Subgeneric diversity of Brasilonema (Cyanobacteria, Scytonemataceae)

Diversidade subgenérica de Brasilonema (Cyanobacteria, Scytonemataceae)

ARTICLES

Subgeneric diversity of Brasilonema (Cyanobacteria, Scytonemataceae)

Diversidade subgenérica de Brasilonema (Cyanobacteria, Scytonemataceae)

Celia Leite Sant'Anna^I,*; Maria Teresa de Paiva Azevedo^I; Marli Fatima Fiore^II; Adriana Sturion Lorenzi^II; Jan Kaštovský^III; Jiří Komárek^III

^IInstituto de Botânica, Núcleo de Pesquisa em Ficologia, Caixa Postal 3005, 01031-970 São Paulo, SP, Brazil

^IIUniversidade de São Paulo, Centro de Energia Nuclear na Agricultura, Av. Centenário 303, 13400-970 Piracicaba, SP, Brazil

^IIIUniversity of South Bohemia, Institute of Botany, Dukelská 135, CZ-37982 Trebon, Czech Republic

ABSTRACT

The recently described scytonematoid cyanobacterial genus Brasilonema is known mainly from tropical and subtropical rain forests (Mata Atlântica) of southeastern Brazil, where it occurs in aerophytic wooden, stony and iron substrates. This genus was defined according to both molecular and morphological criteria. The type species B. bromeliae was described from the specialized habitat: it grows in phytothelmes, epiphytic on both living and died leaves within the rosettes of large bromeliad plants slightly above or in the zone of the water level. The genus Brasilonema is evidently widely distributed in coastal forests of São Paulo State, where it occurs also in remarkable diversity. According to our results, this genus currently comprises seven taxa, which are distinct by different morphology and ecological characteristics.

Key words:Brasilonema, Brazil, new species, subaerophytic habitats

RESUMO

O gênero scytonematóide de Cyanobacteria, Brasilonema, foi descrito recentemente e é conhecido principalmente de florestas tropicais e subtropicais (Mata Atlântica) da região sudeste do Brasil. Ocorre sobre substratos aerofíticos como madeira, pedra e ferro e foi definido de acordo com critérios moleculares e morfológicos. A espécie-tipo, B. bromeliae, foi coletada em um habitat muito específico: epífita em folhas de bromélias, dentro das rosetas, próximo do nível da água, ou ainda em folhas secas destas plantas. O gênero Brasilonema é amplamente distribuído nas florestas costeiras do Estado de São Paulo, onde ocorre em alta diversidade morfológica. De acordo com nossos resultados, este gênero atualmente compreende sete táxons distintos com base nas diferenças morfológicas e características ecológicas.

Palavras chave:Brasilonema, Brasil, espécies novas, habitats aerofíticos

Introduction

The diversity of cyanobacteria in tropical regions, particularly in extreme habitats with specialised environmental conditions, is still insufficiently known. In subaerophytic sites of humid tropical and subtropical forests of southeastern Brazil (Mata Atlântica) it was found a special cyanobacterial type, phenotypically similar to the genus Scytonema, but growing in characteristic erect fascicles and with rare false branching. After combined molecular and phenotype evaluation, this type distinct and different from Scytonema was recognised as a separated generic entity. It was described under the name Brasilonema according to both Bacteriological and Botanical nomenclatoric rules by Fiore et al. (2007).

The type species of Brasilonema, B. bromeliae, has an unusual ecology. It grows in phytothelmes, epiphytic on both living and died leaves inside of the rosettes of large epiphytic species of bromeliads (from the genera Vriesea, Aechmea, Wittrockia, Neoregelia and others), on the limit of the water level. However, the Brasilonema-types also occur on different subaerophytic habitats mainly in humid forests along the coastal mountains in the São Paulo State. They were found on bark of trees and various wetted wooden, stony and iron substrates. The various populations differ by ecology and by morphological deviations, and they represent an interesting cyanobacterial diversified cluster in these tropical and subtropical regions. The various eco and morphotypes should be described as different morphospecies in agreement with the accepted criteria of cyanobacterial taxonomy.

The morphological deviations of these morphotypes were also the reason why Komárek (2003) described one of the species, which belongs evidently to this taxonomic unit, as a member of the similar heterocytous genus Camptylonemopsis (C. sennae Komárek). The similarity to Camptylonemopsis is in the fasciculation of creeping filaments on the substrate and sparse false branching. However, the filaments of Camptylonemopsis are shorter, their creeping by the central parts of filaments is obligatory, and the isopolar filaments have a morphologically middle part distinct from the erect terminal ends (variation in cell form). The akinetes were described in the type species C. lahorense (Ghose) Desikachary (cf. Desikachary 1959, Komárek 2003). The molecular analyses proved the close relationship of C. sennae to the genus Brasilonema (Aguiar et al. 2008).

Another species of this morphotype was characterised by Bourrelly (in Bourrelly & Manguin 1952) from Guadeloupe, originally described as Tolypothrix roberti-lamyi Bourrelly, and later recombined by Komárek (1989) into the genus Schmidleinema. This species has similar initial stages as Schmidleinema, but the fasciculation of filaments, absence of true branching and very rare tolypotrichoid false branching correspond rather to the new Brazilian type. It is different from the diagnoses of Schmidleinema, Tolypothrix, Scytonema and Camptylonemopsis. According to morphological similarity, the identification of Brasilonema populations described under these different generic names must be considered only as preliminary and nomenclatorically invalid in the light of the recent results.

After definition of the genus Brasilonema, seven taxa were recognised. The 16S rRNA gene phylogenetic analysis and the phenotype descriptions are presented in this article.

Material and methods

Twelve samples of the genus Brasilonema collected in São Paulo State, Southeastern Brazil, were used for description of its diversity. Among these samples, eight were collected in different localities from the humid Atlantic Forest (Cardoso Island, Juréia, Paranapiacaba and Campos do Jordão), three in the Botanical Garden of São Paulo, and one in Águas de São Pedro Village. Data of B. octagenarum Aguiar et al. were taken from Aguiar et al. (2008). The samples were collected scraping substrates (stone, concrete, iron and wood) using a spatula. Fresh and preserved materials (dry and in formaldehyde solution) were studied under Zeiss Axioplan-2 optical microscope and photographed with coupled camera. The taxa identification was done based on at least 20 specimens of each sample. The reference strain SPC 951 of the type species B. bromeliae and other strains are cultivated in liquid BG-11 medium (Allen 1968) without inorganic nitrogen source.

The almost complete 16S rRNA gene from the genomic DNA of the B. terrestre CENA116 was amplified by PCR using the oligonucleotide primers 27F1 and 1494Rc (Neilan et al. 1997) corresponding to the Escherichia coli 16S rRNA gene positions 27 to 1494. The PCR amplification and sequencing were carried out as described previously (Fiore et al. 2007). The 16S rRNA sequences of B. bromeliae SPC951, B. sennae CENA114 and B. octagenarum strains UFV-E1 and UFV-OR1 were obtained in earlier study (Fiore et al. 2007, Aguiar et al. 2008). The B. terrestre CENA116 nucleotide sequence obtained in this study has been deposited in GenBank under accession number EF490447. The 16S rRNA gene sequence obtained in this study and reference sequences retrieved from GenBank were aligned, refined and used to generate a phylogenetic tree. The tree was reconstructed using the Neighbor-Joining (NJ) method implemented by the MEGA version 4.0 program package (Tamura et al. 2007) using the Kimura 2-parameter sequence evolution model. The robustness of the tree was estimated by bootstrap percentages using 1,000 replications.

Results and discussion

Six different taxa were recognized within the genus Brasilonema on the basis of Brazilian natural populations and their cultures. One of them (B. terrestre Sant'Anna & Komárek) is presently described also on molecular base (figure 1, table 1). The seventh taxa, B. roberti-lamyi Sant'Anna & Komárek, is a new combination based on Tolypothrix roberti-lamyi Bourrelly from Guadelupe.

Brasilonema bromeliae Fiore et al., J. Phycol. 43:789-798. 2007.

Thallus macroscopic, velvet-like, blackish-green to blackish-violet, densely fasciculated. Filaments 15-17 µm wide, firstly more or less creeping on the substrate, slightly irregularly coiled, cylindrical, later joined in dense erect fascicles. Sheaths firm, cylindrical, slightly lamellated, colorless up to yellowish-brown. Trichomes 14.5-16.8 µm wide, not or very slightly constricted at cross-walls, not attenuated. Cells as long as wide to shorter than wide; cell content slightly granular, greyish-blue, blue-green, olive-green or brownish-violet; vacuole-like structures sometimes in cells of apical (young) parts. Heterocytes solitary, rounded or short cylindrical, 14-19 µm long, 15-16.8 µm wide.

Habitat: in the inner side of bromeliads leaves and on wooden substrates in greenhouse, São Paulo, Brazil.

Brasilonema epidendron Sant'Anna et Komárek, sp. nov.

Typus: Brasilia, Provincia São Paulo, Municipium Campos do Jordão, in sylvis montanosis prope Campos do Jordão (locus classicus), subaerophytice, corticolae lignicolaeque, seape inter lichenes muscisque, XI-2002, M.T.P. Azevedo, J. Komárek et C.L. Sant'Anna, s.n. (icona typica figura nostra 10A-D) Figure 10A-D

 

Thallus cum filametis irregulariter fasciculatis, superficie substrata lignosa repens, interdum stratum leve, griseo-viride formans. Filamenta cylindrica, 20-23 µm lata. Trichoma cylindrica, 17-18µm lata, ad apices non attenuata, ad dissepimenta distincte constricta. Vaginae crassae, lamellosae, superficie regulariter ornamentosae (areolatae), sine colore. Cellulae semper brevior quam latae, contentu plus minusve homogeneo, atro-viridi, breve barriliformes. Heterocystae rotundo, 17-18 µm longis, 3-6 µm lata.

Thallus macroscopic, greyish green, forming irregular fine mats with creeping, partly fasciculated filaments. Filaments 20-23 µm wide. Sheaths thick, lamellated, regularly ornamented, colourless. Trichomes 17-18 µm wide, constricted, not attenuated. Cells short barrel shaped; cell content dark blue-green. Heterocytes rounded, 17-18 µm long, 3-6 µm wide.

Habitat: Subaerophytic, on bark of trees among mosses and lichens.

Locality: Brazil, SP, Campos do Jordão (Horto Florestal), coordinates 22º45'19" S and 45º35'25" W (locus classicus).

Figures 2-8

Figure 9

Brasilonema roberti-lamyi (Bourrelly) Sant'Anna et Komárek, comb. nov.

Basionym: Tolypothrix roberti-lamyi Bourrelly in Bourrelly & Manguin, Algues d'eau douce Guadeloupe. 151. 1952. Figure 11

Thallus macroscopic, forming erect fascicles of ± parallely arranged filaments. Filaments 12-18 µm. Sheaths firm, thick, colourless, finally lamellated. Cells so long as wide or slightly longer or shorter than wide, apical cells barrel shaped to rounded; cell content dark blue-green. Heterocytes discoid to cylindrical. Reproduction by hormogonia with lamellated sheaths (according to Bourrelly & Manguin 1952).

Habitat: Aerophytic, Guadeloupe.

Brasilonema sennae (Komárek) Sant'Anna et Komárek, comb. nov.

Basionym: Camptylonemopsis sennae Komárek, Preslia 75: 225. 2003 Figure 10E-H

Thallus macroscopic, woolly mats, brownish or blackish-green, regular parallely fascicles. Filaments 10-20 µm wide, slightly wavy or coiled, with sparsely tolypotrichoid or scytonematoid branching at free ends. Sheaths thin, firm, later thick, colourless when young, old sheaths lamellated and yellowish-brown. Trichomes 6-12.5 µm wide, not constricted at cross walls, not attenuated. Cells cylindrical, variable in length, particularly in hormogonia and young tri-chomes (from the length 2.2 µm), in old trichomes up to 1.6 longer than wide; cell content blue-green or olive-green. Heterocytes solitary, cylindrical, 6.8-15.4 µm long, 10.2-11.2 µm wide.

Habitat: From wetted subaerophytic stony and iron substrates near springs, several localities are known from the vicinity of Paranapiacaba, SP, Brazil.

Brasilonema terrestre Sant'Anna et Komárek, sp. nov. Typus: Brasilia, Provincia São Paulo, Municipium Cananeia, Insula Cardoso, subaerophytice in substrata saxosa periodice humida, VI-2003, C.L. Sant'Anna, s.n. (SP 399777) (Holopypus SP) Figure 12

Thallus cum filamentis irregulariter in fasciculis consociatis, plus minusve erectis, stratum atrocyaneum macroscopicum formans. Filamenta cylindrica, 12-17 µm lata, dense fasciculata. Trichoma cylindrica, 9-15 µm lata, ad apices leviter attenuata, ad dissepimenta non constricta. Vaginae tenues, firmae, superficie leve granulatae vel lineatae, sine colore. Cellulae plus minusve isodiametricae vel brevior cum latae, praecipue ad apices, contentu griseo-viridi vel atrocyaneae. Heterocystae solitariae, barriliformes vel cylindricae, 6-17 µm longis, 13-14 µm lata.

Thallus macroscopic, dirty-green, forming mats or irregular fasciculated filaments. Filaments 12-17 µm. Sheaths thin, firm, yellow-brown, slightly ornamented at the surface (granular or finely lineated). Trichomes 9-15 µm wide, not constricted, not attenuated. Cells as long as wide, shorter to the ends (up to 1/3 of their width); cell content greyish-green or dirty blue-green. Heterocytes barrel-shaped to cylindrical, 6-17 µm long, 13-14 µm wide.

Habitat: Subaerophytic on concrete.

Localities: Brazil, SP, Cardoso Island, coordinates 25º07'59" S and 47º58' 19" W (locus classicus) and Águas de São Pedro Village.

During the study of subaerophytic cyanobacterial microflora of Atlantic Forest, São Paulo State, SE Brazil, it was recognised a special genus Brasilonema from the family Scytonemataceae. Its separate position from the closest genera (Scytonema, Mastigocladopsis, and Symphyonema) was proved by molecular sequencing as shown in figure 1.

Among the seven Brasilonema taxa, B. bromeliae, B. sennae and B. octagenarum were also characterized based on molecular data (Fiore et al. 2007, Aguiar et al. 2008) and Brasilonema terrestre is presently described. The other morphospecies (B. epidendron, B. ornatum and B. robert-lamyi) differ from all Brasilonema species in dimensions (width of filaments and trichomes), coloration of cells (phycobilin pigment ratio) and sheaths, and ecology (type of substrates) and are presently described. The regular ornamentation of the sheath surface in B. ornatum is specific and unique in cyanobacteria. It was firstly described by Sant'Anna et al. (1983) in an atypical population of Scytonema stuposum (Kützing) Bornet, also from a corticolous habitat (mangrove trees) in the State of São Paulo. The authors mencioned the possibility that the ornamentation could be resultant from a special type of lichenization. This phenomenon is surely interesting and should be explained in future. No hyphae were recognized in the populations presently examined, and the origin (formation) of this sheath ornamentation still remains unclear. Finally, Tolypothrix roberti-lamyi, described from aerophytic habitats in Guadeloupe, belongs evidently also to this genus according to morphological markers, and was taxonomically re-combined. The comparison of phenotype characters of all species is given in table 2.

According to molecular evaluations (Fiore etal. 2007, Aguiar etal. 2008), Brasilonema represents a concise cluster distinctly separated from other groups. The inner diversity is uniform, and the separation in various species was supported according to the formal bacteriological criteria (Wayne et al. 1987, Stackebrand & Goebel 1994) in four species (table 1). The similarity between different morphotypes is usually higher than 96%. However, the different ecotypes are distinguishable according to morphological criteria (table 2) and the occurrence of different stable morphotypes repeats in different localities. It is evidently the same type of diversity, like in other uniform cyanobacterial genera (Microcystis, Planktothrix), where the method of 16S rRNA sequencing cannot separate clearly different stable types. They are distinguished by morphological, ecological, and biochemical characters.

The similarities of 16S rRNA gene sequences among five Brasilonema strains, which characterize four species, are included in table 1. These sequences showed similarities lower than 94% with sequences from Scytonema strains, the closest morphotype at the generic level. However, at the genotype level, Brasilonema strains were close to Symphyonemopsis VAPOR1, which has a true branching morphotype. The phylogenetic analysis also grouped the Brasilonema strains with Symphyonemopsis VAPOR1 (figure 1). The morphology of the strain designated "Symphyonemopsis VAPOR 1" isolated from Spain is unknown and must be explained in future. Thus, further sampling of Symphyonemopsis taxa is required before the phylogenetic position of this group can be solved. However, the relation of this genus with Brasilonema is improbable. The molecular data obtained in this study corroborate earlier results (Fiore et al. 2007, Aguiar et al. 2008) and reinforce the separate position of Brasilonema strains at the generic level.

Acknowledgement - This study was supported by the Brazilian projects Fapesp 2007/07075-5, Fapesp 2002/ 09349-1, CNPq 308086/2004-0 and of the Grant Agency of the Czech Republic no. A6000050704, AV0Z60050516, 206/08/0318 and MSM 6007665801. C.L. Sant'Anna (308086/2004-0) and M.F. Fiore (308299/2009-4) would also like to thank CNPq for the research fellowships. The authors are indebted to Keith Edwards for language correction.

References

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(received: December 09, 2009; accepted: January 20, 2011)

Publication Dates

• Publication in this collection
  11 Apr 2011
• Date of issue
  Mar 2011

History

• Received
  09 Dec 2009
• Accepted
  20 Jan 2011