Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales, Streptophyta) with special reference to cell division and thallus formation

Representatives of the closely related genera, Interfilum and Klebsormidium, are characterized by unicells, dyads or packets in Interfilum and contrasting uniseriate filaments in Klebsormidium. According to the literature, these distinct thallus forms originate by different types of cell division, sporulation (cytogony) versus vegetative cell division (cytotomy), but investigations of their morphology and ultrastructure show a high degree of similarity. Cell walls of both genera are characterized by triangular spaces between cell walls of neighbouring cells and the parental wall or central space among the walls of a cell packet, exfoliations and projections of the parental wall and cap-like and H-like fragments of the cell wall. In both genera, each cell has its individual cell wall and it also has part of the common parental wall or its remnants. Therefore, vegetative cells of Interfilum and Klebsormidium probably divide by the same type of cell division (sporulation-like). Various strains representing different species of the two genera are characterized by differences in cell wall ultrastructure, particularly the level of preservation, rupture or gelatinization of the parental wall surrounding the daughter cells. The differing morphologies of representatives of various lineages result from features of the parental wall during cell separation and detachment. Cell division in three planes (usual in Interfilum and a rare event in Klebsormidium) takes place in spherical or short cylindrical cells, with the chloroplast positioned perpendicularly or obliquely to the filament (dyad) axis. The morphological differences are mainly a consequence of differing fates of the parental wall after cell division and detachment. The development of different morphologies within the two genera mostly depends on characters such as the shape of cells, texture of cell walls, mechanical interactions between cells and the influence of environmental conditions.

Combining data obtained by different methods (morphology, ultrastructure, molecular phylogeny) is important in algal taxonomy.
The Klebsormidiales (Streptophyta) contains the filamentous genera Klebsormidium P.S. Silva, Mattox & Blackwell, Hormidiella M.O.P. Iyengar & Khantamma and Entransia E.O. Hughes . Klebsormidium are typical filamentous algae, with cells dividing vegetatively Pickett-Heaps, 1972;Sluiman et al., 1989;Van den Hoek et al., 1995;Honda & Hashimoto, 2007;Katsaros et al., 2011). The Klebsormidiales also includes the genus Interfilum Chodat which is characterized by unicells and the formation of short filaments, dyads, packets or branched pleurococcoid filaments (Mikhailyuk et al., 2008). According to the original description, Interfilum paradoxum Chodat & Topali is an easily disintegrating filamentous alga, with cells surrounded by bipartite cell walls, dividing by vegetative cell division, forming chains of cells connected by 'threads' of unknown nature (Chodat & Topali, 1922). The cells divide by a process similar to sporulation; the remnants of parental walls form cap-like structures on the cells or thread-like structures between them, so cell walls appear bipartite (Mikhailyuk et al., 2008).
We conducted a detailed morphological and ultrastructural investigation of representatives of Interfilum and Klebsormidium, with emphasis on the protoplast and cell wall, the 'behaviour' of the cell wall during cell detachment, and the morphology of cell division, in an attempt to understand how different morphologies develop in two closely related genera.

Strains and culture conditions
About 100 strains of Interfilum and Klebsormidium from the Sammlung von Algenkulturen, University of Göttingen, Germany (SAG: Friedl & Lorenz, 2012;www.epsag.unigoettingen.de), the Culture Collection of Algae and Protozoa (CCAP, Gaсhon et al., 2007;www.ccap.uk), the collection from the project 'Biota of South Africa' (Büdel et al., 2009), the collection of Klebsormidium strains from Alpine soil crusts Holzinger et al., 2011;Kaplan et al., 2012;Karsten & Holzinger, 2012), some of our own isolates, and samples of field material were used for this study. Information about these strains is included in previous papers (Mikhailyuk et al., 2008;Rindi et al., 2011;Karsten et al., 2013). Information about the 30 strains presented here is summarized in Supplementary  Table 1.
Light, fluorescent and laser scanning confocal microscopy, morphological characterization and vital staining Young (2-3-week-old) and old (2-3-month-old) cultures of Klebsormidium and Interfilum, as well as field-collected material, were morphologically characterized using Olympus BX60 and Olympus IX70 light microscopes (Olympus Europe Holding, Hamburg, Germany) with Nomarski differential interference optics. The morphology of algae was documented with the attached Olympus ColorView III and II cameras (Soft Imaging System GmbH, Münster, Germany) using the imaging software Cell^D and analySIS (Soft Imaging System GmbH). An Olympus IX70 microscope equipped with a fluorescent lamp was used for investigation of mitochondria. A Leica TCS SP2 AOBS laser scanning confocal microscope (Leica Microsystems, Germany) was used for chloroplast morphology and mitochondria.
Mucilage was stained with an aqueous solution of methylene blue at different concentrations. For mitochondrial observations, cells were stained overnight with about 0.75 μg MitoTracker Green FM (Molecular Probes, Eugene, Oregon, USA) dissolved in 100 μl medium (3NBBM) with algal cells. The autofluorescence of chlorophyll was used to observe chloroplast structure (excitation at 488 nm, emission at 610-660 nm).

Transmission electron microscopy
Samples from different phylogenetic clades according to Rindi et al. (2011) Massalski et al. (1995) or Holzinger et al. (2009). For TEM, ultrathin sections were prepared, counterstained with uranyl acetate and Reynold's lead citrate, and investigated in Zeiss LIBRA 120 or Tesla BS 500 transmission electron microscopes at 80 kV. Images were captured with a ProScan 2k SSCCD camera (Proscan Electronic Systems, Lagerlechfeld, Germany) and further processed using Adobe Photoshop software (Adobe Systems Inc., San José, California, USA).
Investigation of the Interfilum cell wall by light microscopy and mucilage staining showed the presence of cap-and ring-like structures as well as exfoliations of the parental wall, forming bridges between cells (Figs 10-13). H-like fragments of the cell wall were found occasionally (Fig. 14). Spaces between neighbouring cells were observed in packet-forming strains (Fig. 15). All these characters were related to the presence of individual walls in each cell and a parental wall.
Klebsormidium observed with light microscopy showed the rare presence of biseriate parts of filaments, packet-and branch-like structures in some strains, especially in old cultures . Caplike structures (Figs 20,21), H-like fragments of cell walls (Figs 22,23,25,27,28), and exfoliations of  parental walls (Figs 24,26), as well as triangular spaces between walls of neighbouring cells (Fig. 29), were frequently present. These structures were most obvious in field-collected material of Klebsormidium, which showed dense cell walls (Figs 30,31).

Confocal laser scanning and fluorescence microscopy
Images obtained with confocal laser scanning and fluorescence microscopy showed that Interfilum and Klebsormidium strains contained the same type of variously lobed plate-shaped chloroplasts (Figs 32-35).
Vital staining of cells of both genera revealed the presence of several polymorphic mitochondria, which were located around the nucleus and along the chloroplast lobes .

Transmission electron microscopy
TEM investigations showed a similar pyrenoid ultrastructure in both genera 48,52,53). Starch grains formed one or several layers around the pyrenoid body, arranged in parallel rows. Several to many parallel single thylakoid membranes penetrated the pyrenoid body, which determined the orientation of the starch grains. One peroxisome was located between the chloroplast and the nucleus (Figs 43,44,48). Mitochondrial profiles were located close to the chloroplast (Fig. 43).
Depending on the lineage, the Interfilum cell wall differed ultrastructurally.  discontinuity of the Klebsormidium cell wall, and thus the presence of individual walls in each cell and parental wall. Cross-walls of Klebsormidium were bi-or multilayered, lacked plasmodesmata, and often showed different thicknesses within the same filament (Figs 51-53).
Ultrastructural analysis of cell walls in different lineages of Klebsormidium indicated overall similarity, but also some differences in detail (Figs 59-66). Samples from clades B/C KUE1 and ASIB V100 (Figs 59, 60) exhibited bilayered outer cell walls covered by a clearly distinguishable mucilage layer. The outer cell walls were~0.16 µm thick in KUE1 and~0.22 µm in ASIB V100, which together with the mucilage layer was~0.4-0.5 µm in thickness (Figs 59, 60). The cell cross-walls were sometimes separated in KUE1, while in ASIB V100 they were rather thick and multilayered (Figs 59,60). Representatives of clades D and G, PIT1 and 14613.5e (Figs 61,62) showed bilayered outer cell walls with the outer layer less obvious in 14613.5e. The cell walls in PIT1 were~0.17 µm thick, and in 14613.5e~0.2 µm. In both strains, triangular spaces between the cross-walls and the outer cell wall were detectable (Figs 61, 62). The cross-walls were layered in 14613.5e, while they appeared rather smooth in PIT1. The thickest outer cell walls, in a Klebsormidium strain from clade F (K. crenulatum (Kützing) Ettl & Gärtner, SAG 2415), were up tõ 0.6 µm thick, clearly layered and corrugated (Fig.  63). The cross-walls were also layered and clearly separated from the outer cell walls (Fig. 63). In many cases, triangular spaces between the outer cell walls and the cross-walls were visible (Figs 52-54). In contrast, Klebsormidium samples from clade E (K. nitens (Meneghini in Kützing) Lokhorst (SAG 2417), K. cf. fluitans (Gay) Lokhorst (BOT3) and K. dissectum (Gay) Ettl & Gärtner (SAG 2416), Figs 64-66) had rather thin cell walls, reaching 0.26 µm thickness in SAG 2417, 0.15 µm in BOT3 and 0.17 µm in SAG 2416. The cross-walls were thin and clearly separated from the outer cell wall in SAG 2416 (Fig. 66). Triangular spaces were observed only in BOT3 (Fig. 65). Layering of the cell walls and a mucilage layer were observed in all strains of clade E (Figs 64-66).

Discussion
Protoplast structure of Interfilum and Klebsormidium According to our observations and to previously published data, protoplasts of Interfilum and Klebsormidium are characterized by common morphological and ultrastructural features: similar structures of the chloroplast and pyrenoid, position of the nucleus in a cytoplasmic bridge between the two terminal vacuoles, fibrous mucilage structure, shape and position of the single peroxisome, and mitochondria Silverberg, 1975;Lokhorst & Star, 1985;Morison & Sheath, 1985;Honda & Hashimoto, 2007;Mikhailyuk et al., 2008). The protoplast structure of some related streptophycean algae, e.g. members of Entransia, Hormidiella, Chlorokybus Geitler and Coleochaete Brébisson, is mostly similar as well (Rogers et al., 1980;Sluiman, 1985a;Lokhorst et al., 2000;Cook, 2004). Some conjugating green algae (Holzinger et al., 2009) and Anthocerotae mosses (Cook, 2004) have similar pyrenoid starch envelope structure. A large single peroxisome located between the chloroplast and the nucleus is also characteristic of Mesostigma Lauterborn, Chaetosphaeridium Klebahn, Chlorokybus, Coleochaete and Hormidiella (Rogers et al., 1980;Sluiman, 1985a;Melkonian, 1989;Van den Hoek et al., 1995;Lokhorst et al., 2000). Multiple peroxisomes of other shapes, closely adjacent to the chloroplast, are found in Nitella C. Agardh (Silverberg & Sawa, 1973), Micrasterias C. Agardh ex Ralfs (Tourte, 1972), photosynthetic cells of Polytrichum (Proctor et al., 2007) and vascular plants (Raven et al., 2005). The peroxisome, nucleus, chloroplast and mitochondria form a specific structural complex, which has been suggested to be a diagnostic feature for streptophycean green algae (Massalski, 2002;Massalski & Kostikov, 2005). The close arrangement of these organelles in the cell is considered to be an evolutionarily progressive character, because it guarantees rapid metabolic exchange processes during photorespiration (Raven et al., 2005). In addition, the enzyme composition of peroxisomes of streptophycean algae and embryophytes is similar, and differs from other algae (Gross, 1993). The structural complex plays an important role in cell division of streptophycean algae (see below). It is likely that the presence of this structural complex reflects the evolutionary success of this algal group (worldwide distribution in terrestrial ecosystems; Rindi et al., 2009), and hence is partially retained in embryophytes (Raven et al., 2005;Proctor et al., 2007).
Mitochondria are probably components of the structural complex as well. There are abundant TEM data on mitochondrial profiles in sections of Klebsormidium cells Silverberg, 1975;Lokhorst & Star, 1985;Morison & Sheath, 1985;Honda & Hashimoto, 2007), but little information about their spatial organization is available. Our data indicate that the location of mitochondria is always similar and strictly ordered. This spatial distribution may support the proposal that streptophycean algae contain a structural complex consisting of the peroxisome, nucleus, mitochondria and chloroplast. Small differences in mitochondrial location among different strains mostly depend on the cell shape and details of chloroplast morphology.

Structure of cell walls and possible origin of cell wall remnants
Originally Interfilum was described as a genus with bipartite cell walls, closely related to the genus Radiofilum Schmidle (Chodat & Topali, 1922). Detailed morphological investigations by Fritsch & John (1942) showed that each Interfilum cell has its own integral cell wall, which is formed inside the parental wall during cell division. The parental wall ruptures in the middle during cell growth and detachment, resulting in cap-like structures closely associated with daughter cell walls. The origin of cap-, ring-and thread-like structures from parental wall remnants was shown in the present study and in a previous publication (Mikhailyuk et al., 2008).
Some cell wall structures observed here in Klebsormidium (H-like and cap-like structures, exfoliations and projections of the parental wall, triangular spaces between the parental wall and the cell walls of neighbouring cells) indicate that the cell wall is heterogeneous and includes daughter walls with closely adhering remnants of the parental wall. The H-like fragments represent structures homologous to the cap-like remnants characteristic of Interfilum: two cap-like structures connected by their tops. H-like fragments are observed in some filamentous algae with bipartite (Microspora Thur., Tribonema Derbès et Solier) or separated (consisted of daughter and parental walls) cell walls (Binuclearia Wittrock, Cylindrocapsa Reinsch) (Sluiman et al., 1989;Massjuk, 1993;Sluiman, 1985b;Van den Hoek et al., 1995), and probably in Klebsormidium as well. Cap-like structures characteristic of Interfilum were recently reported in some strains of Klebsormidium by Škaloud & Rindi (2013).
H-like fragments of cell walls are well known in Klebsormidium (Starmach, 1972;Moshkova, 1979;Ettl & Gärtner, 1995;Van den Hoek et al., 1995;Lokhorst, 1996), but their origin has not been explained satisfactorily. Lokhorst (1996) indicated that H-fragments are remnants of the parental wall, but did not explain how they are formed in an alga with vegetative cell division (only cross-walls are formed during this kind of cell division, as described below, without formation of separate daughter cell walls). Some proposed explanations indicate occasional formation of akinetes or hypnospores in Klebsormidium (Moshkova, 1979;Morison & Sheath, 1985). In this case, the cell protoplast forms its own cell wall, and the resting cell remains enclosed inside the parental wall. However, according to other authors (Klebs, 1896;Lokhorst, 1996), specialized resting cells are not formed in Klebsormidium because cells neighbouring H-fragments are usually not distinguishable (judging by the presence and location of cellular organelles) from other vegetative cells of the filament. Jane & Woodhead (1941) reported that field-collected filaments of Ulothrix Kützing and Klebsormidium contain many H-fragments of cell walls. Detailed microscopic investigation of the filaments showed that their cell walls are discontinuous, with distinct inner and outer layers. Microchemical tests revealed that these layers differ in chemical composition: whilst the inner layer consists of cellulose, the outer layer represents an intermediate stage in the degradation of cellulose to mucilage. Consequently, it is possible to interpret these layers as the daughter cell wall and gradually degrading remnants of the parental wall.

Formation of packets, biseriate parts of filaments and branches in Interfilum and Klebsormidium
Some Interfilum strains are able to form packets, cubic cell aggregations and branched pleurococcoid thalli. Cells divide in several planes and remain enclosed within widened parental walls; the central space between cells of a packet observed in this study indicates that each daughter cell has its own cell wall.
The formation of packets, biseriate parts of a filament and branching have rarely been reported in Klebsormidium (Ettl & Gärtner, 1995;Lokhorst, 1996). The latter author interpreted these structures mostly as the formation and further germination of aplanospores, which formed one per cell and often remained inside the sporangial wall. Germination of the aplanospores led to the formation of branches or cell complexes. Lokhorst (1996) expressed some doubts concerning this interpretation, but proposed that this ability of Klebsormidium might be the first step in the development of definite side-branching.
Some examples of this branching are actually caused by germination of cells originating from zoospores (hemizoospores/aplanospores) inside a sporangial wall. Germination of these cells inside the sporangium leads to the formation of young filaments growing from a parental filament. This type of pseudobranching is clearly visible in some published figures (Lokhorst, 1996, figs 36, 75, 97, 186;Škaloud, 2006, fig. 8). Pseudobranches originating from reproductive cells are evident in micrographs of Entransia fimbriata E. O. Hughes (Cook, 2004, figs. 5e, f), which is closely related to Klebsormidium. This observation provides clear evidence that small branches can originate from reproductive cells, as the tip (characteristic of germinating zoospores of E. fimbriata and absent in Klebsormidium) is clearly visible on the branches.
However, the formation of biseriate parts, packetlike structures and some other kinds of branches in Klebsormidium has another origin, i.e. the division of vegetative cells occurs in several planes. This behaviour was observed in the present study and has been reported in the literature (Ettl & Gärtner, 1995, fig. 203d;Lokhorst, 1996, figs 188, 203, 207, 224). It seems that the cells divide in the same way as packet-forming strains of Interfilum.
It is known that the division of chloroplasts, together with the closely adhered peroxisome, occurs before division in vegetative cells of Klebsormidium and some other streptophycean algae (Coleochaete) Pickett-Heaps et al., 1972;Lokhorst & Star, 1985;Van den Hoek et al., 1995;Honda & Hashimoto, 2007). Furthermore, the septum dividing two protoplasts forms at the same position in the cell where cleavage of these organelles had occurred: the central part of the chloroplast and the centre of the structural complex, where the pyrenoid is located. Therefore, the site of septum formation is strongly correlated with the chloroplast position. Chloroplasts of Klebsormidium are usually located laterally, near longitudinal walls of cylindrical cells and parallel to the filament axis. Chloroplasts are maximally exposed to light in this position. Therefore, a septum dividing two daughter cells is formed in the plane perpendicular to the filament axis (Fig. 67a), resulting in a chain of cells. Cells of packet-forming members of Interfilum range from nearly spherical to broadly ellipsoid. Chloroplasts in the cell dyad are usually located perpendicular to the dyad axis. This part of the cell wall is the longest, and the chloroplasts are maximally exposed to light. The septum is formed similarly to that in Klebsormidium, but parallel to the dyad axis and perpendicular to the previous cross-wall (Fig. 67b). A four-celled packet is formed as a result.
Therefore, the location of the chloroplast (or structural complex) and, partly, the shape of the cell determine the plane of cell division in Interfilum and Klebsormidium. Interestingly, some fundamental rules concerning cell division in embryophytes indicate that the plane of cell division is correlated with the shape of a cell: new cell walls normally form perpendicularly to the axis of growth of a cell (Dupuy et al., 2010). Therefore, the cross-dividing walls are normally formed in long cells, and longitudinal walls in short cells. Division in several planes does not occur in Interfilum strains with long cells. Occasional formation of biseriate parts was observed in Klebsormidium with short cells, i.e. K. crenulatum (Lokhorst, 1996) and K. montanum (Hansgirg) S. Watanabe (Ettl & Gärtner, 1995). We usually observed these structures in old cultures of Klebsormidium, when the cells became shorter. Sometimes the formation of biseriate parts of filaments is related to the deformation of cells, causing a curvature of the filament. The chloroplast has too little space for lateral dislocation in short or deformed cells, and hence sometimes turns from the short lateral wall to the longer cross-wall, or locates obliquely. The septum forms according to the usual rule, but perpendicularly or obliquely to the filament axis (Fig. 67c), in this case forming biseriate parts, packet-like structures or branches. Cell division in several planes in both Interfilum and Klebsormidium shows clearly that many of the properties of dividing plant cells are influenced physically or mechanically (Dupuy et al., 2010).

Type of cell division in Interfilum and Klebsormidium
Various modes of cell division of algae have been described. However, at present two main types of division can be distinguished among green algae with rigid cell walls: sporulation (cytogony, eleutheroschisis) and vegetative cell division (cytotomy, desmoschisis) (Ettl, 1988a, b;Sluiman et al., 1989). The mechanism of cytokinesis of the two types differs substantially and leads to the formation of different division products: specialized reproductive cells (spores or gametes) or young vegetative cells.
The fundamental morphological characters distinguishing sporulation and vegetative cell division are distinctive features in the formation of daughter cell walls. The cross-wall is always formed de novo, but longitudinal cell walls of parental cells are preserved and become part of the daughter cell wall during vegetative cell division (Ettl, 1988a, b;Massjuk, 1993;Van den Hoek et al., 1995). Therefore, the cell wall of a thallus is integral, since each cell is part of the multicellular organism. Pores with plasmodesmata are often characteristic of cross-walls of multicellular algae, in which separate cells are united in an integrated organism (Van den Hoek et al., 1995). The main characteristic of sporulation is the formation of cell walls by each cell independently within the parental (sporangial) wall (Ettl, 1988a, b;Sluiman et al., 1989). The protoplast of the parental cell divides into several parts, each forming its own cell wall during sporulation. The wall of the parental cell is transformed in different ways: it may degrade, releasing the cells to form unicellular organisms, or remain intact, holding the cells in colonies and forming extracellular structures. The cell walls are discontinuous and the cells are not connected by plasmodesmata, although they are united in multicellular complexes that represent colonies of unicellular organisms (Sluiman et al., 1989).
The type of cell division of many algae is easy to attribute to sporulation because it leads to the formation of a typical unicellular state. Cell division of packet-forming (sarcinoid morphotype) and filamentous algae traditionally was regarded as vegetative cell division (Fritsch, 1935;Smith, 1955). However, the morphological classification created by Ettl (1988a, b) and supported with some ultrastructural characters by Sluiman et al. (1989) determined that cell division of many filamentous and sarcinoid algae is actually a kind of sporulation. A complex of ultrastructural and morphological features (mostly accompanied by the presence of partially reduced flagellar structures in daughter cells, specific origin of the septum plasma membrane and a post-cytokinetic circumferential deposition pattern of extracellular material) were used as diagnostic characters indicating cell division via sporulation. The ultrastructural characters of the above-mentioned spores are completely reduced in some algae. The last character (circumferential deposition of the new cell wall) is the most important, as it indicates the formation of a daughter cell wall within the parental wall and attributes cell division to the sporulation type. Cell division of some packetforming (Chlorosarcinopsis Herndon, Tetracystis R. M. Brown & H.C. Bold, Trebouxia Puymaly) and filamentous algae (Geminella Turpin, Binuclearia, Cylindrocapsa, Nannochloris Naumann, Marvania F. Hindák, Microspora, Stichococcus Nägeli, Oedogonium Link ex Hirn) was determined to be sporulation on the basis of these characters (Sluiman, 1985b;Sluiman & Reymond, 1987;Sluiman et al., 1989;Yamamoto et al., 2007). The next conclusion derived from this classification was a fundamentally different concept of thallus organization in these algae: colonies of unicellular organisms (packets or pseudofilaments) and their vegetative cells are spores, according to their origin (Ettl, 1988a, b;Sluiman et al., 1989). Although this classification system of cell division is generally accepted (Van den Hoek et al., 1995), there are also contradictory arguments (Massjuk, 1997(Massjuk, , 2000Massjuk & Demchenko, 2001), i.e. that the products of the cell division are not spores or gametes, but rather vegetative cells. A wider concept of vegetative cell division was proposed later: 'During vegetative cell division the parental cell wall does not rupture or gelatinize, but is used for organization of daughter cell walls, which leads to the formation of vegetative (somatic, not specialized reproductive) cells and growth of thallus' (Massjuk, 2000). This concept has some debatable points as well. Although parental cell walls in pseudofilamentous algae usually represent support for the daughter cell and are involved in the formation of a thallus, they degrade via rupture or gelatinization and transform into remnants (Sluiman, 1985b;Sluiman & Reymond, 1987;Yamamoto et al., 2007).
The cell division of packet-like and pseudofilamentous algae is a transitional type between sporulation and vegetative cell division. The mechanism of this cell division must be attributed to sporulation: the daughter cell wall forms within the parental wall, and the parental wall represents different stages of degradation. The products of cell division (somatic cells, not spores or gametes) reflect vegetative cell division. The parental cell wall is partially retained after this cell division and participates in the formation of the thallus. In our opinion, the best definition of cell division within packet-like and pseudofilamentous algae is 'desmoschisis'sensu Groover & Bold (1969), as previously proposed by Massjuk (2000). However, this term is not generally accepted in modern phycological literature, and is considered a synonym of classical vegetative cell division (Sluiman et al., 1989).
Although cell division in Interfilum initially was determined to be vegetative cell division (Chodat & Topali, 1922), Fritsch & John (1942) reported a sporulation-like type of formation of daughter cell walls. The latter authors even considered I. paradoxum to be a colonial member of the Chlorococcales. The characters of Interfilum cell walls obtained in the present study are arguments for the formation of daughter cell walls within parental walls. Therefore, a sporulation-like type of cell division is characteristic for Interfilum.
Vegetative cells of Klebsormidium divide by vegetative cell division according to the traditional point of view Pickett-Heaps, 1972;Lokhorst & Star, 1985;Sluiman et al., 1989;Van den Hoek et al., 1995;Lokhorst, 1996). The cell cross-walls form by cleavage furrow and lack plasmodesmata (Lokhorst & Star, 1985;Van den Hoek et al., 1995;Lokhorst, 1996). However, different kinds of remnants of parental cell walls and triangular spaces between parental walls and cell walls of neighbouring cells are structures characteristic of the sporulation-like type of cell division, and are homologous to structures observed in Interfilum. Therefore, the type of cell division within the two genera must be the same. In addition, the presence of cross-walls with an H-like appearance (Microspora-type) is an argument for cell division by the sporulation-like type (Sluiman et al., 1989).
Transformation of parental wall and probable structure of thalli in Interfilum and Klebsormidium Gelatinization of parental walls leads to the formation of a mucilage envelope and disintegration of the thallus to the unicellular state. The cell walls of these Interfilum strains are usually thin and homogeneous because they represent a daughter cell wall. Preservation of parental walls around daughter cells caused the formation of densely layered walls, retaining cellular packets. The cell walls of these Interfilum strains are thick and layered, because they consist of a daughter cell wall and several generations of parental walls and their remnants. Partial rupture of the parental walls leads to the formation of cap-, ring-and thread-like structures. The left part of the scheme (Fig. 68b-g) shows that different routes of transformation of Interfilum parental walls lead to the formation of a specific thallus structure.
Vegetative cells of Klebsormidium usually divide in one plane, with subsequent formation of filamentous thalli. The scheme of a filament typical for other algae with the sporulation-like type of cell division (Geminella, Binuclearia, Cylindrocapsa;Massjuk, 1993;Van den Hoek et al., 1995) was chosen as a model of Klebsormidium filaments. The parental walls of Klebsormidium are partially preserved around two daughter cells during cell division (Fig. 68a). Further division of these cells proceeds in the same way, i.e. a chain of cells surrounded by many generations of parental walls is formed (Fig. 68a, h, j). Generations of parental walls are transformed in different ways (ruptured or gelatinized) because of the pressure of growing cells and remnants over time. This model corresponds to some characters of a Klebsormidium thallus and explains the formation of H-and capfragments of the cell wall. The different widths of cross-walls within the same filament of Klebsormidium is a further confirmation of this scheme; i.e. the cell walls are thin and bilayered between freshly divided cells, and thick and multilayered between groups of dividing cells.
The morphological diversity within Klebsormidium reflects different routes of further transformation of parental walls. The preservation of these parental walls around daughter cells leads to the formation of dense filaments, often with H-fragments (Fig. 68h). The mechanism for the formation of an H-fragment is as follows: the cell cross-wall is pressed from opposite sides by two growing neighbouring cells, and the layers of parental cell walls are compressed. Longitudinal cell walls, in contrast, are stretched and become thinner. The filaments are ruptured first in the middle of the longitudinal walls, and the cross-wall is preserved and forms an H-fragment (Fig. 68i). Hfragments are usually formed within the thickest cross-walls, i.e. between groups of dividing cells.
A second route of transformation of the Klebsormidium parental wall leads to gelatinization and formation of a delicate mucilage envelope around the filament (Fig. 68j). The filament can easily disintegrate into short filaments, and the unicells are sometimes morphologically similar to unicellular strains of Interfilum. These Klebsormidium strains sometimes form H-fragments, but this phenomenon is observed rarely because their parental walls are thin and delicate, and gelatinization of cross-walls leads to easy separation of cells. Biseriate and packet-like parts of filaments occur occasionally in Klebsormidium because of an atypical position of the chloroplast in the cell (Fig. 68l).
Investigations of the ultrastructure of Klebsormidium cell walls of different phylogenetic lineages showed a high structural similarity, with some differences in details. These details are the width and degree of lamination of the cell walls, as well as the presence or absence of mucilage. Therefore, the diversity of structures and textures of Klebsormidium cell walls probably represents two main types: (1) dense filaments without mucilage (mostly characteristic of representatives of xerophytic lineages, i.e. clades D, F and G according to Rindi et al., 2011;Fig. 69), and (2) filaments with gelatinized cell walls that easily transform into short filaments or into the unicellular state (characteristic of mesophytic and hydrophytic lineages, i.e. clades E and partially B and C; Fig. 69).
In summary, the data presented here indicate a high similarity in the morphology and ultrastructure of vegetative cells and cell walls of Interfilum and Klebsormidium, and this similarity is in concordance with results of a recent phylogenetic analysis (Rindi et al., 2011). The different morphology of these genera is mostly a consequence of the different 'behaviour' of parental walls after cell division and detachment, as well as of the shape of the vegetative cells. Therefore, the presence of different morphotypes within the two genera depends on shape of cells, mechanical interactions between cells and the influence of environmental conditions.  exhibit similar characters to those found in Interfilum and Klebsormidium. Triangular spaces between daughter and parental walls are visible in TEM micrographs of vegetative cells of E. fimbriata (Cook, 2004, figs. 6c, 7g). H-fragments of the cell wall are characteristic for this species (Cook, 2004). H-fragments are unknown for H. attenuata Lokhorst (Lokhorst et al., 2000), but we observed these structures in strain CCAP 329/1 cultivated on agar medium (data not shown). Consequently, cell division in Entransia and Hormidiella seems to be similar to that in Interfilum and Klebsormidium.
Triangular spaces between cells and the clear parental wall surrounding cell packet are visible in TEM micrographs of another streptophycean alga, Chlorokybus atmophyticus Geitler (Lokhorst et al., 1988, fig. 5). This species was mentioned as the sole representative among species formerly assigned to Charophyceae that shows a type of cell division intermediate between vegetative cell division and sporulation (Sluiman et al., 1989).
Investigation of species of Coleochaete cultivated under conditions simulating the terrestrial habitat (on solid medium and on sand grains) showed the formation of packet-like cell aggregates instead of the typical radial thalli (Graham et al., 2012). Cell packets are formed because of preservation of the parental walls (Graham et al., 2012, fig. 3c). It seems that vegetative cells of Coleochaete divide by the sporulation-like type because of the presence of the parental wall. However, the ultrastructural characters of cytokinesis in Coleochaete are completely different (formation of the cell plate in a phragmoplast) from those seen in the members of Klebsormidiales or Chlorokybales (cleavage furrow) Van den Hoek et al., 1995). These data show that a transition between two fundamentally different thallus structures or morphotypes (radial plate and sarcinoid packet) occurs in nature more often than was previously thought, and depends on environmental conditions. In general, the formation of cell packets and cubic aggregations is typical for many terrestrial algae, and is related to a reduction of the cell surface area subject to evaporation (Nienow, 1996;Karsten et al., 2010). The example of Coleochaete shows that the formation of a packet-like morphotype might be an adaptation to terrestrial conditions. The tendency for cells to easily transition to divide in three planes and form packet-like cell aggregates is probably typical for the sporulation-like type of cell division. This phenomenon was observed within the clade 'Prasiola', including taxa with pseudofilamentous (Stichococcus) and pleurococcoid packetlike thalli (Desmococcus F. Brand, Diplosphaera M. N. Bialosuknia) (Pröschold & Leliaert, 2007;Friedl & Rybalka, 2012). Various morphotypes among different lineages of algae are not the result of their multiple origins, but result from tiny morphological changes that dramatically influence their gross morphology.