002).
1.5.4.6 Depth
Characeae are often the deepest inhabitants of lakes (Dale 1986), and there can be a high
diversity of species in individual lakes, zoned in relation to depth (de Winton et al. 1991).
They have been recorded to 30 m in USA (Stross et al. 1995), 15m depth in New Zealand (de
Winton et al. 1991; Casanova et al. 2007) and 12m depth in Tasmanian lakes (T. Dugdale,
personal communication). Classical studies relating characeae distribution to depth have
generally held light attenuation to be responsible for the determining the lower depth limits of
characeae communities (Wood 1950; Spence 1982; Andrews et al. 1984; Vant et al. 1986).
Some species exhibit definite biomass maxima at certain depths (de Winton et al. 1991;
Asaeda et al. 2007), and allocate more biomass to sexual reproduction at shallower depths
than in deeper water (Casanova 1994; Asaeda et al 2007). Depth can influence allocation of
resources to, and therefore the size and morphology of oospores (Boszke and Bociag 2008).
Depth, along with light availability, can affect the development of internodes and shoot
lengths, with thalli collected from deeper water exhibiting elongated internodes and
branchlets compared to thalli collected from shallower parts of the same water bodies
(Asaeda et al. 2007).
1.5.5 Culturing characeae
In many cases characeae can be harvested from the field and introduced into informal culture
with very little effort. Shoots, along with water from their habitat, can be placed in a vessel,
and given adequate light (e.g. on a window-sill; Karling 1924; Casanova and Brock 1996),
will persist, and sometimes grow, for weeks, if not months. Simple aeration of the water
through a bubbler can prevent the proliferation of bacteria and production of anoxic
conditions, a particular problem for species from brackish and saline water-bodies. This
method can provide simple, if undefined, cultures of characeae for experimental purposes.

26
Some species produce vegetative reproductive organs that can be used to initiate cultures
(e.g. starch-filled branchlet whorls: Starling et al. 1974; bulbils: Fritsch 1948).
Another very common method of developing long-lived laboratory cultures of characeae is to
collect the sediment or soil from under characeae stands, place it in a tank or culture vessel
and flood it with rainwater or distilled water, allowing characeae to germinate under
controlled light and temperature regimes. Drying the sediment prior to flooding can enhance
germination and establishment of characeae (Casanova and Brock 1990, Casanova and Brock
1999a), even when drying is not a common event in the natural habitat (de Winton et al.
2004).
Unialgal, axenic or sterile cultures can be attempted, but success is by no means certain.
Surface sterilised oospores have been used to initiate cultures (Imahori and Iwasa 1965).
Artificial culture conditions that have been used in the past include simple distilled water
with no physical substrate (Ross 1959), soil-water cultures (Proctor 1960), soil-nutrient
solution cultures (Clabeaux and Bisson 2009), and sand (particle size < 710 µm in size) in
distilled water with the addition of organic salts (Andrews et al. 1984). Nutrient agar (0.5 %)
in distilled water can provide good conditions for oospores to germinate (Forsberg 1965; Ray
and Chatterjee 1989; Casanova et al. 1998), but probably doesn’t support growth once the
oospore reserves are exhausted (Casanova and Brock 1994). Amino acids, gibberellic acid
and other plant hormones have been found to promote or inhibit the growth of characeae in
culture (Imahori and Iwasa 1965) and further examination of the factors that affect characeae
growth in vitro are currently under examination (Clabeaux and Bisson 2009).
Wild populations of characeae are frequently home to by a variety of herbivores, and can
be relatively free of epiphytes. Epiphytic growth can be a nuisance in cultures, and can be
reduced by decreasing nutrient concentrations, lowering light intensities or introducing
herbivores (e.g. snails or tadpoles) into the culture.
1.5.6 Life History
The plants that are grown and used in physiological studies are the haploid (gametophyte)
generation in these algae. The mature antheridia release the spermatocytes which swim to the
oogonium, enter it and fuse with the egg cell to form the oospore, which is the only diploid
(sporophyte) cell in the life history. The timing of germination, growth, reproduction and
senescence varies among species (Casanova and Brock 1999a). Some species are monocarpic

27
annuals (i.e. reproducing once in their lives), germinating in either Spring or Autumn,
growing, producing sexual organs once, then dying. These species are highly dependent on
oospore production and storage in a seed bank for survival (Casanova and Brock 1994; Bonis
and Grillas 2002). Other species are polycarpic perennials (i.e. reproduce several times in
their lives) which can survive inclement periods either through dormancy or production of
vegetative reproductive structures (bulbils). Such species are less dependent on oospore
germination for the maintenance of populations. It is likely that many species maintained for
physiological studies are perennial species, because these can persist in culture. Whether a
species is monoecious (with both sexes on a single plant) or dioecious (with separate male
and female plants) can modify the timing of life history events such as germination and
timing of reproduction (Casanova and Brock 1999a). Monoecious annual species can
germinate and grow rapidly, completing their life cycle in a relatively short period of time.
Dioecious annuals can be slower to germinate and grow, and dioecious perennials can have
very specific germination requirements. All these life history types maximise reproductive
success in a variety of habitats (Casanova and Brock 1999a).
1.5.7 Population ecology
Studies into the ecology of characeae populations relies on examination of the transition rates
between the states of propagule dormancy, germination, growth, reproduction, perenniation,
senescence and death. The processes of inter- and intra-specific competition and loss to
predation or herbivory can modify the development of populations. In established populations
charophytes deposit their oospores into the soil or sediment, forming a bank of viable
propagules that can become both diverse and dense. Oospores can be dispersed to new
habitats by water birds, through their digestive systems (Proctor 1962) or attached to the feet
and coverings of animals and people. Oospores in the seed bank can be readily germinable
(Casanova and Brock 1990), or more frequently, dormant (Skurzynski 2009). The dormancy
can be innate or enforced, depending on the adaptations of the species, and the type of habitat
in which they occur. Enforced dormancy is defined as a maintenance of viability when
conditions are not adequate for germination. Innate dormancy is defined as the failure of a
viable oospore to germinate when exposed to conditions that are adequate for germination
(Harper 1980).

28
There is often a high degree of innate dormancy in charophyte oospores. Germination of
fresh or field-collected oospores is typically less than 10%, and often less than 1% of the total
oospore pool (Bonis and Lepart 1994; Skurzynski 2009). Rarely germination rates in excess
of 40 % have been recorded (Casanova and Brock 1996; Dugdale et al. 2001). The role of
such a uniformly high degree of dormancy is to ensure that populations remain viable, even if
the habitat becomes unsuitable for characeae growth for long periods of time. A possible
additional role is to allow transport of oospores long distances within soil, or the intestinal
tracts of herbivores (Proctor 1962). The conditions that break dormancy vary for different
species, so that species that germinate in Spring and Summer can require chilling, or
stratification before germination will occur, and other require conditions that simulate winter
inundation (Casanova and Brock 1996). The timing of germination in relation to dormancy
breakage can vary as well. The high degree of dormancy and variability in the conditions
required to break that dormancy make germination ‘on demand’ for laboratory experiments
difficult to achieve.
Characeae growth rates vary in relation to species (Blindow 1988; Casanova 1994; Casanova
and Brock 1999a) and season (Blindow et al 2002) and an extension rate of 5 mm day -1 in the
field is not exceptional. When water levels are constant some populations have been shown to
maintain their position in the water through apical growth while the basal portions of the
plant decay, initiating new rhizoids from sequentially higher nodes as the plant sinks through
the water (Andrews et al 1994). Where water levels increase apical growth can maintain the
upper photosynthetic parts of the thallus in the photic zone, and where water levels fall the
thallus can lean over in the direction of water movement and initiate rhizoids at the nodes that
are in contact with the substrate (Casanova 1994). In contrast, characeae can be found almost
emergent at the edges of shallow and temporary water bodies, often dying back as the
exposed shoots dry. In some exceptional circumstances populations of characeae can exist in
a film of water in moist terrestrial habitats.
Initiation of sexual reproduction in characeae populations can be a consequence of time since
germination (e.g. for monocarpic annuals) or as a response to environmental conditions or
resources (Casanova 1994). Polycarpic perennial species, such as Chara australis can
produce oospores every year through the warmer months (Casanova 1994). Annual species
can produce reproductive organs on the first whorl of branchlets before significant vegetative

29
growth has taken place (e.g. Chara muelleri). Other species establish a vegetative thallus
before fertile shoots are produced (e.g. Nitella leonhardii).
Although there are few studies on the interactions among characeae and between characeae
and animals, the dense structure of characeae stands indicates that competition for light could
affect growth, and the occurrence of characeae meadows that exhibit an abrupt, unexplained
growth boundary (in plant height or depth distribution) suggest the presence of biotic
interactions or controls.
Characeae species vary with respect to their palatability to animal herbivores (Proctor 1999),
so that the existence of perennial beds of characeae could be explained by their resistance to
herbivory, and the absence of particular species to their susceptibility to herbivory (Proctor
1999). In general, interactions with herbivores are difficult to detect except through direct
observation and experimentation. However characeae would be exceptional (i.e. unlike all
other plants) if herbivory or creation of structures or behaviours to resist herbivory did not
play a role in their ecology.
1.5.8 Application of ecological knowledge
Knowing something of the ecology of characeae can have at least two positive consequences
for physiological research: 1) the development of enhanced techniques and protocols for
culturing and maintaining characeae for laboratory work, and 2) as a stimulus for
physiological research. Observations of characeae in the natural environment can lead us to
asking ‘why is it so?’, and physiologists and physicists will look for answers in relation to the
physiology and biophysics of characeae plants and cells. Observations of the distribution of
characeae in relation to environmental constraints can allow development of better
germination and growth protocols, and a better understanding of the relationships among
species, as photosynthesisers, as food for herbivores and as part of an integrated community
of plants and animals. The rest of this book contains information about some of the studies
that have been undertaken on the physiology of characeae plants, informed by a knowledge
of charophyte morphology, cytology and ecology.

30
1.6 Figure and Table captions
Figure 1.1 Thallus of a characeae plant (Chara sp. r862). a) stem showing whorls of
branchlets, scale bar = 10 mm. b) reproductive organs, scale bar = 1 mm. c) Scanning
electron micrograph of an oospore of Chara sp. (r862), scale bar = 200 µm.
Figure 1.10 Tolypella glomerata (p798), a) whole plant showing basal axis, first nodal
complex with elongate simple branchlets, and numerous fertile branches. b) a single fertile
node with 8 branchlets in a whorl, each of which has a primary node with multicelluar rays
and gametangia. Oogonia and antheridia are clustered at the branchlet nodes, c) oogonium
prior to fertilisation with coronula intact, d) oogonium after fertilisation when the coronula
has fallen and the spiral cells have swollen.
Figure 1.11 Nitellopsis obtusa (t810). a) Axis of a female plant, with six branchlets in whorls
at the nodes, and elongate bract cells at the branchlet nodes. scale bar = 5 cm. b) Oogonium at
the branchlet node, subtended by bract cells, with a 5-celled coronula. Oogonia are usually
heavily calcified with the oospore developing within a calcareous lime-shell or gyrogonite. c)
Apex of a shoot showing absence of stipulodes and simple branchlets with elongate bract
cells. d) vegetative reproductive organ characteristic of Nitellopsis (after Fritsch 1948).
Figure 1.12 Morphology of Nitella. a) whole plant and b) corounula at the top of the
oogonium, c) terminal branchlet segments with central antheridium and bicellulate dactyls, d)
terminal branchlet segments with lateral oogonia and a central antheridium.
Figure 1.13 Diagrammatic representation of a transverse section through the lowest three
cells of a corticated branchlet of Chara. Cells are bounded by a cell wall, the cytoplasm is
bounded by the plasma membrane. The peripheral cytoplasm contains numerous discoid
chloroplasts (along with mitochondria, nuclei, golgi apparatus etc.) surrounding a central
tonoplast-bound vacuole. The branchlet structure consists of intermodal cells and nodal
complexes with cortical cells, bract cells, and gametangia arising at the nodes (after Fritsch
1948, Pickett-Heaps 1975 and Moestrup 1970).
Figure 1.14 Structures associated with spermatogenesis in characeae. a) spermatid of Nitella
with two flagellae, mitrochondria, nucleus and plastids (after Pickett-Heaps 1975 and
Moestrup 1970), b) part of the contents of an antheridium after removal of the shield cells c)
four of the eight shield cells.

31
Figure 1.15 The characeae oospore: a) gyrogonite of Lamprothamnium macropogon, b)
oospore of Chara sp. with flanged striae, c) oospore of Chara sp. with thickened (pachygyra)
striae, d) oospore of Nitella lhotzkyi with a verrucate ornamentation, e) oospore of Nitella
tasmanica with a reticulate ornamentation, f) oospore of Nitella hyalina with fibrous oospore
wall construction, g) basal view of the oospore of Chara sp showing the single, pentagonal
basal cell impression. h) basal view of the oospore of Nitella sonderi showing the two basal
cell impressions, i) apical view of the oospore of Chara sp. showing the ‘w-shaped’ suture
where the striae meet.
Figure 1.16 The relationships among processes that contribute to the size of the seed bank of
characeae in lakes and wetlands.
Figure 1.17 Diagram of a simplified model of the relationships and processes leading to
characeae abundance or phytoplankton abundance in shallow lakes, according to the
Alternative Stable States model (Scheffer et al. 1993). Arrow type indicates whether the
effect is positive (solid) or negative (dashed). The size of the circle indicates the relative
abundance of each element. a) High water clarity leads to establishment of high characeae
biomass (which enhances water clarity by stabilising sediment, removing nutrients available
for phytoplankton growth and providing habitat for cladoceran herbivores). b) High nutrient
availability leads to high phytoplankton abundance which reduces charophyte abundance by
decreasing water clarity. Absence of charophytes increases sediment resuspension, further
decreasing water clarity and decreases the habitat available for cladoceran herbivores, which
further enhances phytoplankton biomass.
Figure 1.2 Characeae axial nodes. Scale bar = 1 mm. a) Chara sp. (r822) node with two rows
of stipulodes, short cortical cells and small spine cells. b) Chara canescens (r020) node with
longer stipulodes and spines cells. c) Lamprothamnium sp. (r028) with no cortication,
oogonium and stipulodes below the base of the whorl, and spreading bract cells at the
branchlet nodes.
Figure 1.3 Whorls of characeae branchlets viewed in cross-section, with the branchlets
spread around the sectioned node. a) Chara gymnopitys whorl with 12 branchlets in a whorl,
12 stipulodes at the base of the whorl, long bract cells at each branchlet node and branchlets
terminated by a group of bract cells. b) Lamprothamnium sp aff. macropogon with 7
branchlets in a whorl, no apparent stipulodes, long bract cells at the branchlet nodes and an
oogonium at the base of the whorl. c) Nitella sp. with 7 branchlets in a whorl, the branchets

32
divided into 3-5 secondary rays and 3-5 tertiary rays. d) Nitella hyalina with two whorls of
branchlets at the node (a longer, primary whorl of 8 branchlets, and a smaller, inner,
secondary whorl of 7 branchlets), with the branchlets divided into 3-6 secondary rays, 3-5
tertiary rays and 3-4 quaternary rays. Scale bars = 5 mm.
Figure 1.4 Branchlet morphology in the different genera of charophyte plants. a) Chara, b)
Lamprothamnium, c) Nitella d) Tolypella.
Figure 1.5 Branchlet nodes and gametangia of Chara spp. In Chara the oogonium (female) is
above the antheridium, and bract cells, bracteoles and cortical filaments arise from the nodal
complex.
Figure 1.6 General morphology of Chara australis (r186, r612, r692). a) whole plant
showing main axis and whorls of branchlets. b) whorl of branchlets from a male plant with 6
branchlets in a whorl and antheridia at the branchlet nodes. c) whorl of branchlets from a
female plant with 6 branchlets in a whorl with oogonia at the base of the whorl and on the
branchlet nodes. d) oogonium with coronula of appressed cells, and spiral cells surrounding
the internal egg cell. e) branchlet node with bract cells and two oogonia, f) base of branchlet
whorl with relatively elongate stipulodes, g) base of whorl with obscure stipulodes and two
basal oogonia.
Figure 1.7 Lamprothamnium macropogon (r809, r822) a) whole thallus showing whorls of
branchlets, downward pointing stipulodes and verticillate bract cells on the branchlets. b)
fertile whorl with antheridia on the branchlets and oogonia inside the base of the whorl, or
beside the antheridia. c) internal view of whorl with a single antheridium squeezed among the
oogonia. d) branchlet and bract cell morphology. e) Lamprothamnium papulosum (r729) with
conjoined gametangia, the antheridium above the oogonium.
Figure 1.8 Lamprothamnium sp aff. succinctum from Australia (r 272). a) whole plant
showing elongated internodes and few, small stipulodes and bract cells. b) whorl of
branchlets with antheridia and oogonia on the branchlets. c) detail of basal gametangia,
oogonia within the whorl, a single antheridium below the whorl in the place of a stipulode. d)
antheridium at a branchlet node with two bract cells. e) two young oogonia at a branchlet
node with three bract cells.
Figure 1.9 Lamprothamnium inflatum (r901, r902, r905). a) Shoot of highly inflated
specimen with almost spherical second segments on the branchlets, obscure stipulodes and
contracted apicies. b) elongated shoot. c) whorl of fertile branchlets with oogonia and

33
antheridia clustered at the base of the whorl and on the first branchlet nodes. d) dissected base
of a whorl showing stipitate antheridia below the whorl and stipitate and clustered oogonia
within the whorl.
Table 1 Names and synonyms of characeae species used in physiological studies.

34
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36
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