Revista de Biología Tropical ISSN: 0034-7744 [email protected] Universidad de Costa Rica Costa Rica 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 Available in: http://www.redalyc.org/articulo.oa?id=44925650034 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative True branched nostocalean cyanobacteria from tropical aerophytic habitats and molecular assessment of two species from field samples 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 col- lection, 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