True branched nostocalean cyanobacteria from tropical aerophytic habitats and molecular assessment of two species from field samples

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.

True branched nostocalean cyanobacteria (formerly Stigonematales) are the most evolved prokaryotes regarding thallus organization and cell differentiation (Golubić 1976).These organisms are common in terrestrial habitats and the inner taxonomy is problematic, especially when tropical populations are included.Anagnostidis & Komárek (1990) classified the true branching cyanobacteria in the order Stigonematales with eight families and 48 genera.The order was characterized by the occurrence of true branched filaments and heterocytes and the below order taxa were defined by filament structure, true branching type, heterocyte position and reproduction strategies.
The classification system proposed by Hoffmann et al. (2005) to Cyanobacteria merged the former Nostocales and Stigonematales into Nostocales, after several studies have pointed out that branching type has no phylogenetic value for distinguishing orders.The presence of heterocytes, however, supports phylogeny and indicates the monophyly of the group (Seo & Yokota 2003, Gugger & Hoffmann 2004).Further contributions to the classification of cyanobacteria (Komárek 2006(Komárek , 2010) ) have supported the systematic arrangement proposed by Hoffmann et al. (2005) for this group of organisms.
The taxonomy of cyanobacteria is now based on polyphasic approach including morphological characters (e.g., cell division and polarity, branching type and thallus morphology), molecular data, ultrastructural arrangement (mainly thylakoids) and ecological parameters, which are combined to define groups.However, the developmental biology of the true branched cyanobacteria is still in the early stages of knowledge (Finsinger et al. 2008).The problems related to the artificial cultivation of most of true branched cyanobacteria samples (e.g.environmental exigencies not totally understood, slow growth and low competitive ability under artificial conditions prior to isolation) difficult the assessment of morphological variability and molecular sequencing by the most common and regular methods.
Taxonomic studies on true branched cyanobacteria are not abundant.The few papers devoted to the knowledge of these organisms include just some genera (e.g., Capsosira, Chlorogloeopsis, Fischerella, Nostochopsis, Stigonema and Westiellopsis) with reduced number of species, mainly about populations from temperate regions, and, in fact, this is considered not sufficient to solve the evolutionary history of the true branched cyanobacteria (Tomitani 2004).
This paper aims to improve the taxonomic knowledge of an ecologically and evolutionary important group of organisms and to describe morphological and habitat data of species present in a tropical region.In addition, the molecular assessment from field samples constitutes a complementary objective in order to improve the characterization of the populations found.

MATERIALS AND METHODS
Detailed information on sampling area, as well as on collection, preservation and characterization of sites, samples and taxonomic study are described in Branco et al. (2009).Briefly, samples of true branched cyanobacteria were collected in the rainforest region along the Northern littoral area of the São Paulo State (Table 1).Visible growths of cyanobacteria on soil, tree trunks and rocky substrates were randomly collected with spatula or penknife.The samples were stored in paper bags and dried at room temperature (usually for 24 to 48h).For each collection (sometimes different samples-coded SAMA-were taken from the same site), data about global position and altitude (GPS Garmin, eTrex Summit), air and plant-mass temperature (as close as possible to the growths) (digital thermometer with a 11cm long metal probe), air and plant-mass humidity (ThermoHygrometer Hana Instruments, HI8564), total and relative irradiance (Li-Cor Quantameter, with spherical sensorthe relative values are a relation between the absolute value measured as close as possible to the mass by the absolute value measured at an open area) and substratum pH (Merck pH strips-pH was measured through a solution with distilled/deionized water and a sample of the substratum) of collection sites and samples were recorded.
In the laboratory, samples were rehydrated and then examined under stereoscope (Olympus SZX7) and light (Olympus BX-50) microscopes and microphotographs were taken by using a digital camera (Olympus DP71) coupled to the light microscope.Specimens were studied according to their taxonomic features and were identified based on specific literature on cyanobacteria.The classification system by Hoffmann et al. (2005), with the modifications proposed by Komárek (2006), was adopted.
Field samples were selected for molecular analysis and the DNA extractions were made using the "Ultra Clean Microbial DNA Isolation" Kit (MoBio Laboratories).The products were amplified using the primers 27F1 and 1494Rc (Neilan et al. 1997) and the "Pure Taq Ready To-Go PCR Beads" kit (GE Life Science).Reactions were cycled with an initial denaturation step at 94ºC for 4min, followed by 30 cycles of denaturation at 94ºC for 20sec, annealing at 50ºC for 30sec, extension at 72ºC for 2min and final extension step at 72ºC for 7min.PCR products were purified with Wizard PCR Preps DNA Purification System (Promega) and cloned by using the "pGEM ® -T Easy Vector Systems" Kit (Promega).Vector with the insert was introduced by thermal shock (Sambrook et al. 1989) in competent Escherichia coli DH5a cells.Plasmids with the interested insert were purified by alkaline hydrolysis (Birnboim & Doly 1979) and amplified with "DYEnamic ET Terminator Cycle Sequencing" Kit (Amersham Biosciences, Piscataway, NJ, EUA).M13F and SP6R vector primers and 357F, 357R, 704F, 704R, 1114F and 1114R internal primers (Lane, 1991) were used to select DNA sequences of about 1 500bp.
Products were sequenced (in triplicates and both directions) in an ABI PRISM® 3100 Genetic Analyzer Applied Biosystems) and the nucleotide sequences obtained were aligned, refined and edited using the software BioEdit v 7.0.5 (Hall 1999).The two sequences produced in this study were compared to other 19 sequences of 16S rRNA gene retrieved from GenBank (NCBI), mostly representing taxonomically similar genera.
Phylogenetic trees were constructed by neighbor-joining (NJ; using SeaVIEW4 software), maximum likelihood (ML; PhyML 3.0) and Bayesian inference (BI; Mr. Bayes 3.12).Bootstrap values for neighbor-joining and maximum likelihood were generated on the basis of 1 000 replicates and Bayesian probability was based on two million generations.The evolution model used was GTR as suggested by Treefinder (version March 2011).

RESULTS
Environmental data: Values of the parameters were variable, but the ranges of species occurrence were mostly overlapped (Table 2).Values of pH ranged from 4-5, but reached up to 10; the values higher than five were observed for the lime painted hard substrates (rocks or cement curbs) where some populations were growing on.Total irradiance values oscillated from 300-2 740µmol photons/m 2 /s and the relative values (2-100%) showed the occurrence of populations in open to shaded sites.Air relative humidity was usually high, ranging from 60-92%, but always slightly lower than the values observed close to the plant masses (62-94%).
Floristic survey: Nine true branched nostocalean species, pertaining to three families, were found in the 20 samples from 15 distinct sites along the studied area (Table 1).
Occurrence: n=1, SAMA 01.Note: This population corresponds well to the description of Hapalosiphon aureus,  but differs from it by its shorter cells in the branches and by its biotope characteristics.H. aureus is known from aquatic habitats, inhabiting standing water bodies, while the studied population is aerophytic on soil in tropical rainforest.
Occurrence: n=1, SAMA 45.Note: this population resembles Hapalosiphon welwitschii in filament diameter, however heterocytes are rare and, mainly, the branches are short and tapered in this species.
Note: The species is commonly referred in literature with distinct morphotypes.In addition, populations identified as S. minutum are recorded occurring in very distinct climate regions, from temperate (type material) to tropical or even polar areas.Species revision is probably needed.
Note: This is one of the most commonly recorded species of the genus Stigonema and the literature data indicate a cosmopolitan distribution, occurring in very distinct types of habitats.As well as, morphological and metrical ranges of S. ocellatum are quite wide and some authors (Geitler, 1932) suggest that it is a species complex instead a well-defined taxonomic entity.More detailed studies, including molecular approaches, are surely required to a more precise evaluation.
Occurrence: n=1; SAMA 38.Note: This population is close to Stigonema hormoides, however the structure of the filament is distinct between them (cylindrical cells in the studied population and usually nearly spherical in S. hormoides).In addition, metrics are not fully compatible.

Molecular characterization:
Sequencing was tried for all species surveyed, but positive results were obtained only for Hapalosiphon sp.(three clones, 1 390bp) and Stigonema ocellatum (one clone, 1 352bp).GenBank accession codes for the new sequences are GQ354274 and GQ354275, respectively.
Phylogenetic analysis on the basis of Hapalosiphon sp. and S. ocellatum sequences and 19 strain sequences retrieved from Gen-Bank resulted in a consensus tree (Fig. 10) with different bootstrap/probability clade values according to the method employed.
The phylogenetic tree shows that both species grouped with heterocytous cyanobacteria, as expected.Stigonema ocellatum grouped with another strain of the same species (SAG 48.90, GenBank access code AJ544082, 98% sequence similarity) constituting a well-supported cluster (C1) with bootstrap/probability values of 89.2%, 100% and 1 (respectively ML, NJ and BI).The two S. ocellatum strains are close to a Nostoc commune strain.
Hapalosiphon sp. was similar to H. welwitschii (access code AY034793, 98% sequence similarity) and, according to NJ test (77% bootstrap), grouped with three other species of the genus (C2), but this group is not formed in ML and BI tests.However, a well-supported clade constituted by Hapalosiphon species and one strain of Westiellopsis prolifica (C3; cluster bootstrap/probability of 82.7%, 100% and 1 for ML, NJ and BI, respectively) was consistently formed in all tests performed.This Hapalosiphon/Westiellopsis clade is nested in a major group (C4; 77% and 0.96 for NJ and BI, respectively) that includes other morphologically similar genera (Fischerella, Nostochopsis and Chlorogloeopsis) spread in the clade.

DISCUSSION
Results on spatial distribution reveal that some species were well distributed throughout the sampling area (Stigonema ocellatum, 12 different samples, nine distinct sites; S. hormoides, nine samples, six sites), while some are geographically more restricted (Spelaeopogon somierii, Hapalosiphon cf.aureus, Hapalosiphon sp., Sigonema informe and Stigonema sp., all of them occurring in just one sample).The ecological data obtained, however, do not permit to address any conclusion on the species environmental preferences or tolerances.As most of measurements are punctual and too variable (e.g.light) or reasonably homogeneous in rainforest area (e.g.temperature, humidity), those data must be used as a reference for habitat characterization, but not as habitat definition.Nevertheless, the more widespread species occurred under wider ranges of variation for most parameters evaluated, as obviously expected.
All the species with occurrence in two or more samples were found growing on different substrate types (soil and rock) and substrate fidelity was not observed for any species recorded.Even though there are species with restricted dependence or preference on a determined substrate, frequently it is not possible to establish a clear relationship between the species and the substrate, especially for the most widespread and morphologically variable species.Unfortunately, there were not found studies in literature developing this subject in detail and more robust conclusions and comparisons cannot be addressed.
Interestingly, it is the very common joint occurrence of different species of Stigonema in a same sample or within a centimeter spatial scale.In sample SAMA 38, for instance, four species (S. hormoides, S. informe, S. ocellatum and Stigonema sp.) were found growing together.Stigonema hormoides, S. ocellatum and S. turfaceum were all present in the samples SAMA 34, SAMA 35 and SAMA 36.Thus, it is possible to speculate that many species have similar environmental requirements, but a more robust evaluation would only be supported with additional data from populations of the same and other ecologically distinct habitats.
Considering biogeography, only two species found, Stigonema hormoides and S. minutum, were also recorded by Hoffmann (1986) for Luxemburg and this low number of common species among the surveys is probably connected to the distinct climatic areas studied.In comparison with the data presented by Couté et al. (1999), for New Caledonia, five species (Stigonema hormoides, S. minutum, S. ocellatum, S. informe and S. turfaceum) were common to both studies, possibly reflecting environmental similarities among the areas.Sant 'Anna et al. (1991) found, in a small cave, four species (Spelaeopogon sommierii, Stigonema hormoides, S. minutum and S. ocellatum) also recorded in this study and this relatively low similarity is surely due to the size of the area studied.
Although it is possible to reach specific identification for most of populations according to the classical monographs on cyanobacteria, the criteria used for species circumscription seem to be very wide.Many recorded species in literature perfectly correspond to original and further descriptions of such taxon, but more detailed analyses of illustrations reveal distinct filament structures and cell arrangements.However, the exact value of each taxonomic criterion used on this group is still to be proved, probably with the help of additional tools.
Some contributions to the cyanobacterial classification system (Komárek & Anagnostidis 2000, 2005, Hoffmann et al. 2005, Komárek 2006, 2010) emphasize the necessity of also taking into account the environmental and geographical characteristics in the identification process.If on one hand it is a well-supported concept because it probably reflects species requirements, it also creates a practical problem on the other.During this work, these premises were considered to improve the taxonomic evaluation but it is still very difficult to assure that two very similar (or identical) morphotypes are different because the existence of some ecological deviation from the type.The central problem is that it is still not clear if identical genotypes can support distinct environmental pressures without phenotypic changes or in what extension these changes occur.The concept of ubiquitous occurrence of most species has been discarded (Komárek 1994), but few data on genetic variability of true branched complex cyanobacteria have been produced to provide a solid theoretical background about the biogeography of the species.
The results of the molecular assessment obtained from this study suggest that populations identified as same species can be also similar at genetic level even they are from distinct climatic regions.The S. ocellatum SAG 48.90 is a cultivated strain from specimens collected in a Sphagnum peat bog in Germany and the paper of Gugger & Hoffmann (2004) presents a brief morphological account of the specimens.In general, there is good metrical correspondence and the identification as S. ocellatum seems to be correct for all.However, the structure of filaments is not always quite similar (cell size in the main filament, cell size in branches, stratification), but it is difficult to evaluate if the variations are really meaningful.A critical review of different Stigonema ocellatum descriptions and illustrations found in literature (Frémy 1930, Geitler 1932, Skinner & Entwisle 2001, Komárek & Hauer 2011, this paper) corroborates that a very wide morphological range is recorded for the species, even though most of the populations can be correctly identified on the basis of the species original description.The present results from morphological and molecular studies and the available data in literature allow considering that S. ocellatum is probably a complex of species rather than a well delimited species.As it is a very widespread "species", more detailed molecular and morphological studies with populations from distinct geographical and ecological habitats are strongly recommended.
Hapalosiphon welwitschii AY034793, which is the closest sequence to Hapalosiphon sp., is a strain from Australian soils, but, unfortunately, no morphological data could be found for further comparison to the Brazilian material.The phylogenetic tree demonstrates that the clade formed by different species of Hapalosiphon, Westiellopsis, Fischerella and Nostochopsis constitute a mixed and much supported group and that the distinction among the different genera and species needs to be improved.
The molecular approach of the heterocytous true branched cyanobacteria group is still at the very initial stage.The usual techniques for DNA extraction require considerable amount of unicyanobacterial growths (cultures), that have been proved to be very uncommon to be achieved, mainly for species of Stigonema or Hapalosiphon, for instance.They apparently have a special set of environmental requirements to grow and their growth rate is usually low, favoring the establishment of cyanobacteria from other groups prior to the final isolation.New methods to study directly from nature have been developed (e.g.DNA sequencing from one or few cells), but they need to be improved and extended.Some time will be taken until we can access a reasonable amount of data to incorporate to the taxonomical approach.Meanwhile, it is important and relevant to record the morphological and molecular (when possible) diversity of populations with special geographical occurrence and ecology.
The cyanobacterial systematics is still confused and its reorganization on the basis of traditional morphological characters seems to be not adequate (Garcia-Pichel et al. 1996, Palinska et al. 1996).Instead, a polyphasic approach has been employed to better define the taxonomic levels (Zehr et al. 2003, Rajaniemi et al. 2005, Marquardt & Palinska 2007) and the modern taxonomy is becoming a synthetic science that incorporates several tools to achieve a more robust evaluation of the biodiversity.However, mainly in tropical areas, there is an enormous contingent of still not described morphotypes that is extremely relevant for the understanding of evolutionary relationships and producing a more natural classification system for this important group of organisms.

Fig. 10 .
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 *.

TABLE 1
Global position and altitude of the collection sites and their respective samples