Thermal hot springs are unusual environments, distributed in widely separated geographical areas, characterized by different physical and chemical features of the waters that severely limit the survival of photoautotrophic organisms. In the acidic, high temperature conditions around hot springs (pH < 3) the presence of photoautotrophic organisms is restricted to the Cyanidiales, a group of asexual unicellular red algae (Ciniglia et al
), but in hot springs with alkaline and neutral pH, cyanobacteria are dominant. In the latter environments, cyanobacterial diversity is rather low, since most species are not able to tolerate temperatures higher than 50–60°C (Edwards et al
; Miller & Castenholz, 2000
; Balme et al
), apart from some unicellular Synechococcus
spp. that can live at 73–74°C (Castenholz, 1969
; Miller & Castenholz, 2000
; Steunou et al
). Moreover, most thermotolerant cyanobacteria from neutral and alkaline pH waters are excluded by high (>1 mg l−1
) soluble sulphide concentrations (Castenholz, 1976
The specificity of these thermal environments makes the cyanobacteria living there endemic (Castenholz, 1996
; Papke et al
; McGregor & Rasmussen, 2008
). Therefore, apart from a few cosmopolitan thermophilic cyanobacterial species (e.g. Mastigocladus laminosus
Cohn) (Castenholz, 1996
; Miller et al.
), most cyanobacteria described from this environment are new operational taxonomic units (OTUs) (Ward et al
; Taton et al
; McGregor & Rasmussen, 2008
During recent studies to evaluate the biodiversity of the cyanobacterial mats of the Euganean Thermal Springs (Padua, Italy), an Oscillatorialean Leptolyngbya-like cyanobacterium was found in several tanks with mud temperatures ranging from 26 to 59°C. Denaturing gradient gel electrophoresis (DGGE) analyses, carried out on the cyanobacterial mats of different spas of the Euganean Thermal District, revealed that this is one of the commonest organisms.
To ascertain the taxonomic position of this organism, a study was undertaken to compare its morphological, ultrastructural and biochemical features with those of strain PCC 8501, presently listed as Geitlerinema
sp. (= Phormidium laminosum
Gomont ex Gomont strain OH-1-p Cl 1 = Leptolyngbya laminosa
(Gomont) Anagnostidis et Komárek 1988), isolated from Hunter's Hot Spring, Oregon, USA. This strain was previously identified by Castenholz (1970
) as the same taxon as CCMEE 5345, isolated from the Abano Terme (Padua) in 1969.
Accordingly, the genetic similarity and phylogenetic relationships of the Euganean strain (ETS-08) and the Oregon strain (PCC 8501) were investigated by sequencing the rbcL gene, encoding the large subunit of the ribulose 1,5-bisphosphate carboxylase-oxygenase, the rpoC1 gene, encoding the γ subunit of RNA polymerase, the 16S rRNA gene and the 16S–23S internal transcribed spacer (ITS) region. Based on our results, ETS-08 could either be a very similar sister species to PCC 8501 or an ecotype of the same species in a currently undefined clade within the paraphyletic Leptolyngbya group.
The morphological and ultrastructural features observed in the Euganean strain, ETS-08, and the Oregon strain, PCC 8501, would place them in the family Pseudanabaenaceae, sub-family Leptolyngbyoideae, genus Leptolyngbya sensu
Komárek & Anagnostidis (2005
). These features are long, thin, slightly constricted, cylindrical trichomes, surrounded by individual thin sheaths, phenotypic traits common to several Leptolyngbya
species from a variety of aquatic and sub-aerial environments (Albertano & Kovacik, 1994
; Bellezza et al.
; Asencio & Aboal, 2004
; Casamatta et al.
; Komárek & Anagnostidis, 2005
; Li & Brand, 2007
). Among Leptolyngbya
species typical of thermal waters with <1 µm wide trichomes, ETS-08 and PCC 8501 are quite similar to L. granulifera
(Copeland) Anagnostidis and L. laminosa
(Gomont) Anagnostidis et Komárek, but differ in having a round apical cell and lacking granules on either side of the cross walls (Komárek & Anagnostidis, 2005
), and to L. thermalis
Anagnostidis in Anagnostidis & Komárek (1988
), which lacks cell constrictions. There is also low sequence identity (88.44%, 71 substitutions) with the 614 bp of the 16S rRNA gene sequence of a L. thermalis
strain from extreme seawater environments of Mexico (GenBank accession no. AF410932) and ETS-08.
The 16S rRNA phylogenetic analysis confirms the results of the most recent phylogenetic analyses on Leptolyngbya
(Casamatta et al.
; Yoshida et al
; Bruno et al
). Furthermore, most of the sequences reported in GenBank for strains identified as Leptolyngbya
species have not been accompanied by morphological, ultrastructural, biochemical or eco-physiological data (Komárek & Anagnostidis, 2005
; McGregor, 2007
). In addition no sequences of L. granulifera
are available for comparison and the partial 16S rRNA gene sequence (628 bp, GenBank accession no. EU057151) of the sole strain identified as L. laminosa
(Sorokovikova & Belykh, pers. comm), isolated from the hot springs of Baikal Rift Zone, showed very low sequence identity (89.33%, 67 substitutions) with the corresponding sequence of ETS-08.
Thus, the inclusion of ETS-08 and PCC 8501 in Leptolyngbya
is unlikely. Similarly the inclusion of ETS-08 in the genus Geitlerinema
, sub-family Pseudanabaenoideae, is unconvincing given its slow motility in natural samples and on agar. This gliding behaviour differs from that described for PCC 8501 (http://www.pasteur.fr/recherche/banques/PCC/docs/pcc8501.htm
). However, it should be noted that reduction or lack of intense motility, observed in ETS-08 and PCC 8501 (Castenholz, 1970
), might be a loss occurring during growth in liquid medium as reported for Pseudanabaena
, field material or freshly isolated cultures of which show active gliding (Wilmotte & Herdman, 2001
; Castenholz et al
). The nine sequences available for marine Geitlerinema
strains of clade C had showed 87.8–89.0% sequence identities and are therefore evolutionarily distant, assuming a 95% threshold for the inclusion in the same genus (Ludwig et al.
; Řeháková et al.
The <95% sequence similarity with representatives of Leptolyngbya in clade B, Geitlerinema in clade C, and Halomicronema in clade D, will allow us to consider clade A, containing ETS-08, PCC 8501, and five other unidentified thermophilic filamentous cyanobacteria, as a new OTU within the family Pseudanabaenaceae.
The six thermal strains, Ob05, from Luzon Island in the Philippines (Lacap et al.
), PCC 8501 from Hunter's Hot Spring, Oregon, USA, OS Type I, from Octopus Spring in Yellowstone National Park (Ward et al.
), tBTRCCn 408, tBTRCCn 102 and tBTRCCn 302 from Zerka Mà in Jordan (Ionescu et al.
), grouped in the same clade as ETS-08, suggesting that they could belong to the same genus.
None of the representatives of the other genera in the Pseudanabaenaceae showed >95% sequence identity with our strain. Taking into consideration that the systematics of most cyanobacteria characterized by very thin trichomes still needs a full revision (Johansen et al
), the present work contributes to the definition of a separate OTU, like the recent genera Halomicronema
distinguished on the basis of 16S rRNA sequence identity of <92% and 93–94%, respectively (Abed et al
; Siegesmund et al
Taking 97.5% similarity for 16S rRNA gene sequence identity as the criterion for collapsing strains into a single species (Stackebrandt & Goebel, 1994
; Casamatta et al
; Palińska & Marquardt, 2008
), the high identity (99.17%) between ETS-08 and PCC 8501 for this locus suggests that the two entities could be ecotypes, as reported for different isolates of Mastigocladus laminosus
(Miller et al
). That is they are ‘genetically determined phenotypes of a species that are found as local variants associated with certain ecological conditions’ living in geographically distant areas (Ward et al
). In our case, however, the 70.9% sequence identity of the 16S–23S region suggests greater intraspecific variation between the strains, indicating that ITS is more useful for closely related strains of different geographic distribution (Boyer et al
; Bruno et al
). By combining ecological and evolutionary patterns, it was shown that 16S rRNA gene sequences are too conserved to detect all ecologically specialized populations and that ITS analysis provided evidence of localized geographical patterning, e.g. nine ITS variants within a single 16S rRNA genotype (Ward et al
). However, because of the possible presence of multiple rRNA operons in some cyanobacteria there are potential pitfalls that should be considered when using ITS to infer phylogeny (Boyer et al
). Nevertheless, the agarose gel-electrophoresis analyses of the amplified PCR products from the current study (both 16S rRNA and ITS) consistently produced a single band from the two strains, indicating the presence of a single operon (data not shown).
The results of the 16S–23S ITS sequencing, showing differences between the two strains, support the recognition of different lineages within the clade. This was also found for some Leptolyngbya
species in which ITS sequencing agreed with the 16S rRNA results and allowed discrimination at inter- and intraspecific levels. It also showed the relatedness of Leptolyngbya
strains from hypogean environments with strains from subaerophytic and geothermal environments (Bruno et al.
The rbcL phylogenetic analyses show that L. boryana, which is the type species of Leptolyngbya, is a sister taxon to the clade including ETS-08 and PCC 8501. However the low number of Leptolyngbya rbcL gene sequences in GenBank limits further interpretation of this result.
However, we have found good correlation between ITS region and rpo
C1 phylogenetic analyses that could support the 16S rRNA results. Similar data have been obtained with M. aeruginosa
strains by Yoshida et al
) using a polyphasic approach, combining phenotypic and genetic characterization. This approach is effective for defining distinct lineages and discriminating the complexity of intra-specific populations. Nevertheless, 90.1% rpo
C1 sequence identity between ETS-08 and PCC 8501 does not prove unambiguously either that they belong to the same species or that they should be separated as two species. According to Wilson et al
), the rpo
C1 locus was ineffective for distinguishing among several strains of Cylindrospermopsis raciborskii
with sequence identities from 99 to 100%, while Toledo & Palenik (1997
) recognized different strains of Synechococcus
sp. based on 83% rpo
C1 sequence identity.
ETS-08 might be one of the 80 cyanobacteria reported from the Euganean thermal environment by Trevisan (1870
). Unfortunately, it has not been possible to find dry specimens or isolates for comparison and it is also difficult to establish the synonymy of several Oscillatorialean taxa reported in Komárek & Anagnostidis (2005
Interestingly, ETS-08 differs from two other filamentous cyanobacteria (ItalyCy04 and ItalyCy05) recently found in the Euganean Thermal District and identified by partial 16S rRNA gene sequences (AF505886 and AF505887) (Papke et al.
). There is about 94.3% sequence identity between them and our strain. This suggests that the cyanobacterial composition of phototrophic mats in the rather unusual environment of the Euganean Thermal District is variable, depending on the physico-chemical features of the different spa waters. In fact, recent surveys carried out on ninety spas suggest that the cyanobacterial diversity might be related to the thermal mud processing, which is carried out in different maturation tanks using thermal waters at various temperatures. As expected, while cyanobacterial diversity can range from 2 to 12 taxa between 26 and 40°C, only Leptolyngbya
(ETS-08) and Spirulina
(ETS-02) are able to thrive above 50–55°C (unpublished data).
Based on a polyphasic approach combining morphological, ultrastructural and genetic tools, we have characterized and established the taxonomic position of the Euganean strain, ETS-08, which, together with six other isolates, represents a new OTU in the family Pseudanabaenaceae. In order to erect a new systematic group to which these strains belong, further studies involving comparison of phenotypic features as well as sequences from a wider number of strains from the Leptolyngbyoideae and Pseudeanabaenoideae will be necessary. It will also be necessary to determine what is the representative generitype for any new genus.