Redalyc.True Branched Nostocalean Cyanobacteria from Tropical

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Ferreira, Viviani; Branco, Luis Henrique Z.; Kaštovský, Jan True branched nostocalean cyanobacteria from tropical aerophytic habitats and molecular assessment of two species from field samples Revista de Biología Tropical, vol. 61, núm. 1, marzo, 2013, pp. 455-466 Universidad de Costa Rica San Pedro de Montes de Oca, Costa Rica

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Viviani Ferreira1, Luis Henrique Z. Branco1,2 & Jan Kaštovský3 1. Microbiology Graduation Program, UNESP-São Paulo State University, IBILCE, São José do Rio Preto, SP, Brazil; [email protected] 2. UNESP-São Paulo State University, IBILCE, Department of Zoology and Botany, R. Cristóvão Colombo, 2265, BR-15054-000, São José do Rio Preto, SP, Brazil; [email protected] 3. University of South Bohemia, Department of Botany, Branišovská, 31a, 370 05, České Budějovice, Czech Republic; [email protected]

Received 13-ii-2012. Corrected 10-VIII-2012. Accepted 11-IX-2012.

Abstract: Aerophytic cyanobacteria are commonly found growing on rocks, tree trunks and soil, but the diversity of these organisms is still poorly known. This complex group is very problematic considering the taxonomic arrangement and species circumscription, especially when taking into account tropical populations. In this work, 20 samples of aerophytic cyanobacteria from 15 distinct sampling sites were collected along a tropical rainforest area at the São Paulo State (Brazil). Samples were dried at room temperature after the collection, and once in the laboratory, they were rehydrated and analyzed. The taxonomic study resulted in the record and description of nine species of true-branched cyanobacteria pertaining to the genera Spelaeopogon (one specie), Hapalosiphon (two species) and Stigonema (six species). The similarity of the flora found when compared to surveys conducted in other geographical regions was relatively low. These differences could be addressed to ecological conditions of the habitats, to the extension of the area surveyed or even to taxonomic misinterpretations. The molecular assessment of 16S rDNA on the basis of field material was successful for two morphospecies, Hapalosiphon sp. and Stigonema ocellatum; however, their relationships with other populations and species revealed to be uncertain. The results of the floristic survey and of the molecular approach evidenced the fragile delimitation of some genera and species in the true branched group of cyanobacteria. Rev. Biol. Trop. 61 (1): 455-466. Epub 2013 March 01.

Key words: 16S rDNA, Brazil, Nostocales, diversity, “Stigonematales”, tropical rainforest, taxonomy.

True branched nostocalean cyanobacteria structure, true branching type, heterocyte posi- (formerly Stigonematales) are the most evolved tion and reproduction strategies. prokaryotes regarding thallus organization The classification system proposed by and cell differentiation (Golubić 1976). These Hoffmann et al. (2005) to Cyanobacteria organisms are common in terrestrial habitats merged the former Nostocales and Stigone- and the inner taxonomy is problematic, espe- matales into Nostocales, after several studies cially when tropical populations are included. have pointed out that branching type has no Anagnostidis & Komárek (1990) classified the phylogenetic value for distinguishing orders. true branching cyanobacteria in the order Sti- The presence of heterocytes, however, sup- gonematales with eight families and 48 genera. ports phylogeny and indicates the monophyly The order was characterized by the occurrence of the group (Seo & Yokota 2003, Gugger & of true branched filaments and heterocytes and Hoffmann 2004). Further contributions to the the below order taxa were defined by filament classification of cyanobacteria (Komárek 2006,

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 455 2010) have supported the systematic arrange- Pulvinularia suecica, respectively) occurring ment proposed by Hoffmann et al. (2005) for in Brazilian territory. this group of organisms. This paper aims to improve the taxonomic The taxonomy of cyanobacteria is now knowledge of an ecologically and evolutionary based on polyphasic approach including mor- important group of organisms and to describe phological characters (e.g., cell division and morphological and habitat data of species pre- polarity, branching type and thallus morpholo- sent in a tropical region. In addition, the mole- gy), molecular data, ultrastructural arrangement cular assessment from field samples constitutes (mainly thylakoids) and ecological parameters, a complementary objective in order to improve which are combined to define groups. How- the characterization of the populations found. ever, the developmental biology of the true branched cyanobacteria is still in the early MATERIALS AND METHODS stages of knowledge (Finsinger et al. 2008). The problems related to the artificial cultiva- Detailed information on sampling area, tion of most of true branched cyanobacteria as well as on collection, preservation and cha- samples (e.g. environmental exigencies not racterization of sites, samples and taxonomic totally understood, slow growth and low com- study are described in Branco et al. (2009). petitive ability under artificial conditions prior Briefly, samples of true branched cyanobacte- to isolation) difficult the assessment of mor- ria were collected in the rainforest region along phological variability and molecular sequenc- the Northern littoral area of the São Paulo State ing by the most common and regular methods. (Table 1). Visible growths of cyanobacteria Taxonomic studies on true branched cya- on soil, tree trunks and rocky substrates were nobacteria are not abundant. The few papers randomly collected with spatula or penknife. devoted to the knowledge of these organisms The samples were stored in paper bags and include just some genera (e.g., Capsosira, dried at room temperature (usually for 24 to 48h). For each collection (sometimes diffe- Chlorogloeopsis, Fischerella, Nostochopsis, rent samples-coded SAMA-were taken from Stigonema and Westiellopsis) with reduced the same site), data about global position and number of species, mainly about populations altitude (GPS Garmin, eTrex Summit), air and from temperate regions, and, in fact, this is plant-mass temperature (as close as possible considered not sufficient to solve the evolution- to the growths) (digital thermometer with a ary history of the true branched cyanobacteria 11cm long metal probe), air and plant-mass (Tomitani 2004). humidity (ThermoHygrometer Hana Instru- Some of the most extensive surveys of ments, HI8564), total and relative irradiance tropical aerophytic cyanobacteria that include (Li-Cor Quantameter, with spherical sensor– true branched taxa are those of Gardner (1927), the relative values are a relation between the Kufferath (1929), Golubić (1967), Hoffmann absolute value measured as close as possible (1986), Sarthou et al. (1995), Büdel et al. to the mass by the absolute value measured at (1994, 1997, 2002), Couté et al. (1999), Pat- an open area) and substratum pH (Merck pH tanaik & Adhikary (2002) and Kaštovský et strips-pH was measured through a solution al. (2011). Surveys of the Brazilian flora of with distilled/deionized water and a sample of true branched cyanobacteria (including those the substratum) of collection sites and samples from freshwater habitats) are very scarce and were recorded. practically restricted to the papers of Silva & In the laboratory, samples were rehy- Sant’Anna (1991, 1996). The contributions of drated and then examined under stereoscope Branco & Necchi-Júnior (1999) and Branco (Olympus SZX7) and light (Olympus BX-50) et al. (2010) focused single true branched microscopes and microphotographs were taken freshwater species (Doliocatella formosa and by using a digital camera (Olympus DP71)

456 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 TABLE 1 Global position and altitude of the collection sites and their respective samples

Site Sample Latitude (S) Longitude (W) Altitude (m) 01 SAMA 01 23º35’51” 46º10’08” 800 02 SAMA 02 23º41’07” 46º05’52” 791 03 SAMA 04 23º41’09” 46º05’52” 787 04 SAMA 10 23º46’21” 45º57’14” 22 05 SAMA 13 23º46’14” 45º43’14” 55 08 SAMA 22 23º36’10” 45º26’43” 183 09 SAMA 09 23º30’34” 45°07’56” 0 10 SAMA 23, SAMA 24 23º35’16” 45º26’57” 600 13 SAMA 33 23º32’56” 45º14’47” 23 14 SAMA 34, SAMA 35, SAMA 36, SAMA 37 23º29’39” 45º08’38” 66 15 SAMA 38 23º23’06” 45º07’19” 122 18 SAMA 45 23º22’37” 45º07’07” 309 19 SAMA 48, SAMA 49 23º24’50” 45º01’43” 77 21 SAMA 53 23º10’52” 44º50’00” 1 239 22 SAMA 54 23º09’54” 44º50’21” 1 460

coupled to the light microscope. Specimens hydrolysis (Birnboim & Doly 1979) and ampli- were studied according to their taxonomic fied with “DYEnamic ET Terminator Cycle features and were identified based on specific Sequencing” Kit (Amersham Biosciences, Pis- literature on cyanobacteria. The classification cataway, NJ, EUA). M13F and SP6R vector system by Hoffmann et al. (2005), with the primers and 357F, 357R, 704F, 704R, 1114F modifications proposed by Komárek (2006), and 1114R internal primers (Lane, 1991) were was adopted. used to select DNA sequences of about 1 500bp. Field samples were selected for molecular Products were sequenced (in triplicates analysis and the DNA extractions were made and both directions) in an ABI PRISM® 3100 using the “Ultra Clean Microbial DNA Isola- Genetic Analyzer Applied Biosystems) and the tion” Kit (MoBio Laboratories). The products nucleotide sequences obtained were aligned, were amplified using the primers 27F1 and refined and edited using the software BioEdit 1494Rc (Neilan et al. 1997) and the “Pure Taq v 7.0.5 (Hall 1999). The two sequences pro- Ready To-Go PCR Beads” kit (GE Life Sci- duced in this study were compared to other 19 ence). Reactions were cycled with an initial sequences of 16S rRNA gene retrieved from denaturation step at 94ºC for 4min, followed GenBank (NCBI), mostly representing taxo- by 30 cycles of denaturation at 94ºC for 20sec, nomically similar genera. annealing at 50ºC for 30sec, extension at 72ºC Phylogenetic trees were constructed by for 2min and final extension step at 72ºC neighbor-joining (NJ; using SeaVIEW4 soft- for 7min. PCR products were purified with ware), maximum likelihood (ML; PhyML 3.0) Wizard PCR Preps DNA Purification System and Bayesian inference (BI; Mr. Bayes 3.12). (Promega) and cloned by using the “pGEM®-T Bootstrap values for neighbor-joining and Easy Vector Systems” Kit (Promega). Vec- maximum likelihood were generated on the tor with the insert was introduced by thermal basis of 1 000 replicates and Bayesian probabi- shock (Sambrook et al. 1989) in competent lity was based on two million generations. The Escherichia coli DH5a cells. Plasmids with evolution model used was GTR as suggested the interested insert were purified by alkaline by Treefinder (version March 2011).

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 457 RESULTS in small groups in the mucilage of other cyanobacteria; main filament and branches Environmental data: Values of the para- similar; filaments usually uniseriate, occasio- meters were variable, but the ranges of species nally biseriate, 10.0-11.0μm wide, irregularly occurrence were mostly overlapped (Table 2). T-branched, Y-false branching present; sheath Values of pH ranged from 4-5, but reached up thin, hyaline; trichomes constricted, someti- to 10; the values higher than five were obser- mes not constricted, 8.0-10.0(-13.0)μm wide; ved for the lime painted hard substrates (rocks cells cylindrical to rounded, 10.0-12.0(-15.0) or cement curbs) where some populations were μm long; heterocytes intercalary or apical, 9.0- growing on. Total irradiance values oscillated 16.0μm long, (8.0-)10.0-12.0(-13.0)μm wide. from 300-2 740µmol photons/m2/s and the Occurrence: n=1; SAMA 09. relative values (2-100%) showed the occurrence of populations in open to shaded sites. Mastigocladaceae Air relative humidity was usually high, ran- ging from 60-92%, but always slightly lower Hapalosiphon cf. aureus W. West & G.S. than the values observed close to the plant West, J. Bot. 35: 241, 1897. (Fig. 2) masses (62-94%). Plant mass formed by entangled filaments, Floristic survey: Nine true branched nos- caespitose; main filament and branches similar or with slightly distinct in diameter; filaments tocalean species, pertaining to three families, uniseriate, 7.0-15.0(-17.5)µm wide; branches were found in the 20 samples from 15 distinct usually on one side of the filament; sheath sites along the studied area (Table 1). thin to moderately thick, hyaline to yellowish Borzinemataceae brown; trichomes constricted, 5.0-13.0µm wide; cells 4.0-10.0(-12.0)µm long, 0.4-1.5 Spelaeopogon sommierii times longer than wide; cell content granula- Borzi, Nuovo Giorn. Bot. Ital. 24: 108, 1917. ted, blue-green; heterocytes not abundant, 6.0- (Fig. 1) 9.3µm long, 5.5-6.8µm wide. Occurrence: n=1, SAMA 01. Plant mass formed by entangled filaments, Note: This population corresponds well caespitose, sometimes isolate filaments or to the description of Hapalosiphon aureus,

TABLE 2 Values (or range) of selected ecological parameters of the sites where the species were found

n AT MT ARH MRH Ir Pir pH S Spelaeopogon sommierii 1 * * * * * * * rock Hapalosiphon cf. aureus 1 19.6 19.9 85 90 110 29 5 soil Hapalosiphon sp. 1 22.3 21.5 92 94 43 66 5 soil Stigonema hormoides 9 21.1- 30.6 19.5-38.4 59-92 67-94 35-2740 2-100 4-5 rock, soil S. informe 1 24.6 25.5 80 87 160 51 5 rock S. minutum 4 21.9-27.7 19.5-36.0 55-86 62-89 14-1700 3-100 4-5 rock, soil S. ocellatum 12 19.6-31.0 18.3-38.0 62-83 70-87 25-2740 5-100 4-10 rock, soil S. turfaceum 5 21.8-27.7 20.0-23.9 74-90 75-93 3-330 5-48 4-5 rock, soil Stigonema sp. 1 24.6 25.5 80 87 160 5 5 rock

* data not available. n=number of samples with species record, AT=air temperature (ºC), MT=plant mass temperature (ºC), ARH=relative humidity of the air (%), MRH=relative humidity close to the plant mass (%), Ir=irradiance measured close to the plant mass (µmol photons/m2/s), PIr=percentage of irradiance reaching the plant mass in relation to the irradiance at a near open site (%), S=substratum type.

458 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 Figs. 1-9. (1) Spelaeopogon sommierii. (2) Hapalosiphon cf. aureus. (3) Hapalosiphon sp. (4) Stigonema hormoides. (5) S. informe. (6) S. minutum. (7) S. ocellatum. (8) S. turfaceum. (9) Stigonema sp.

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 459 but differs from it by its shorter cells in the rounded, lateral or intercalar, 4.0-9.6µm long, branches and by its biotope characteristics. 3.2-9.6µm wide; homorgones terminal in the H. aureus is known from aquatic habitats, branches, 25.0-30.0(-70.0)µm long. inhabiting standing water bodies, while the Occurrence: n=9, SAMA 04, SAMA 22, studied population is aerophytic on soil in SAMA 33, SAMA 34, SAMA 35, SAMA 36, tropical rainforest. SAMA 38, SAMA 45, SAMA 54. Note: According to literature data, S. hor- Hapalosiphon sp. (Fig. 3) moides is distributed along tropical and temperate regions and its metrical and morphological Plant mass formed by densely entangled ranges are relatively wide. filaments, prostrate; main filament and branches similar; filaments uniseriate, 6.0-9.0(10.7) Stigonema informe µm wide; branches long, usually on one side of Kützing ex Bornet & Flahault, the filament, not narrowing at the apex; sheath Ann. Sci. nat., 7, 5: 75, 1887. (Fig. 5) thin to moderately thick, hyaline to yellowish brown; trichomes constricted, 4.0-7.6µm wide; Plant mass formed by entangled filaments, cells 3.5-7.0(-10.5)µm long, 0.7-1.3(-2.3) times caespitose, yellowish to yellowish brown; longer than wide; cell content granulated, pale main filament and branches distinct; main green to pale blue-green; heterocytes frequent, filaments with up to four rows of cells, 56.0- cylindrical to sub-quadrate, 4.0-8.5(-10.0)µm 87.4µm wide; branches 24.0-44.0µm wide; long, 5.0-8.0µm wide; arthrospores cylindrical sheath thick, yellowish to yellowish brown; to rounded, sometimes in long chains, usually cells hemispherical to rounded, sometimes with one (or more) big refringent granule (oil?) grouped forming “packs”, 7.2-16.0µm long, inside, 8.0-11.0µm long, 5.5-7.0µm wide, epis- 6.4-12.0µm wide; cell content granulated, blue- pore thick, smooth, dark yellowish brown. green; heterocytes hemispherical, lateral, 7.2- Occurrence: n=1, SAMA 45. 12.0µm long, 3.2-11.2µm wide; homorgones Note: this population resembles Hapalosi- terminal in the branches, short, 16.0-32.0µm phon welwitschii in filament diameter, however long, 6.9-12.0µm wide. heterocytes are rare and, mainly, the branches Occurrence: n=1, SAMA 38. are short and tapered in this species. Stigonema minutum Bornet & Flahault, Stigonemataceae Ann. Sci. nat., 7, 5: 72, 1886. (Fig. 6)

Stigonema hormoides Bornet & Flahault, Plant mass formed by entangled filaments, Ann. Sci. nat., 7, 5: 68, 1887. (Fig. 4) caespitose (main filament prostrate and branches erect); main filament and branches dis- Plant mass formed by entangled filaments, tinct; main filaments with up to three rows of erect, yellowish brown or dark brown, or cells, 20.0-28.0µm wide; branches up to two filaments grouped or growing isolated in gela- rows of cells, not enlarging or tapering towards tinous masses of other cyanobacteria; main to the end, 14.0-18.2(-20.0)µm wide; sheath filament and branches distinct; filaments with thick, yellowish to yellowish brown; cells dis- one row of cells, rarely two, (7.0-)9.0-17.5(- coid to hemispherical, 5.0-12.0(-15.0)µm long, 22.0)µm wide; branches usually short; sheath 5.0-13.0µm wide; cell content granulated, blue- thick, yellowish to yellowish brown; cells green; heterocytes hemispherical, lateral, 7.0- variable, discoid, spherical, oval, squarish, 11.0µm long, 6.0-13.0µm wide; homorgones rounded or irregular, 2.4-9.6(-14.0)µm long, terminal in the branches, short. (-2.5)4.0-12.0µm wide; cell content granula- Occurrence: n=4, SAMA 24, SAMA 36, ted, blue-green; heterocytes hemispherical or SAMA 48, SAMA 54.

460 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 Note: The species is commonly referred similar; filaments with up to four rows of in literature with distinct morphotypes. In cells, (20.0-)26.0-35.0(-40.0)µm wide; bran- addition, populations identified as S. minutum ches slightly tapering to the apex; sheath are recorded occurring in very distinct climate thick, yellowish to yellowish brown; cells regions, from temperate (type material) to rounded, 4.8-12.5µm long, 4.0-11.0µm wide; tropical or even polar areas. Species revision is cell content granulated, blue-green; heterocytes probably needed. hemispherical, lateral; homorgones terminal, 9.5-45.0µm long, 4.0-11.0µm wide. Stigonema ocellatum Occurrence: n=5, SAMA 02, SAMA 34, Thuret ex Bornet & Flahault, SAMA 35, SAMA 36, SAMA 49. Ann. Sci. nat., 7, 5: 69, 1887. (Fig. 7)

Plant mass formed by entangled filaments, Stigonema sp. (Fig. 9) erect or filaments grouped or isolated growing in gelatinous masses of other cyanobacte- Isolated filaments growing mixed with ria; main filament and branches similar; fila- other Stigonema species; main filament and ments with one row of cells, occasionally two, branches similar; filaments with one row of 18.7-45.0µm wide; sheath thick, yellowish cells, 8.0-12.8μm wide; sheath thick, smooth, to yellowish brown; cells variable, irregu- hyaline; trichomes 4.0-7.2μm diam.; cells lar, hemispherical, oval, squarish or, more cylindrical, squarish, 2.4-8.0μm long.; cell commonly, rounded, 3.0-20.0µm long, 4.0- content yellowish green, homogeneous; hetero- 25.6µm wide; cell content granulated, blue- cytes rare, lateral, 3.1-3.5µm wide, 5.0-5.5µm green; heterocytes discoid, hemispherical or long; hormogones not observed. rounded, lateral, 8.7-21.6µm long, 6.4-22.4µm Occurrence: n=1; SAMA 38. wide; homorgones terminal in the branches, Note: This population is close to Stigo- long at maturity, (20.0-)48.0-180.0µm long, nema hormoides, however the structure of the 9.6-20.0µm wide. filament is distinct between them (cylindrical Occurrence: n=12, SAMA 04, SAMA 10, cells in the studied population and usually SAMA 13, SAMA 22, SAMA 23, SAMA 33, nearly spherical in S. hormoides). In addition, SAMA 34, SAMA 35, SAMA 36, SAMA 37, metrics are not fully compatible. SAMA 38, SAMA 53. Note: This is one of the most commonly Molecular characterization: Sequencing recorded species of the genus Stigonema and was tried for all species surveyed, but positive the literature data indicate a cosmopolitan results were obtained only for Hapalosiphon sp. distribution, occurring in very distinct types of (three clones, 1 390bp) and Stigonema ocella- habitats. As well as, morphological and metri- tum (one clone, 1 352bp). GenBank accession cal ranges of S. ocellatum are quite wide and codes for the new sequences are GQ354274 some authors (Geitler, 1932) suggest that it is and GQ354275, respectively. a species complex instead a well-defined taxo- Phylogenetic analysis on the basis of nomic entity. More detailed studies, including Hapalosiphon sp. and S. ocellatum sequences molecular approaches, are surely required to a and 19 strain sequences retrieved from Gen- more precise evaluation. Bank resulted in a consensus tree (Fig. 10) with different bootstrap/probability clade val- Stigonema turfaceum ues according to the method employed. Cooke ex Bornet & Flahault, The phylogenetic tree shows that both spe- Ann. Sci. nat., 7, 5: 74, 1887. (Fig. 8) cies grouped with heterocytous cyanobacteria, as expected. Stigonema ocellatum grouped Plant mass formed by entangled fila- with another strain of the same species (SAG ments, caespitose; main filament and branches 48.90, GenBank access code AJ544082, 98%

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 461 Cyanobium sp. AM710383 *,100,* Prochlorococcus marinus X63140 Phormidium sp. AF218376 Leptolyngbya boryana AF132793 Brasilonema terrestre EF490447 100,100,1 87.9,100,1 Symphyonemopsis sp. AJ544085 83.1,89,0.97 96.2,100,1 88.3,90,1 Iphinoe spelaeobios HM748317 Scytonema ho manii AF132781 Nostoc commune AY577536 *,96,* 99.5,100,1 89.2,100,1 Stigonema ocellatum Brazil GQ354275 C1 95.4,98,1 Stigonema ocellatum AJ544082 Chlorogloeopsis sp. DQ431000 Fischerella sp. AB075986 79,72,1 Fischerella sp. AY039703 *,77,0.96 93.4,100,1 Nostochopsis sp. AJ544081 *,*,* Hapalosiphoin hibernicus EU151900 Westiellopsis prolica AJ544086 C4 *,94,* # Hapalosiphon sp. AB093485 82.7,100,1 $ Hapalosiphon delicatulus AB093484 C2 C3 Hapalosiphon welwitschii AY034793 *,77,* Hapalosiphon sp. Brazil GQ354274 # = *,91,0.9 0.01 substitutions/site $ = *.*.*

Fig. 10. Consensus phylogenetic tree including the sequences Hapalosiphon sp. and S. ocellatum, from Atlantic rainforest, and 19 strains obtained from GenBank (access numbers are indicate after species name). Values at the nodes are bootstraps for maximum likelihood and neighbor joining (1 000 replications; %) and probability for Bayesian inference (two million of generations), respectively; only values higher than 70% or 0.7 are indicated and lower or absent values are indicated by *.

sequence similarity) constituting a well-sup- DISCUSSION ported cluster (C1) with bootstrap/probability values of 89.2%, 100% and 1 (respectively ML, Results on spatial distribution reveal that NJ and BI). The two S. ocellatum strains are some species were well distributed throughout close to a Nostoc commune strain. the sampling area (Stigonema ocellatum, 12 Hapalosiphon sp. was similar to H. wel- different samples, nine distinct sites; S. hor- witschii (access code AY034793, 98% sequence moides, nine samples, six sites), while some are similarity) and, according to NJ test (77% boot- geographically more restricted (Spelaeopogon strap), grouped with three other species of the somierii, Hapalosiphon cf. aureus, Hapalo- genus (C2), but this group is not formed in siphon sp., Sigonema informe and Stigonema ML and BI tests. However, a well-supported sp., all of them occurring in just one sample). clade constituted by Hapalosiphon species The ecological data obtained, however, do and one strain of Westiellopsis prolifica (C3; not permit to address any conclusion on the cluster bootstrap/probability of 82.7%, 100% species environmental preferences or toleran- and 1 for ML, NJ and BI, respectively) was ces. As most of measurements are punctual consistently formed in all tests performed. This and too variable (e.g. light) or reasonably Hapalosiphon/Westiellopsis clade is nested in a homogeneous in rainforest area (e.g. tempe- major group (C4; 77% and 0.96 for NJ and BI, rature, humidity), those data must be used as respectively) that includes other morphologi- a reference for habitat characterization, but cally similar genera (Fischerella, Nostochopsis not as habitat definition. Nevertheless, the and Chlorogloeopsis) spread in the clade. more widespread species occurred under wider

462 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 ranges of variation for most parameters evalua- Although it is possible to reach specific ted, as obviously expected. identification for most of populations accord- All the species with occurrence in two or ing to the classical monographs on cyanobacte- more samples were found growing on different ria, the criteria used for species circumscription substrate types (soil and rock) and substra- seem to be very wide. Many recorded species in te fidelity was not observed for any species literature perfectly correspond to original and recorded. Even though there are species with further descriptions of such taxon, but more restricted dependence or preference on a deter- detailed analyses of illustrations reveal dis- mined substrate, frequently it is not possible tinct filament structures and cell arrangements. to establish a clear relationship between the However, the exact value of each taxonomic species and the substrate, especially for the criterion used on this group is still to be proved, most widespread and morphologically variable probably with the help of additional tools. species. Unfortunately, there were not found Some contributions to the cyanobacterial studies in literature developing this subject in classification system (Komárek & Anagnosti- detail and more robust conclusions and compa- dis 2000, 2005, Hoffmann et al. 2005, Komá- risons cannot be addressed. rek 2006, 2010) emphasize the necessity of also Interestingly, it is the very common joint taking into account the environmental and geo- occurrence of different species of Stigonema graphical characteristics in the identification in a same sample or within a centimeter spatial process. If on one hand it is a well-supported scale. In sample SAMA 38, for instance, four concept because it probably reflects species species (S. hormoides, S. informe, S. ocella- requirements, it also creates a practical problem tum and Stigonema sp.) were found growing on the other. During this work, these premises together. Stigonema hormoides, S. ocellatum were considered to improve the taxonomic and S. turfaceum were all present in the samples evaluation but it is still very difficult to assure SAMA 34, SAMA 35 and SAMA 36. Thus, it that two very similar (or identical) morphoty- is possible to speculate that many species have pes are different because the existence of some similar environmental requirements, but a more ecological deviation from the type. The central robust evaluation would only be supported with problem is that it is still not clear if identical additional data from populations of the same genotypes can support distinct environmental and other ecologically distinct habitats. pressures without phenotypic changes or in Considering biogeography, only two spe- what extension these changes occur. The con- cies found, Stigonema hormoides and S. minu- cept of ubiquitous occurrence of most species tum, were also recorded by Hoffmann (1986) has been discarded (Komárek 1994), but few for Luxemburg and this low number of com- data on genetic variability of true branched mon species among the surveys is probably complex cyanobacteria have been produced to connected to the distinct climatic areas stud- provide a solid theoretical background about ied. In comparison with the data presented by the biogeography of the species. Couté et al. (1999), for New Caledonia, five The results of the molecular assessment species (Stigonema hormoides, S. minutum, S. obtained from this study suggest that popu- ocellatum, S. informe and S. turfaceum) were lations identified as same species can be also common to both studies, possibly reflecting similar at genetic level even they are from environmental similarities among the areas. distinct climatic regions. The S. ocellatum SAG Sant’Anna et al. (1991) found, in a small cave, 48.90 is a cultivated strain from specimens four species (Spelaeopogon sommierii, Stigo- collected in a Sphagnum peat bog in Germany nema hormoides, S. minutum and S. ocellatum) and the paper of Gugger & Hoffmann (2004) also recorded in this study and this relatively presents a brief morphological account of the low similarity is surely due to the size of the specimens. In general, there is good metrical area studied. correspondence and the identification as S.

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 463 ocellatum seems to be correct for all. However, sequencing from one or few cells), but they the structure of filaments is not always quite need to be improved and extended. Some time similar (cell size in the main filament, cell size will be taken until we can access a reasonable in branches, stratification), but it is difficult to amount of data to incorporate to the taxo- evaluate if the variations are really meaningful. nomical approach. Meanwhile, it is important A critical review of different Stigonema ocel- and relevant to record the morphological and latum descriptions and illustrations found in molecular (when possible) diversity of popu- literature (Frémy 1930, Geitler 1932, Skinner lations with special geographical occurrence & Entwisle 2001, Komárek & Hauer 2011, this and ecology. paper) corroborates that a very wide morpho- The cyanobacterial systematics is still con- logical range is recorded for the species, even fused and its reorganization on the basis of tra- though most of the populations can be correctly ditional morphological characters seems to be identified on the basis of the species original not adequate (Garcia-Pichel et al. 1996, Palins- description. The present results from morpho- ka et al. 1996). Instead, a polyphasic approach logical and molecular studies and the available has been employed to better define the taxo- data in literature allow considering that S. ocel- nomic levels (Zehr et al. 2003, Rajaniemi et latum is probably a complex of species rather al. 2005, Marquardt & Palinska 2007) and the than a well delimited species. As it is a very modern taxonomy is becoming a synthetic sci- widespread “species”, more detailed molecular ence that incorporates several tools to achieve and morphological studies with populations a more robust evaluation of the biodiversity. from distinct geographical and ecological habi- However, mainly in tropical areas, there is tats are strongly recommended. an enormous contingent of still not described Hapalosiphon welwitschii AY034793, morphotypes that is extremely relevant for the which is the closest sequence to Hapalosi- understanding of evolutionary relationships phon sp., is a strain from Australian soils, but, and producing a more natural classification unfortunately, no morphological data could system for this important group of organisms. be found for further comparison to the Bra- zilian material. The phylogenetic tree dem- onstrates that the clade formed by different ACKNOWLEDGMENTS species of Hapalosiphon, Westiellopsis, Fisch- The authors thank to Conselho Nacional erella and Nostochopsis constitute a mixed and de Pesquisa e Desenvolvimento Tecnológico much supported group and that the distinction (CNPq), Coordenação de Aperfeiçoamento de among the different genera and species needs Pessoal de Nível Superior (CAPES), Fundação to be improved. de Amparo à Pesquisa do Estado de São Paulo The molecular approach of the hetero- (FAPESP) and the grant LH12100 to JK for cytous true branched cyanobacteria group is the financial support. We also thank to Tomáš still at the very initial stage. The usual tech- Hauer (University of South Bohemia and Insti- niques for DNA extraction require consid- tute of Botany of Academy of Science of Czech erable amount of unicyanobacterial growths Republic) for improving the molecular analysis (cultures), that have been proved to be very and tree construction. uncommon to be achieved, mainly for species of Stigonema or Hapalosiphon, for instance. They apparently have a special set of environ- RESUMEN mental requirements to grow and their growth rate is usually low, favoring the establishment Las bacterias aerofíticas crecen comúnmente en las rocas, troncos de árboles y el suelo, pero la diversidad of cyanobacteria from other groups prior to the de estos organismos todavía es poco conocida. Este com- final isolation. New methods to study directly plejo grupo es muy problématico considerando la dispo- from nature have been developed (e.g. DNA sición taxónomica y la circunscripción de las especies,

464 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 61 (1): 455-466, March 2013 especialmente cuando se toman en cuenta las poblaciones lowlands and the Guyana uplands, Venezuela. Bot. tropicales. En esta investigación, 20 muestras de cianobac- Acta 107: 422-431. terias aerofíticas de 15 distintos sitios de muestro fueron Büdel, B., H.M. Weber, S. Porembski & W. Barthlott. 2002. recolectadas a lo largo de un área de selva tropical en el Cyanobacteria of inselbergs in the Atlantic rainforest estado de São Paulo (Brasil). Las muestras fueron secadas zone of eastern Brazil. Phycologia 41: 498-506. a temperatura ambiente después de su recolecta, y una vez Couté, A., G. Tell & Y. Thérézien. 1999. Cyanophyceae en el laboratorio, se hidrataron y analizaron. El estudio (Cyanobacteria) aérophiles de Nouvelle-Calédonie. taxónomico resultó en un registro y descripción de nueve Cryptogamie, Algol. 20: 301-344. especies de cianobacterias con ramificaciones verdaderas pertenecientes al género Spelaeopogon (una especie), Finsinger, K., I. Scholz, A. Serrano, S. Morales, L. Uribe- Hapalosiphon (dos especies) y Stigonema (seis especies). Lorio, M. Mora, A. Sittenfeld, J. Weckesser & W.R. La similutud de la flora encontrada en comparación con Hess. 2008. Characterization of true-branching cya- las encuestas realizadas en otras regiones geográficas fue nobacteria from geothermal sites and hot springs of relativamente baja. Estas diferencias podrían deberse a Costa Rica. Environ. Microbiol. 10: 460-73. las condiciones ecológicas de los hábitats, a la extensión Frémy, P. 1930. Les Myxophycées de l’Afrique équatoriale de la zona de estudio o incluso a malas interpretaciones française. Archives de Botanique Mémoire 3: 1-508. taxonómicas. 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