Flagellate green algae from four water bodies in the state of Rio de Janeiro, Southeast Brazil

Floristic survey of flagellate green algae (Chlorophyceae and Prasinophyceae) from four water bodies in the state of Rio de Janeiro, southeast Brazil. Samples were collected bimonthly from September 1988 to August 1991. Thirty-five taxa of which 31 in Chlorophyceae (24 Chlamydomonadales and seven Volvocales) and four in Prasinophyceae (Polyblepharidales) were identified. Twenty-three taxa are new records for Brazil and 31 are registered for the first time for Rio de Janeiro State. On basis of cell shape, location of the nucleus and degree of longitudinal striations on the chloroplast surface a new combination, Vitreochlamys lefevrei (Bourr.) Menezes & C. Bicudo, is proposed. Integrating studies among morphological analysis on natural/cultured material and characterizing of reproduction/life cycle as well the correlating between the occurrence of the species and environmental conditions are needed in order to better knowledge the degree of phenotypic plasticity within the members of the flagellate green algae in the country.


Introduction
Few floristic and taxonomic investigations have been carried out on the pigmented flagellate algae in Brazilian freshwater systems. Information concerning these algae is dispersed in general floristic´ surveys and plankton studied ecology. Many of these publications, however, include neither illustration nor description of the material studied. To complicate matters, many of the latter publications just refer to the flagellate genus name or, even worse, simply mention them as 'phytoflagellates'.
Strict taxonomic studies of Brazilian phytoflagellates started effectively at the beginning of the last century with the works of Cunha (1913Cunha ( , 1914, who recorded altogether 90 pigmented taxa including descriptions of nine new species of euglenoid flagellates.
Somewhat recently, other phytoflagellate groups have become the object of floristic and taxonomic studies. Knowledge of these organisms in Brazil, however, is still very scarce. Despite of being just a few, such studies have shown there to be a very rich and interesting Brazilian flagellate algal flora, especially in the tropical part of the country. Some of the latter publications include descriptions of new taxa (Castro et al. 1991, Conforti 1993a, b, 1994, Menezes 1996, Menezes & Huszar 1997.
Seventy-six papers have already published on the pigmented flagellate algal flora of Brazil, of which 70% have focused on the Euglenophyceae. Regarding the other flagellate classes, publications can be summarized as follow: nine publications on Chrysophyceae and Synurophyceae, five on the Dinophyceae, three on the Raphidophyceae and Prasinophyceae, and three on the Chlorophyceae and the Cryptophyceae.
The present study is part of the floristic survey of the pigmented flagellate algae of Brazil. It includes the Chlorophyceae and Prasinophyceae from four selected water bodies located in the state of Rio de Janeiro. The paper is aimed at increasing knowledge of the flagellate algal flora from tropical Brazil, and includes critical taxonomic remarks on several taxa, information about species composition of each system, as well as notes on the ecology and geographical distribution of each group.

Material and methods
All water bodies investigated are located in the southern part of the municipality of Rio de Janeiro, state of Rio de Janeiro, southeast Brazil (figure 1). The area includes a coastal plain region and some escarpments from the Precambrian Complex (Serra do Mar). Altitude is around 800 m above the sea level, and relief ranges from plane to slightly waved.
Local climate is tropical with warm and rainy summers and dry winters. The mean annual temperature varies between 18 and 24 °C and the annual rainfall from 1,200 to 2,000 mm. Dominant soils are hydromorphic latosols, which are associated with litosols and podzols. The major part of the coastal plain consists of lagoons, the Jacarepaguá-Marapendi lagoon complex, that are interconnected by sandy strands. Due to its inclination, the lowland is crossed by diverging rivers which are broken close to the sea by penetration of tides raising pools, swamps, and mangroves. In this wet lowland dominates humid vegetation, dunes, and sandy coastal plains ("restingas"). Escarpment areas are found continuously throughout the coastal strand, where remnants of the Atlantic rainforest can be seen (Golfari & Moosmayer 1980, RADAMBRASIL 1983.
The four water bodies investigated can be described as follows: 1. Camorim Reservoir: located on the Pedra Grande massif, at approximately 430 m altitude (23°03'S and 43°27'W). The reservoir has total surface area of about 22 km², maximum depth 3 m, and alkaline, mesotrophic waters. This reservoir is the only source of water supply for the District of Jacarepaguá.
2. Unnamed oxbow pond: located in the Jacarepaguá lowland in front of the km 42 mark at the roadside of Avenida das Américas (22°50'00"S and 43°38'55"W). It has total surface area of approximately 0.1 km², maximum depth between 1 and 2 m and alkaline, eutropic waters. Extensive growth of macrophytes and mainly of Typha domingensis Person can be observed along its entire shoreline and the water surface is mostly covered by Salvinia sp.
3. Lagoinha: a coastal lagoon located in the Biological Reserve of Jacarepaguá (23º02'23"S and 43º28'97"W). It has total surface area of approximately 0.7 km², and mean depth of about 2.4 m. The bottom is covered with organic sediment mainly of plant origin, and the water is slightly acidic, oligohaline, eutrophic. Along the entire shoreline there is extensive macrophyte vegetation growth represented mostly by Typha domingensis Person, and its water surface is mostly covered with Pistia stratiotes Linnaeus. 4. Lake at the Bosque da Barra: a natural lake located in a preservation "restinga" area at Barra da Tijuca (22º59'02"S and 43º23'30"W). It has total surface area of around 0.2 km², mean depth of about 2 m, and oligotrophic, brown, acidic waters due to the presence of humic substances. Specimens of Nymphaea sp. and Utricularia sp. may sporadically occur along its shore.
Samples were collected from September 1988 to August 1991, every two months, from the shore by filling bottles directly with water and/or sediments using a 25 µm mesh size plankton net as well as by gathering by hand macroscopic filamentous algae and cutting off submerged parts of macrophytes. In every case, material was kept in bottles and immediately examined alive under the light microscope. Three or four days after collection material was invariably fixed and preserved with 3%-5% neutralized formalin solution.
All taxa were described in detail. Taxonomic comments, whenever necessary, are given with illustrations and information on the geographic distribution of each taxon identified in Brazil.
The circumscription of classes is according to Round (1981). The systematic arrangement of orders and families essentially follows that of Ettl (1983). Although the groups names proposed by Ettl (1983) for the genus Chlamydomonas are not to be formed according International Code of Botanical Nomenclature these names have been adopted in this work aiming a more didactic presentation. The concept the species applied was the morphospecies from natural samples. When possible, the delimitation of taxa was complemented by works based on combined analysis of morphology by light microscopy and molecular biology, among them of ), Nozaki (2003, Nozaki & Kuroiwa ( 1992), Nozaki & Itoh (1994), Pröschold et al. (2001). For more information on the techniques used see Menezes (1994).

Results
The total flagellate green algal flora of the four investigated water bodies was 35 species of which 31 belong to the Chlorophyceae (24 Chlamydomonadales and seven Volvocales) and four to the Prasinophyceae (Polyblepharidales). Twenty-three taxa are new records for Brazil and 31 are registered for the first time for Rio de Janeiro State. unequal rays, pyrenoid 1, spherical or elliptical, slightly displaced towards the posterior cell end. Nucleus central, located above the pyrenoid. Stigma elliptical, at the upper part of cell. Contractile vacuoles 2, apical. Flagella 2-2.5 times longer than the cell diameter.
Carteria lohammari belongs to a species-complex that is characteristic by having cells somewhat circular in outline, with a stelloid, cup-shaped chloroplast with one pyrenoid. The main diagnostic features to sort out species in this complex are the cell free-swimming or epibiont habit and the number of chloroplast incisions. Sometimes, secondary features like cell shape and size, presence of an anterior papilla, pattern of wall decoration, and distance between wall and protoplast can also be used.
According to Ettl (1983), C. lohammari and C. conochlii Skuja belong to the group of epibiontic species. However, C. lohammari has 12-16 chloroplast incisions and a rounded anterior cell pole, whereas C. conochlii has eight chloroplast incisions and a truncate anterior cell pole.
Twenty individual free-swimming cells were studied. The fact of consistently having spherical cells with smooth wall and never a papilla at the anterior cell pole, led us to the conclusion that all Brazilian populations examined could be C. abiscoensis Skuja sensu Peterfi (1968), young cells of C. disclusa Skuja, or with free-swimming cells of C. conochlii and C. lohammari. Material presently examined showed cell dimensions and number of chloroplast incisions that did not fit exactly any of the above species, but the one most closely matching was C. lohammari. So, despite having never found individual cells attached to any substrate, we believe the material from the state of Rio de Janeiro most closely corresponds to the latter species.
Recorded only for Sweden up to now. New to Brazil. Found in plankton samples from Bosque da Barra Lake and Camorim Reservoir.

Figures 9-13
Cells 16-24 µm long, 12-19 µm broad, broadly ovoid or rounded. Cell hyaline, wall with a hemispherical anterior papilla, sometimes cells enclosed in a mucilaginous sheath. Chloroplast cupshaped, smooth, sometimes with radial, peripheric incisions, basal thickening occupying nearly 1/2 of the cell, pyrenoid 1, spherical or elliptical, basal. Stigma elliptical, in the upper cell part, ca. 2 µm long. Contractile vacuoles 7-10, at the anterior part of cell. Flagella 1.5-2 times the cell length. Gloeocystis-like stage with thick mucilaginous sheath surrounding each cell, all cells inside of a common yellowish mucilaginous envelope, 23-25 µm diameter. Isogametes, zygote globose, with a reddish verrucose wall, 15-19 µm in diameter. World-wide distributed, reported from Africa, Asia, Europe, and North America. New to Brazil. Chlamydomonas pertyi was a common species in the area studied, being found in all collecting sites both in plankton and metaphyton samples. Ettl,Bot. Tidsskr. 74(4): 212, pl. 15, fig. 1 This species was only recorded from Denmark. New to Brazil. Present in one plankton sample from an oxbow pond.

Chlamydomonas tetravacuolata
Material studied differs from the original description in Ettl (1980) by having a much smaller basal thickening of the chloroplast and sometimes a rectangular pyrenoid. Chlamydomonas tetravacuola shows basal thickening of the chloroplast occupying 1/2 of cell and elliptical pyrenoid.  Chlamydomonas pseudotarda, C. ambigua Gerloff var. minor L.S. Peterfi, and C. saccus Pascher morphologically closely resemble each other. Their circumscriptions are very poorly delimited and based on cell shape and size, stigma morphology, and number of pyrenoids.

Chlamydomonas pseudotarda
According to their original descriptions, C. ambigua var. minor is distinct from C. pseudotarda and C. saccus mainly in having smaller cell dimensions (12-16 × 9-11 µm) and lesser number of pyrenoids (1-2). Also, in its sometimes ovoid cells. Chlamydomonas saccus has obovoid-oblong cell and the stigma is greater than in the other two species. Chlamydomonas pseudotarda differs from C. saccus only by having elliptical cell. Regarding cell dimensions and pyrenoid number there are overlap between these two species, i.e. cells 22-30 × 13-20 µm and 4 pyrenoids in C. saccus; cells 26 × 13 µm and 4-6 pyrenoids in C. pseudotarda. Ettl (1976) distinguished C. pseudotarda from C. saccus and C. ambigua var. minor on the basis of pyrenoid size. Chlamydomonas pseudotarda has pyrenoids with different sizes while C. saccus and C. ambigua var. minor have pyrenoids of the same size.
Populations presently studied showed variable cell shape and pyrenoid number and size, which could relate to all three taxa above. As similar situation was recorded by Jakubiec (1988) in material from Poland, i.e. cells oblong, oblong-elliptical to elliptical, with 2-13 pyrenoids. The specimens with oblongelliptical to elliptical cells had 4-5 differently-sized pyrenoids (figures 18-20), except for those with 2 pyrenoids that had equally-sized pyrenoids (figure 21). Individual oblong-celled organisms had differentlysized pyrenoids that varied in number from 7 to 13 (figure 16).
We decided to attribute the present material from the state of Rio de Janeiro to C. pseudotarda due to the high frequency of cells with more than four pyrenoids, and because we never found specimens typically of C. saccus.

Figures 22-25
Cells 13-17 µm long, 7-11 µm broad, elliptical. Cell wall hyaline, with an anterior conical papilla 1.5-2 µm long. Chloroplast cup-shaped, smooth, lateral thickening with 1 spherical pyrenoid, sometimes angular in outline, in the middle of the chloroplast. Nucleus in the middle of the cell or slightly displaced to the posterior. Stigma not observed. Contractile vacuoles 2, apical. Flagella about same length as the cell.
Material studied: BRASIL. Rio de Janeiro: Rio de Janeiro, Camorim Reservoir, 19- VII-1990 This species is very similar to C. pseudogloegama Gerloff, from which it differs in the lack of a stigma. Gerloff (1940) had, however, referred to the sporadic occurrence of a pale yellowish stigma in the original description of C. typhlos. All Brazilian specimens presently studied never showed a structure that could be a stigma.
Reported from Germany. New to Brazil. Found in just one plankton sample unit from the Camorim Reservoir.

Figures 26-27
Cells 20-25 µm long, 11-14 µm broad, elliptical, cylindrical-elliptical or ovoid. Cell wall hyaline, with a conical-truncated, double, anterior papilla. Chloroplast tubular, outer side with vertical ribbons, small rectangular space in the cell middle in which lies the nucleus. Pyrenoids 2, spherical, above and below the space. Stigma elliptical, in the upper part of the cell or behind the nucleus. Contractile vacuoles 2, apical. Some individuals (5% of total examined) were found, which were different from the original description of C. parisii by having ovoid-shaped cell and the stigma located behind the nucleus (figure 27).
Recorded from France. New to Brazil. The species was found in the plankton of Bosque da Barra Lake.

Figures 28-30
Cells 18-21 µm long, 12-14 µm broad, elliptical or oblong. Cell wall hyaline, with a conical, truncated anterior papilla 1.3-2 µm long. Chloroplast tubular, smooth, rectangular outline to space in middle of cell, in which lies the nucleus. Pyrenoids 2, spherical, located below and above the space. Stigma elliptical, in the upper portion of cell, 3-4 µm long. Contractile vacuoles 2, apical. Flagella 0.5-1.5 times the cell length. Regarding cell shape, part of the material studied is similar to C. pertusa Chodat. However, the structure of the papilla was a very stable characteristic that led us to consider the Brazilian specimens identical to C. pseudopertusa.
Reported from Asia, Europe, New Zealand. New to Brazil. This species was collected only from the plankton of Camorim Reservoir. Material from the state of Rio de Janeiro differs from the original description of the species in its homogeneous and thick cell wall and, sometimes, in the occurrence of a cup-shaped chloroplast. Our material also differs from of C. glans in having a conical rounded sometimes truncated anterior papilla.
Recorded from Europe. New to Brazil. This species was collected from the Bosque da Barra Lake plankton. Part of the present material differs from the original description of C. virgata in that the outer side of the chloroplast is interrupted by incisions that produce lobes of different extent. However, we are of the opinion that localization, extensions, and number of chloroplast incisions and lobes are not good taxonomic characters. Similar conclusions also apply to Carteria lohammarii Skuja that was also identified in the present study.

Chlamydomonas virgata
Reported from Europe. New to Brazil. This species was found in plankton and metaphyton samples from the Camorim Reservoir.

Figures 36-37
Cells 12-15 µm long, 5-7 µm broad, dorsiventral, elliptical in frontal view, ventrally curved, convex on dorsal side, convex in anterior 2/3 or retuse in its mid portion. Cell wall hyaline, with a hemispherical anterior papilla. Chloroplast dorsal, laminar, smooth, with a spherical pyrenoid in the middle of the dorsal side, sometimes below the cell equator. Nucleus central. Stigma elliptical, in the upper part of the cell, ca. 1.8 µm long. Contractile vacuoles 2, apical. Flagella about the same length as the cell.

Figures 41-46
Cells free-swimming or an epibiont in the mucilage of plankton algae, 7-9 µm long, 2.8-3.2 µm broad, ovoid or club-shaped, sometimes curved, dorsal side convex, ventral side flattened or concave in the anterior third of cell, without papilla or with a small anterior protrusion similar to a membranaceous hemispherical papilla. Chloroplast dorsal, laminar, smooth, with a spherical pyrenoid in the middle of the dorsal part. Stigma punctiform, in the upper part of the cell, dorsal, near to the vacuoles. Contractile vacuoles 2, apical. Flagella 1-1.5 times the cell length. Asexual reproduction while cell is actively motile, by division into 4 daughter cells.
Chlamydomonas rattuli is closely related to C. gloeophila Bourr. Both species are epibiontic in the mucilage of rotifers and plankton algae when vegetative and differ from each other by the presence of papilla in C. gloeophila. Bourrelly (1951) described and sketched C. rattuli with an anterior papilla, and considered C. gloeophila a synonym of the former. Ten years latter, Huber-Pestalozzi (1961) maintained C. rattuli sensu Korš. and considered C. rattuli described by Bourrelly (1951) to be synonymous with C. gloeophila.
We sometimes found in the same sample on the mucilage of Botryococcus braunii Kützing coexisting with some others that were actively motile. At other times, they were observed free-swimming in the plankton. Epiphytic cells were mostly attached to the substrate by their flagella. The latter were ovoid in shape, with a rounded anterior pole that did not touch the host surface (figures 41-42, 44). Club-shaped cells with their anterior pole attached to the substrate were also observed, which presented a slight short protuberance that was well delimited from the rest of the cell body (figures 43, 45).
Approximately the same variation above was also observed in populations of free-swimming specimens. Very often, epiphytic vegetative cells released from the substrate later on formed zoospores which kept themselves in a motile stage (figure 46).
Reported from Asia, Europe. New to Brazil. This species was found solely in the Camorim Reservoir plankton.

Figure 47
Cells 20-23 µm long, 11-18 µm broad, ovoid. Cell wall hyaline, with a hemispherical or truncate anterior papilla. Chloroplast cup-shaped, dissected into discoid portions. Nucleus central. Stigma elliptical, at the upper portion of cell, 6-7 µm long. Contractile vacuoles 2, apical. Flagella about the same length as the cell. Our material differs from of C. vesterbottnica in having hemispherical sometimes truncate papilla. Chloromonas vesterbottnica shows a spherical-conical anterior papilla (Ettl 1983) Reported from Denmark, Rumania, Sweden. New to Brazil. Present species was found in one plankton sample from the oxbow pond. Cells 15-20 µm long, 11-14 µm broad, ellipticalovoid, rounded poles. Cell membrane hyaline, wide. Protoplast 11-13 µm long, 6-8 µm broad, ovoid, elliptical-ovoid or pear-shaped widely separated from the membrane. Chloroplast cup-shaped, smooth, basal thickening occupying 1/3 of protoplast, with a spherical pyrenoid. Nucleus central, slightly displaced to the anterior cell end. Stigma elliptical, at the upper part of cell, ca. 1 µm long. Contractile vacuoles 2, apical. Flagella about the same length as the cell, penetrating the cell membrane at both sides of papilla, surrounded by short tubes at the base. Some specimens were morphologically close to S. alpina Pascher & Jahoda (=Vitreochlamys alpina?) and V. ordinata (Skuja) Nakazawa, respectively, since having the protoplast at a much shorter distance from the membrane and the elliptical-ovoid protoplasts.

Gloeomonas
Reported from Hungary, India, Japan, North America, Rumania, Scandinavia, Ukranie. New to Brazil. Material studied was planktonic and collected from the Bosque da Barra Lake.

Figures 56-57
Cells 25-28 µm long, 12-15 µm broad, oblong, poles rounded. Cell membrane hyaline, wide. Protoplast 15-18 µm long, 7-9 µm broad, elliptical or elliptical-ovoid, widely distant (about 3,5 µm) from the membrane. Chloroplast cup-shaped, smooth, basal thickening occupying almost 1/2 of protoplast, with a spherical basal pyrenoid. Nucleus anterior, median, sometimes shifted from the longitudinal axis. Stigma The genus has been revised by Nazakawa et al. (2001) based upon strains isolate from different parts of Japan, the Czech Republic, Slovak Republic and Germany using comparative light and electron microscopy and rbcL (large subunit of Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase) gene phylogeny. The results of this work showed that the delimitation of the six studied species-V. fluviatilis (Stein) Batko, V. gloeocystiformis (Dill) Nakazawa, V. ordinata (Skuja) Nakazawa, V. nekrassovii (Korš.) Nakazawa, V. aulata (Pascher) Batko, and V. pinguis Nakazawa based on the morphological differences used in the traditional taxonomic system were consistent with molecular data: cell and protoplast shape, numbers of contractile vacuoles and pyrenoids, form and position of the stigma, location of the nucleus, degree of longitudinal striations on the chloroplast surface. Besides of these characters Nakazawa et al. (2001) included as criteria for species delimitation the ultrastructure of the pyrenoid and stigma.
Although our material have not been analyzed under electron microscopy the populations previously identified as S. lefevrei belong to the group of species of Sphaerellopsis/Vitreochlamys with chloroplast surface smooth or with inconspicuous striations and nucleus sometimes shifted from the longitudinal axis. However, it can be distinguished from these species by clearly its oblong cell.
Reported from Austria, France and Switzerland. New to Brazil.This species has been found in the plankton of an oxbow pond.

Figures 60-61
Cells (15-)19(-25) µm long, (11-)13(-17) µm broad, elliptical, rounded poles. Cell membrane hyaline, wide, lacking papilla. Protoplast 12-15 µm long, 7-8.5 µm broad, ovoid, distant from the outer membrane (ca. 3-5 µm), with a hyaline anterior end forming a conspicuous mammillae projected, which is distant from the outer membrane of about 2 µm. Coarse radiating protoplasmic processes branched or not, 13-14 hyaline cytoplasmic strands frequently branched and connecting the protoplasmic processes to the outer membrane. Chloroplast cup-shaped, smooth, basal thickening occupying almost 1/2 of protoplast, with a spherical basal pyrenoid. Stigma elliptical, near to the pyrenoid, ca. 1 µm long. Contractile vacuoles numerous, irregularly distributed. Flagella about the same length as the cell, penetrating the cell membrane at both sides of papilla, surrounded by short, divergent tubes at the base. Cells (30-)33(-37) µm long, (20-)23(-26) µm broad, elliptical, rounded poles. Cell membrane hyaline, wide, sometimes with a 2-lobed papilla. Protoplast (22-)26(-32) µm long, (20-)23(-26) µm broad, elliptical or ovoid, apart 5-7 µm from the outer cell membrane, anterior end hyaline, projecting into a conspicuous conical-rounded mammilla which is 1.2-3.5 µm from the outer cell membrane. Coarse radiating protoplasmic processes branched or not, mostly with 11 branched hyaline cytoplasmic strands connecting protoplasmic processes with the outer membrane, sometimes cytoplasmic strands absent and the protoplasmic processes touching the cell membrane. Chloroplast tubular, with a central space rectangular in outline, smooth, pyrenoids 2, one anterior, the other one posterior to the space. Hematochrome granules numerous, disperse in the cytoplasm. Nucleus in the space of the chloroplast. Stigma elliptical, in the space of chloroplast, ca. 1 µm long. Contractile vacuoles numerous, irregularly distributed. Flagella about the same length as the cell, penetrating the cell membrane at both sides of papilla, surrounded by short tubes at the base. Some individuals studied showed some features of H. zimbabwiensis Pocock, such as longer flagellar tubes ( figure 63) and a small papilla at the anterior protoplast pole ( figure 64). Furthermore, about 1% of individual cells examined lacked cytoplasmic strands, the protoplasmic processes being more developed and sometimes touching the cell membrane ( figure  65). Latter specimens do not agree with Pocock's (1960) circumscription of the species that states that presence of cytoplasmic strands is a diacritic character for Haematococcus.
Reported from Hungary, South Africa. In Brazil it was previously reported for the Amazonas State (Bittencourt-Oliveira 1993). The species was collected from the Camorim Reservoir plankton.
Material studied: BRASIL. Rio de Janeiro: Specimens from the state of Rio de Janeiro showed variation in cell shape that partially agreed with the one registered by Peterfi (1965Peterfi ( , 1968 for P. pseudoangulosa L.S. Peterfi. Some specimens resembled those of P. angulosa Lemm. in having one or both sides of the cell with a median concave depression (figures 71, 77-78). Some other ones were similar to P. cordiformis Lemm. in having a flagellar tube (figures 80-81). In all individuals examined variation was detected in the number of pyrenoids, from one to five, as well as in their location in the cell.
After fixation, some cells showed wall surface somewhat waved (figure 82) that, when the specimens had two or more pyrenoids, were very much similar to those of P. aequiciliata (Gicklhom) Bourr.
Material studied: BRASIL. Rio de Janeiro: Rio de Janeiro, Lake at Bosque da Barra, 1-X-1988, M. The presence of an extra cellular matrix of the gelatinous parental envelope, together the maximum numbers of cells in a colony and modes of sexual reproduction have been considered important morphological features for delimiting volvocacean genera based on cultures, ultrastructure and molecular phylogeny studies. Pandorina Bory have up to 16 vegetative cells without the cellular envelopes of the matrix with isogamous reproduction without sperm packets. Within genera with up to 32 cells per colony, like Eudorina Ehrenb. and Yamagishiella Nozaki, each vegetative cell is enclosed by the cellular envelope. Eudorina has anisogamous sexual reproduction with sperm packets while Yamagishiella shows isogamous sexual reproduction without sperm packets (Nozaki & Kuroiwa 1992, Nozaki et al. 1997, 2000, Coleman 2001).
Although we have not observed sexual reproduction in the Brazilian populations, these can be assigned to the genus Eudorina in having more than two contractile vacuoles. These reject the possibility that material belongs to the genus Yamagishiella which has only two contractile vacuoles (H. Nozaki, unpublished data).
Colonial envelope varied both in the young and adult colonies, being smooth, waved, with the posterior pole sometimes truncate. Mammillae at the posterior pole of colonial matrix were only observed in adult colonies, where they became more evident after fixation (figure 94). Frequently, in asexual reproduction the cells showed division not synchronous resulting in an irregular distribution and differentiation of daughter colonies development in a colony ( figure 91).
Populations studied always showed individual cells having gelatinous sheath that was hexagonal in outline. Sheaths were clearly evident in adult colonies without staining (figure 95). In young colonies, however, sheaths were evident only after staining ( figure 96).
World-wide distribution. In Brazil it was previously recorded from North, Southeast, and South areas (Kammerer 1938, Thomasson 1971, Bittencourt-Oliveira 1990. This species was collected from the plankton of an unnamed roadside oxbow pond.
Material studied: BRASIL. Rio de Janeiro: Rio de Janeiro, Lake at Bosque da Barra, 1-X-1988 (Goldstein 1964, Nozaki et al. 1997. However, the occurrence of more than two contractile vacuoles led us to consider the Brazilian specimens as E. unicocca.
Cells in colonies may or may not have individual gelatinous sheaths. Individual sheaths clearly observed without staining were detected in adult colonies, which also had more developed posterior mammillae.
Reported from Central and North America, Switzerland. In Brazil, it was previously recorded from North, Southeast, and South areas (Uherkovich & Rai 1979, Stankiewiez 1980, Huszar 1984, 1985, Dewes & Huszar 1986, Bittencourt-Oliveira 1993. This species was found in plankton, neuston and metaphyton samples from Bosque da Barra Lake and from an unnamed roadside oxbow pond.
According to Starr (1962), V. steinii Playfair differs from V. pringsheimii Starr mainly in the presence of pyrenoids in the vegetative cells of the latter species. However, light and electron microscopy studies carried out by Nozaki (1982) and Nozaki et al. (1987) have shown that V. pringsheimii has pyrenoids always at the bottom of the chloroplast, and pyrenoids do not vary with culture age. Vegetative cells of V. steinii, however, may also have pyrenoids located at the chloroplast edge but only in some culture conditions. Nozaki and collaborators also observed that during the daughter colonies formation of V. pringsheimii the parental pyrenoid divided and was distributed to each one of the cells of the newly formed colonies. In V. steinii, pyrenoids do not divide but remain in one of the daughter cells of newly formed colonies. Moreover, V. pringsheimii has two contractile vacuoles while V. steinii shows two to eight (Ettl 1983).
In the Rio de Janeiro State material, some colonies were similar to those of V. pringsheimii in having the colonial sheath distinct from the cellular one. However, in all colonies studied cells with 4-6 contractile vacuoles without pyrenoids were observed, what led us to identify the Brazilian material with V. steinii.
World-wide distribution. In Brazil, it was previously reported from Amazonas State (Gessner 1931, Bittencourt-Oliveira 1993. This species was found in the plankton and sediments from an unnamed roadside oxbow pond.

Yamagishiella Nozaki
Yamagishiella sp.  (Angeler 1998, Nozaki & Kuroiwa 1992. The presence of this gelatinous (extracellular) matrix of vegetative colonies and the two contractile vacuoles indicated that Rio de Janeiro material can be assigned to Yamagishiella unicocca or even represent a new species of this genus. Since we have not worked with cultures and not observed sexual reproduction it was impossible to define a better circumscription of Brazilian populations (see comments of E. elegans).
Reported from Europa, Asia, South America, North America China, Finland, Chile, Austria, Germany, Ukraine, Japan, and North America. The material was collected from the plankton of Camorim Reservoir and metaphyton of a roadside oxbow pond.
As far as we now, M. ophistostigma is only reported from Europe; new to Brazil. This species was collected from the plankton of Camorim Reservoir. Cosmopolitan in distribution. In Brazil the species was previously recorded from a brackish coastal lagoon in the state of Rio de Janeiro (Menezes & Domingos 1994, Domingos & Menezes 1998. Pedinomonas minutissima was found only once in a plankton sample from the roadside oxbow pond.

Figures 115-116
Cells 5.5-9 µm long, 6-12 µm broad, flattened laterally, outline globose in front view, sometimes elliptical, anterior end round, with a apical swelling. Chloroplast cup-shaped, taking half of the cell, pyrenoid 1, spherical, basal. Contractile vacuole 1, apical. Stigma small, near the insertion of flagella. Flagella heterodynamyc, unequal in size, the shorter one about the cell length, the longer one ca. 1.5 times as long as the cell.

Figures 117-118
Cells 20-24 µm long, 12-16 µm broad, dorsiventrally flattened, broadly elliptical in frontal view, kidney-shaped in lateral view, anterior end rounded, with a deep apical depression. Chloroplasts 2, lateral, laminar, filling the entire cell, without pyrenoid. Two irregular rows of rounded trichocysts extending towards the posterior part of cell, others dispersed in the cytoplasm periphery. Stigma elliptical or rounded, short distance below the cell equator. Contractile vacuoles 2, apical. Flagella about as long as the cell.
Reported from Europe; new to Brazil. This species was gathered just once, from the plankton of Bosque da Barra Lake.

Discussion
The circumscription of unicellular and colonial volvocacean species is problematic because most of their original descriptions were based on light microscope of a few specimens from natural samples without information of the intraspecific variation of morphological characters or of the life history   (Pröschold et al. 2001, Nozaki et al. 2006. Several species have been recently analyzed using combined morphology, reproduction, and molecular data, however, a reasonable part of these results are divergent with respect to their taxonomy and many questions still remain concerning their revisions that must be solved before accepting their new generic names for clades (Colemann 2002, Hoham et al. 2002, Pollio et al. 2005, Nozaki et al. 2006.
In this work morphological characteristics used to distinguish species of flagellate green algae were, in general, often adequate for that purpose and the majority of examined populations were morphologically well defined and easy to distinguish under light microscope. However, the variability detected for some morphological features only from field observations and the absence of reproductive aspects becomes confusing and a clear delineation of some taxa was not possible like Chlamydomonas aff. glans, Chloromonas cf. vesterbottnica and Yamaguishiella sp. Possibly many of these organisms here identified can represent new taxa for the science or only morphotypes but would be necessary to analyze better the phenotypic living population characteristics from field observations and cultured material to determine the range of their sexual reproduction, size and shape.
Observation of cell habit did not help in the identification of Carteria lohammari and Chlamydomonas rattuli, both species being described in the literature as epibiontic on zooplankton or phytoplankton organisms. Skuja (1948) had already reported free-swimming vegetative cells of C. lohammari forming zoospores in the absence of substrate and for other epibiontic species of Chlamydomonas, especially during the warm season of the year. According to Burkholder (1996), such a kind of interaction apparently is not obligatory since the alga may also be epizoic. This could be well interpreted as a dispersal mechanism or adaptation strategy to increase the organism's access to nutrients, to escape predators, or to get an advantageous location for growing. Then, it is possible to understand the epibiontic habit as a facultative phase that would constitute an adaptation process under stressed environmental conditions. Va r i a t i o n i n t h e n u m b e r a n d e x t e n d of the chloroplasts in Carteria lohammari and Chlamydomonas virgata, as well as in the pyrenoid number and size in Chlamydomonas pseudotarda needs to be re-evaluated. Apparently, these characteristics could be related to the growth and/or physiology of the organism. However, in the present study increase of pyrenoid number was closely related to the increase of cell dimensions, especially with the cell breadth. Concerning size, different pyrenoid development stages in the same specimen might correspond to the formation of secondary pyrenoids in adult cells, as it was already observed in some Hematoccocaceae .
In relation to occurrence of identified taxa in different communities and environments our results agreed with literature data based on morphospecies criteria. Among the present unicellular species of Chlorophyta (24 of Chlorophyceae and four of Prasinophyceae), all 27 were always found in the plankton, mainly in Camorim Reservoir. Colonial volvocacean, in particular species of Gonium and Volvulina, were more frequent in the plankton, metaphyton, and sediments of the oxbow pond. Flagellated green algae are found in a variety of differing aquatic habitats. In such environments their life cycle provides for rapid colonization and further growth (Desnitski 2000(Desnitski , 2002(Desnitski , 2003. Particularly, Chlamydomonadales and colonial representatives of the Volvocales are frequently associated with small, mild alkaline, shallow water bodies. The latter group (Volvocales) is also often associated with high solar radiation (Menezes 1999, Pocock 1933a, b, Reynolds 1997 hot dry weather, warm water temperatures (Znachor & Jezberova 2005).
From the geographical distribution view based on phenotypic approach available in literature the flagellate chlorophyte flora (taxa size < 0,15 mm) was represented mainly by taxa that are common in temperate climates and that constituted about 57% of the total number of identified taxa. 17% of identified taxa were common to temperate and tropical climates, and 12% were typical of warmer climate. Cosmopolitan and worldwide distributed taxa accounted for 14% of total taxa identified.
Even retaining the concept of morphospecies our findings did not support the main idea about the distribution of microrganisms, "cosmopolitan hypothesis", which predicts that free-living microbes will be present in all kinds of environments where they can live due to unrestricted dispersal capabilities (Finley 2004, Fenchel 2005. This means that freeliving microbes have a lack of biogeography pattern and a low global diversity (Logares 2006). This capacity of ubiquitous dispersal is driven by huge population sizes leading to low probability of local extinction and, then the most microorganisms with size smaller than 1 mm have worldwide distribution while those larger than 10 mm are much less abundant and rarely cosmopolitan (Fenchel & Finlay 2003, Finlay & Fenchel 2004. Although some molecular studies support the "cosmopolitan hypothesis" (Daugjberg et al. 2000) for microalgae this idea is controversy because more recently available data suggest that micro-organisms (including protists and algae) have distribution patterns similar to those known from higher plants and animals, and that these patterns reflect historical (Gondwanan/Laurasian), ecological (tropical/temperate), and continental/local conditions (Coleman 2002, Foissner 2006, Luo et al. 2006.
In summary, our results may reflect, just how little is known about the chlorophytes flora from tropical and subtropical regions of the World. It is important to highlight the limited sampling of tropical species in the studies encompassing more complete description of intraspecific variation on natural and cultured material and/or more advanced tools like molecular phylogenetic.
Then, further investigations of Brazilian green flagellates should proceed, at least, to integrate morphological analysis on natural and cultured material in order to analyze the degree of phenotypic plasticity, to observe the characteristics of reproduction/life cycle, and correlate them to environmental conditions.