Cyanobacteria 16 S

Molecular Phylogenetics and Evolution 105 (2016) 15–35
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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev
Detection of the new cosmopolitan genus Thermoleptolyngbya

(Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and
16S–23S ITS region
Katia Sciuto ⇑, Isabella Moro
Department of Biology, University of Padova, via U. Bassi 58/B, 35131 Padova, Italy
a r t i c l e i n f o a b s t r a c t
Article history: Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal
Received 24 March 2016 springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sam-
Revised 19 July 2016 pled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be
Accepted 15 August 2016
polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S–
Available online 18 August 2016
23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the
This paper is dedicated to Prof. Patrizia separation of new genera from Leptolyngbya and to the description of new species inside this genus
Albertano who devoted a great part of her and in other related groups.
research career to the study of In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S–23S ITS region
Leptolyngbyaceae. were performed alongside 16S rRNA and 16S–23S ITS secondary structure analyses on cyanobacteria of
the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal
Keywords: springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions
16S rRNA
showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct
16S–23S ITS
from Leptolyngbya. The 16S–23S ITS secondary structure results supported the separation of this cluster.
Cryptic taxa
Secondary structures
A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were
Thermal environments described inside this new taxon: T. albertanoae and T. oregonensis.
Thermoleptolyngbya Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Antarctica and thermal springs (e.g., Anagnostidis and Komárek,

1988; Albertano and Kovacik, 1994; Komárek and Anagnostidis,
Cyanobacteria are complex prokaryotes that are among the 2005). In thermal environments, where cyanobacteria generally
most widespread organisms due to a series of features and adap- represent a conspicuous part of microbial communities, strains
tive strategies. While some cyanobacteria are strictly related to morphologically ascribable to the genus Leptolyngbya are often
given ecological niches, other taxa are found in a wide range of the most abundant (e.g., McGregor and Rasmussen, 2008; Coman
environments (Sciuto and Moro, 2015). Among the most common et al., 2013; Mackenzie et al., 2013; Amarouche-Yala et al., 2014).
taxa is the genus Leptolyngbya Anagnostidis and Komárek, which Identification of Leptolyngbya-like strains based solely on mor-
includes freshwater, halophilic and marine, epiphytic and endo- phology is difficult due to the scarcity of diagnostic features and
gloeic, aerophytic and subaerophytic, and hypogean species and the overlapping characters for many taxa attributable to this genus
which is found in vastly different extreme environments, such as or to related groups. Indeed, the current morphological definition
of Leptolyngbya is very simple: non-heterocytous thin cyanobacte-
Abbreviations: BI, Bayesian inference; BIC, Bayesian information criterion; BT, ria with less than 3.5 lm wide trichomes, each one surrounded by
bootstrap; INSD, international nucleotide sequence database; 16S–23S ITS, internal an individual sheath, and showing parietal thylakoids inside the
transcribed spacer between 16S rRNA and 23S rRNA ribosomal genes; LM, light cells (Komárek and Anagnostidis, 2005; Komárek, 2007; Johansen
microscopy; ML, maximum likelihood; MP, maximum parsimony; MCMC, Markov
chain Monte Carlo; NJ, neighbor joining; PP, posterior probabilities; rbcL, gene
et al., 2011). Several phylogenetic studies based on the 16S rRNA
coding for the large subunit of RuBisCo; rpoC1, gene coding for the c subunit of the gene have demonstrated that the genus Leptolyngbya is a poly-
cyanobacterial RNA polymerase core; SEM, scanning electron microscopy; TEM, phyletic group that requires revision (e.g., Komárek and
transmission electron microscopy. Anagnostidis, 2005; Taton et al., 2006; Moro et al., 2010; Sciuto
⇑ Corresponding author.
et al., 2011; Johansen et al., 2011).
E-mail address: [email protected] (K. Sciuto).
http://dx.doi.org/10.1016/j.ympev.2016.08.010
1055-7903/Ó 2016 Elsevier Inc. All rights reserved.
16 K. Sciuto, I. Moro / Molecular Phylogenetics and Evolution 105 (2016) 15–35
The 16S rRNA gene is the most commonly used marker in 2000) and the second used the primer pair 1070F (Ferris et al.,
cyanobacterial systematics and has been proven to be an invalu- 1996)-23R (Papke et al., 2003). In both cases, the PCR protocols
able tool to confirm/reject morphology-based taxa and to detect were performed in 50 ll aliquots with the Taq DNA polymerase
new clades. However, some studies have noted that its resolution (Fisher Molecular Biology, Trevose, PA, USA) according to the man-
power at the species level can vary in different lineages and is often ufacturer’s recommendations. The thermocycling conditions were
limited (e.g., Engene et al., 2010; Eckert et al., 2015). Recently, as follows: 1 cycle at 95 °C for 5 min; 35 cycles of 95 °C for 45 s,
another genomic region has been re-evaluated in cyanobacterial 52 °C for 45 s, and 72 °C for 50 s; and a final step at 72 °C for
systematics through the analysis of secondary structures: the 8 min. Approximately 80 ng of template DNA was used per
16S–23S ITS region (Johansen et al., 2011). The joining of 16S rRNA reaction.
gene phylogenies with 16S–23S ITS secondary structure analysis If a single band was observed on the electrophoretic gel, the
has proven to be a very useful method to detect new cryptic spe- corresponding amplification product was purified with the QIA-
cies and to circumscribe new cyanobacterial genera. In the last quick PCR Purification kit (Qiagen, Hilden, Düsseldorf, Germany)
few years, new genera were separated from Leptolyngbya and prior to sequencing. In the presence of non-specific products, the
new species were described both inside this genus and in other band corresponding to the expected amplification product was
genera of the family Leptolyngbyaceae using this molecular excised from the electrophoretic gel and purified with the DNA
approach (e.g., Johansen et al., 2011; Perkerson et al., 2011; Gel Extraction Kit (Millipore, Billerica, MA, USA).
Zammit et al., 2012; Osorio-Santos et al., 2014; Dvořák et al., DNA sequencing was performed at the BMR Genomics Sequenc-
2015; Vaz et al., 2015; Miscoe et al., 2016). ing Service (University of Padova) with the same primer pairs used
Here, we give taxonomic recognition to a monophyletic lineage in the amplification reactions.
that has been highlighted in previous phylogenetic studies (Moro The final consensus sequences were assembled using the Seq-
et al., 2010; Coman et al., 2013; Mackenzie et al., 2013; Peng Man II program from the Lasergene software package (DNAStarÓ,
et al., 2013; Andreote et al., 2014; Gaisin et al., 2015) by erecting Madison, WI, USA) and then compared with the sequences avail-
the new genus Thermoleptolyngbya, which is distinct from Leptolyn- able at the INSD using the BLAST tool (Altschul et al., 1990).
gbya sensu stricto, and by describing two species belonging to this
new taxon: the generitype Thermoleptolyngbya albertanoae Sciuto
& Moro and Thermoleptolyngbya oregonensis Sciuto & Moro. 2.3. Sequence analysis and dataset construction
The obtained 16S–23S ITS sequences were checked for the pres-
2. Materials and methods
ence of tRNAs with tRNAscan-SE (Lowe and Eddy, 1997; Schattner
et al., 2005). The conserved domains (D1-D10 , D2, D3, boxA, and
2.1. Information on cyanobacterial strains ETS-08 and PCC 8501
D4) and the variable regions (V2, boxB, and V3) of this locus were
detected following Iteman et al. (2000).
Strain ETS-08 was collected from thermal mud in the Euganean
Separate datasets were constructed for the 16S rRNA gene and
Thermal District, Montegrotto Terme, Padova, Italy in February,
16S–23S ITS using the sequences of strains ETS-08 and PCC 8501,
2004. A detailed description of the collection site, including the
sequences found through a BLAST search, and other published
chemical composition of the water and physical data, was reported
sequences of strains belonging to taxa considered relevant to the
in Moro et al. (2010). Strain ETS-08 has been maintained in culture
present study, especially members of the genus Leptolyngbya sensu
with BG11 liquid medium (Rippka et al., 1979) at 30 °C under a
stricto and other genera belonging to the family Leptolyngbyaceae.
light intensity of 35 lmol photons m2 s1 with a 12 h:12 h
The 16S rRNA and 16S–23S ITS multiple alignments were gen-
light-dark cycle.
erated with MUSCLE (Edgar, 2004).
Strain PCC 8501 was isolated from Hunter’s Hot Spring (now
For each 16S rRNA sequence retrieved from the INSD, the posi-
renamed ‘‘Geyser Hot Springs”), Lake County, Oregon, USA, in
tion of the variable domains H15, H17, H21, H22–H23, H41, and
1966 and was originally identified as Phormidium laminosum strain
H44 was detected by comparison with the published sequence of
OH-1-p by Castenholz (1970). This strain is currently named Geit-
Nostoc commune WY1KK1 (EU586733), whose secondary structure
lerinema sp. PCC 8501 at the Cyanobacteria Collection of Institut
is depicted in its entirety by Řeháková et al. (2014).
Pasteur (PCC); however its ascription to the genus Geitlerinema
The 16S–23S ITS sequences retrieved from the INSD were
(Anagnostidis and Komárek) Anagnostidis is clearly incorrect and
checked for the presence of tRNAs, conserved domains, and vari-
misleading. Indeed, one of the main diacritical and obligatory fea-
able regions as described above.
tures that characterize the genus Geitlerinema is the absence of
The 16S rRNA dataset included 74 sequences, and the final
sheath (Anagnostidis, 1989; Komárek and Anagnostidis, 2005)
alignment contained 1025 aligned positions. A genomic region
and this unquestionably distinguishes the members of this genus
from 9 conserved nucleotides upstream of the H15 domain begin-
from strain PCC 8501, which has a thin sheath surrounding the fil-
ning (GGGGAATTT) to a conserved sequence between the H41 and
aments (e.g., Moro et al., 2010).
H44 domains (CGTTCCCGGGCCTTG) was considered in the analy-
ses. The H44 domain was excluded since it was not present in
2.2. DNA extraction, amplification, and sequencing the shorter sequences. Sequences of strains belonging to the genus
Gloeobacter were used to root the 16S rRNA phylogenetic
A culture aliquot of strain ETS-08 was ground in a mortar with reconstruction.
liquid nitrogen and genomic DNA was extracted using the Genomic For the 16S–23S ITS, 21 sequences were included in the dataset
DNA Purification Kit (Fermentas International, Burlington, ON, for a final alignment of 561 positions. A genomic region from the
Canada) according to the manufacturer’s instructions. For strain beginning of the D1 domain (ARGGRGACC) to the end of tRNAAla
PCC 8501, previously extracted and stored genomic DNA was used. gene (ACCTCCAC) was considered in the analyses. Sequences of
The 16S–23S ITS was amplified to verify the identity of strain representatives of the genera Haloleptolyngbya and Nodosilinea
ETS-08 kept in laboratory culture conditions and to confirm the (inside the family Leptolyngbyaceae) were used to root the trees
previously obtained sequences for both strains. Two different due to the higher variability of this molecular locus and based on
amplification reactions were performed for each strain; the first the results obtained with the 16S rRNA phylogenetic
amplification used the primer pair 322F-340R (Iteman et al., reconstruction.
K. Sciuto, I. Moro / Molecular Phylogenetics and Evolution 105 (2016) 15–35 17
2.4. Molecular and phylogenetic analyses default conditions. In the case of the 16S rRNA gene, when neces-
sary, the structures obtained with RNAstructure were constrained
Phylogenetic analyses were performed with MEGA version 5 in some parts to bring them in line with published structures
(Tamura et al., 2011) by applying the NJ and MP methods. The (Cannone et al., 2002; Řeháková et al., 2014).
16S rRNA dataset had 248 parsimony-informative sites in 1025 The obtained secondary structures were visualized with VARNA
aligned positions, and the 16S–23S ITS dataset had 139 version 3.9 (Darty et al., 2009) and then made suitable for publica-
parsimony-informative sites in 561 aligned positions. tion with CorelDRAW X4.
ML analyses were performed with PHYML version 3.065
(Guindon and Gascuel, 2003) and BI analyses were carried out 2.6. Microscopy analyses of strains ETS-08 and PCC 8501
using MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003).
For ML and BI analyses, the models that best fit our data were Strains ETS-08 and PCC 8501 were examined with a Leica 5000
found using jModelTest version 0.1.1 (Posada and Crandall, 1998; microscope (Wetzlar, Germany), equipped with a digital image
Posada and Buckley, 2004; Posada, 2008) under the BIC criterion acquisition system.
(Schwarz, 1978). For the 16S rRNA dataset, the model that best fit Specimens of strain ETS-08 and PCC 8501, earlier set up for a
the data was TPM3 + I + G and the following parameters were imple- previous paper (Moro et al., 2010), were observed with a scanning
mented: substitution rate matrix with A-C substitutions = 0.6787, electron microscope Cambridge Stereoscan 260 (Cambridge, UK),
A-G = 2.3213, A-T = 1.0000, C-G = 0.6787, C-T = 2.3213, and G- operating at 25 kV, and with a transmission electron microscope
T = 1.0000; proportion of sites assumed to be invariable = 0.4970 FEI Tecnai G2 (Hillsboro, Oregon), operating at 100 kV.
and gamma shape = 0.4610. For the 16S–23S ITS dataset, the model
that best fit the data was TPM2uf + G and the following parameters
2.7. Confocal laser microscopy analyses of strains ETS-08
were implemented: nucleotide frequencies as freqA = 0.2643,
freqC = 0.1750, freqG = 0.2490, freqT = 0.3116; substitution rate
Strain ETS-08 was examined with a Leica SP5 confocal laser
matrix with A-C substitutions = 6.4029, A-G = 18.6309, A-
scanner system (Wetzlar, Germany) mounted on an inverted
T = 6.4029, C-G = 1.0000, C-T = 18.6309, G-T = 1.0000; proportion
microscope. The excitation laser wavelengths were 406 and
of sites assumed to be invariable = 0; gamma shape = 0.2230.
488 nm for the blue region, 515 and 543 nm for the green region,
The nexus files for the BI analyses were generated through Mes-
and 635 nm for the red region. The fluorescence emission was col-
quite version 2.71 (Maddison and Maddison, 2009). The analyses
lected at 400–480 nm in the blue, 490–530 nm in the green, and
included two separate concurrent MCMC runs, each composed of
590–800 nm in the red-far red regions.
four chains (three heated and one cold). Each Markov chain ran
for 10 106 generations for the 16S rRNA dataset and 2 106 gen-
erations for the 16S–23S ITS dataset, with trees sampled every 100 3. Results
generations. At the end of each run, we considered the sampling of
the posterior distribution to be adequate if the average standard 3.1. Molecular and phylogenetic results
deviation of the split frequencies was 60.01. The first 25,000 trees
for the 16S rRNA locus and 5000 trees for the 16S–23S ITS were The amplification and sequencing of the 16S–23S ITS verified
discarded as burn-in as determined by the stationarity of the lnL that strain ETS-08 had not been substituted while it was main-
assessed using Tracer version 1.5 (Rambaut and Drummond, tained in culture and confirmed the sequences previously obtained
2007). Consensus topologies and PP values were calculated from for strain ETS-08 and strain PCC 8501, with both the used primer
the remaining trees. pairs. In all cases, a single band of the expected size was visualized
Nonparametric BT re-sampling (Felsenstein, 1985) was used to on the electrophoretic gel with the exception of the amplification
test the robustness of the NJ, MP, and ML tree topologies (1000 BT reaction performed with the primer pair 322F-340R for strain
replicates). ETS-08, for which a lighter band was visible in addition to the
Additional NJ, MP, and ML phylogenetic analyses based on the expected amplicon. This was, subsequently, revealed to be a spuri-
16S rRNA gene were performed with MEGA version 5 to investigate ous product. Only ribosomal operons with both the tRNAIle and
more Leptolyngbya-like strains from thermal environments, but tRNAAla genes inside the ITS region were recovered from strains
shorter sequences were included in the alignment (431 aligned ETS-08 and PCC 8501.
positions); the considered genomic region ranged from 9 con- The phylogenetic reconstruction based on the 16S rRNA gene
served nucleotides upstream of the H15 domain beginning resolved thirteen well-supported clades (Fig. 1), nine of which cor-
(GGGGAATTT) to a more variable, but alignable, sequence down- responded to previously described genera: Oculatella Zammit et al.,
stream of the H22–H23 domain (CCCHRTWSTYY). The ‘‘shorter” Phormidesmis Turicchia et al., Alkalinema Vaz et al., Plectolyngbya
16S rRNA dataset had 104 parsimony-informative sites in 431 Taton et al., Leptolyngbya Anagnostidis & Komárek (hereafter
aligned positions and the model of evolution used for the ML anal- referred to as Leptolyngbya sensu stricto), Pantanalinema Vaz et al.,
ysis, found by ModelTest implemented in MEGA, under the BIC cri- Halomicronema Abed et al., Nodosilinea Perkerson & Casamatta,
terion, was K2 + G. and Gloeobacter Rippka et al. (this last genus was the outgroup of
All tree topologies were visualized with NJplot (Perrière and the tree). The genera Planktolyngbya Anagnostidis & Komárek, Scy-
Gouy, 1996) and drawn using CorelDRAW X4 (Corel Corporation, tolyngbya Song et al., and Haloleptolyngbya Dadheech et al. were
Ottawa, ON, Canada). also present, each one represented by a sequence. The remaining
clades did not coincide with previously described taxa, and of these
2.5. Prediction and drawing of secondary structures three were named clade A, clade B, and clade C.
The last undescribed clade included 16 sequences of both
The hypothetical secondary structures of the 16S rRNA and uncultured and cultivated cyanobacteria that were all sampled
16S–23S ITS were estimated as reported in Johansen et al. from thermal environments (Table 1). Although most of the
(2011); i.e. each structure was individually folded after its position strains belonging to this cluster had been originally attributed
in the sequence was identified. to the genus Leptolyngbya, the group was placed far from the
Secondary structures were determined with the program genus Leptolyngbya sensu stricto. Therefore, this monophyletic lin-
RNAstructure version 5.6 (Reuter and Mathews, 2010) using eage represented a new cyanobacterial genus and was named
Fig. 1. Phylogenetic reconstruction of Leptolyngbyaceae based on 16S rRNA gene analyses. The NJ topology is depicted and the numbers associated with nodes indicate
support values for NJ, MP, ML, and BI analyses, respectively. Only bootstrap supports P50% and posterior probabilities P0.70 are reported. Values for nodes that obtained
support in only one of the performed phylogenetic analyses were omitted. Sequences of strains common to the 16S–23S ITS phylogenetic reconstruction are in boldface font.
Genera, including Thermoleptolyngbya gen. nov., and clades A, B, C are indicated by vertical bars. Horizontal bar represents expected number of nucleotide substitutions per
site.
Thermoleptolyngbya gen. nov. The percent identities of the 16S Table 1 and only 16S rRNA percent identities between representa-
rRNA gene sequences were calculated for the Thermoleptolyngbya tives of Thermoleptolyngbya and the focus taxa are reported in
clade and other focus taxa in the 16S rRNA tree (Table S1). For Table 2. The 16S rRNA within-clade percent identities ranged from
clarity, only 16S rRNA percent identities between strain ETS-08 96.03 to 99.90% inside the Thermoleptolyngbya clade, 98.77–
and the other members of Thermoleptolyngbya are reported in 99.90% inside the Leptolyngbya sensu stricto clade, and 96.83–
Table 1
Strains attributable to the new genus based on the 16S rRNA analyses. For each strain, the percent identity with the sequence of strain ETS-08, the collection site, and literature
reference are reported. Near each percent identity, the alignment length between that comparison sequence and the sequence of ETS-08 is indicated inside brackets, with a
number followed by ‘‘aln”. Strains are listed in order from that with the highest to that with the lowest sequence identity with ETS-08. A dash denotes that no data are available.
Strain Sequence identity with Isolated from References

ETS-08
ETS-08 100% (1437 aln) Euganean thermal District, Padova, Italy Moro et al. (2010)
Uncultured cyanobacterium clone ST7S_1CY 99.67% (1203 aln) Meskoutine hot spring, Guelma, Algeria Amarouche-Yala et al. (2014)
PCC 8501 99.24% (1438 aln) Hunter’s Hot Spring, Oregon, USA Castenholz (1970), Moro et al.
(2010)
Leptolyngbya sp. O-77 98.72% (1403 aln) Hot spring in Kumamoto, Aso-Kuju National Park, Japan Nakamori et al. (2014)
Uncultured Leptolyngbya sp. clone 98.71% (1399 aln) Tsenher hot spring, Mongolia –
Tsenher12otu4-2
Uncultured Leptolyngbya sp. clone 98.68% (1361 aln) Alla hot spring, Buryatia, Russia Gaisin et al. (2015)
Alla11otu1-1
Alla11otu1-4
Uncultured Leptolyngbya sp. clone 98.58% (1407 aln) Tsenher hot spring, Mongolia –
Tsenher12otu4-1
Uncultured cyanobacterium clone OB05 98.58% (1406 aln) Wonder Lake geothermal location, Laguna, Luzon Island, Lacap et al. (2007)
the Philippines
Alla11otu1-5
Uncultured Leptolyngbya sp. 98.13% (1337 aln) Tapovan hot spring, Ladakh, India –
TPB_GMAT_SPRING12_32
Leptolyngbya sp. NgrLPT40 97.74% (1418 aln) Nigrita hot spring, Serres, Macedonia, Greece Bravakos et al. (2016)
Thermophilic cyanobacterium tBTRCCn 408 97.42% (1357 aln) Zerka Ma’in thermal springs - Jordan Oren et al. (2009)
Uncultured cyanobacterium OS Type I 97.42% (1278 aln) Octopus spring, Yellowstone National Park, USA Weller et al. (1992)
Uncultured cyanobacterium clone 9B-56 97.37% (1371 aln) Gumingquan hot spring, Rehai geothermal area, Peng et al. (2013)
Tengchong, China
Leptolyngbya sp. CENA538 97.26% (1388 aln) Saline-alkaline lake, Nhecolândia, Pantanal wetland, Brazil Andreote et al. (2014)
Table 2
16S rRNA percent identities (based on the alignment of 1025 positions used for phylogenetic analyses) among sequences of key-strains belonging to the genus Thermoleptolyngbya
(ETS-08, PCC8501, CENA 538) and sequences of representative strains belonging to the genus Leptolyngbya sensu stricto or to focus taxa in the 16S rRNA phylogenetic
reconstruction.
Strain Sequence identity with ETS- Sequence identity with PCC Sequence identity with CENA
08 8501 538
Leptolyngbya sp. PltLPT2 94.95% 94.75% 94.46%
Clade A Leptolyngbya sp. 1T12c 93.57% 93.47% 93.27%
Leptolyngbya frigida ANT.LMA.1 93.27% 93.27% 92.78%
Leptolyngbya frigida ANT.L70.1 93.27% 93.27% 92.78%
Clade B Leptolyngbya antarctica ANT.LG2.5 93.67% 93.57% 94.26%
Leptolyngbya antarctica ANT.L18.1 93.67% 93.57% 94.26%
Leptolyngbya antarctica ANT.L67.1 93.57% 93.47% 94.16%
Clade C Leptolyngbya sp. Greenland_7 92.37% 92.37% 93.06%
Strain tBTRCCn 407 92.37% 92.37% 93.06%
Leptolyngbya sp. IkmLPT16 92.97% 92.97% 92.77%
Oculatella Oculatella atacamensis ATA3-4Q-KO2 93.77% 93.67% 94.16%
Oculatella cataractarum GSE-PSE-MK49-07D 92.88% 92.98% 93.37%
Oculatella neakameniensis Kovacik 1990/54 92.87% 92.87% 93.07%
Oculatella subterranea VRUC135/ 92.68% 92.78% 93.08%
Albertano1985
Leptolyngbya Leptolyngbya angustata UTCC 473 90.60% 90.50% 90.01%
sensu Leptolyngbya boryana PCC73110 90.68% 90.58% 90.09%
stricto Leptolyngbya corticola CCALA 085 90.80% 90.70% 90.11%
Leptolyngbya foveolarum Komárek 1964/112 91.10% 91.00% 90.60%
Leptolyngbya tenerrima UTCC 77 90.89% 90.79% 90.30%
99.51% inside the Oculatella clade. The clade A strains showed tolyngbya, Leptolyngbya sensu stricto, and Alkalinema. Haloleptolyng-
identities of 98.62–100%, the clade B strains shared identities of bya alcalis KR2005/106 and Nodosilinea nodulosa UTEX 2910
99.90–100%, and the two strains forming clade C had 100% clustered with high statistical support and rooted the tree. Clade A
identity. was represented by Leptolyngbya frigida ANT.LMA.1 and L. frigida
The phylogenetic reconstruction based on the 16S–23S ITS ANT.L70.1 and showed high support values. For the genus Ther-
(Fig. 2) was in agreement with the previous tree, although fewer moleptolyngbya, only sequences of strain ETS-08 and strain PCC
sequences were included in the analyses. Five clades corresponding 8501 were available; these sequences clustered with good support
to described genera were detected: Oculatella, Phormidesmis, Plec- values.
Fig. 2. Phylogenetic reconstruction of Leptolyngbyaceae based on the 16S–23S ITS region analyses. The NJ topology is depicted and the numbers associated with nodes
indicate support values for NJ, MP, ML, and BI analyses, respectively. Only bootstrap supports P50% and posterior probabilities P0.70 are reported. Values for nodes that
obtained support in only one of the performed phylogenetic analyses were omitted. Sequences of strains common to the 16S rRNA phylogenetic reconstruction are in boldface
font. Genera, including Thermoleptolyngbya gen. nov., and clade A are indicated by vertical bars. Horizontal bar represents expected number of nucleotide substitutions per
site.
3.2. 16S rRNA secondary structures each of the considered sequences and three structural types were
found, two of which (structural types 2 and 3) in line with other
Hypothetical secondary structures of the 16S rRNA gene vari- published H17 structures (Cannone et al., 2002; Řeháková et al.,
able domains H15, H17, H21, H22–H23, H41, and H44 were esti- 2014). The structural type 1 of H17 helix as predicted by RNAstruc-
mated for strain ETS-08, strain PCC 8501, and other ture corresponded to the alternative stable structure reported for
representative strains of focus clades in the 16S rRNA tree. The Leptolyngbya boryana in Fig. 3H of Řeháková et al. (2014); it was
obtained hypothetical structures are depicted in Figs. 3–5 and constrained and brought in line with other published H17 struc-
the different structural types found for each domain are reported tures. However, also for the other two obtained structural types,
in Table 3. the G in position 13 had to be constrained to bind with the C in
For H15 domain, only one structure was generated by the pro- position 24 (structural type 2) or 23 (structural type 3) to repro-
gram RNAstructure for each of the considered sequences and only duce the residue motif that interacts with the ribosomal protein
one structural type was found, common to all of the analyzed taxa S16 in E. coli (Powers and Noller, 1995; Řeháková et al., 2014). Lep-
(Fig. 3A). At the primary structure level, only a nucleotide substitu- tolyngbya sensu stricto showed structural type 1 of H17 helix, strain
tion was observed and it fell in the terminal hairpin loop, at posi- Leptolyngbya sp. PltLPT2 had structural type 2, the genus Oculatella
tion 14; all but one (KF246502) of the Thermoleptolyngbya showed structural types 1 and 2, and Thermoleptolyngbya had
sequences had C in position 14, whereas all the other analyzed structural types 1 and 3 (Table 3).
sequences of the family Leptolyngbyaceae had A. Considering the Also for H21 domain only one structure was obtained for each
16S rRNA ‘‘short” dataset, two further Thermoleptolyngbya of the considered sequences and six structural types were found
sequences (HE979753, HE979754) had A in position 14 of H15 among the focus taxa (Fig. 3C). Where possible, the A in position
domain. 9 was constrained to bind with the U in position 57 (structural
The structural types obtained for H17 helix are depicted in types 1, 2, 3) or 58 (structural type 5) to reproduce the residue
Fig. 3B. For this domain, RNAstructure produced one structure for motif that interacts with the ribosomal protein S8 in E. coli
A
(A) (B)
A C C
U
A
A
G
C
U A
A U
G C
H15 H17 - type 1 H17 - type 2 H17 - type 3
U
(C)
A U
G C
U C
U
C G
U
U
A U G A
U A G U A
G A U C
G G
H21 - type1 H21 - type 2 H21 - type 3 H21 - type 4 H21 - type5 H21 - type6
Fig. 3. Hypothetical secondary structures of the 16S rRNA H15 (A), H17 (B), and H21 (C) helices. Structures of a same helix are inscribed in a box and the structural type is
reported under each structure. In paired regions, A-U pairings are represented with a single line, G-C pairings with a double line, and unconventional pairings with a line
interrupted by a circle. Nucleotide substitutions for a given structure are illustrated by double-headed black arrows plus circles inscribing nucleotide symbols near the
interested nucleotide positions. Compensatory base changes are highlighted by yellow circles with red border. For H17 and H21 helices, the residue motif that interacts with
the ribosomal protein S16 and S8, respectively, in E. coli (Powers and Noller, 1995) is evidenced by a red dotted line box. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
(Powers and Noller, 1995; Řeháková et al., 2014). Structural type 1 helix to maintain the secondary structure (i.e. compensatory base
of H21 helix was common to Leptolyngbya sensu stricto, strain Lep- changes) were observed in structural type 1 among the different
tolyngbya sp. PltLPT2, and some Thermoleptolyngbya strains, struc- taxa (Fig. 3C).
tural types 2 and 3 were found solely in the genus Oculatella, and For H22–H23 domain, RNAstructure generated one structure for
structural types 4, 5, 6 were present only in the genus Thermolep- each sequence of the genera Leptolyngbya sensu stricto and Ocu-
tolyngbya (Table 3). Several nucleotide changes in both sides of the latella and for strain Leptolyngbya sp. PltLPT2. For the genus Ther-
(A)
H22-H23 - type 1 H22-H23 - type 2
(B) 7 14 15 16 17 18 65 67 69 80 81 82 83 84 89 90
Leptolyngbya A U A A C A C A C U G U U A G U
strain PltLPT2 A C A A G G U G U C C U U G G U
Oculatella G C A G A G C G U C U C U G G C
Thermoleptolyngbya G U Y A G G C G U C C U G A A C
Fig. 4. Hypothetical secondary structures of the 16S rRNA H22–H23 helix (A). The structural type is reported under each structure. In paired regions, A-U pairings are
represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by a circle. Red boxes evidences primary structure variable
nucleotide regions that were conserved inside each of the analyzed focus taxa. H22–H23 primary structure haplotypes for the focus taxa (B); red rectangles correspond to the
red boxes highlighted on the structure pictures (A). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
moleptolyngbya two hypothetical structures were generally pro- between Leptolyngbya sensu stricto and Oculatella to maintain
duced and in one case four structures. With the exception of some structural type 1 of H41 helix (Fig. 5A).
portions, none of the structural types predicted by RNAstructure For H44 domain, the program RNAstructure generated 3–5
matched the crystallography proofed structure of H22–H23 helix hypothetical structures for each of the considered sequences; thus,
available on the Comparative RNA Web (CRW) Site (Cannone the lowest free energy structure is shown. All of the obtained struc-
et al., 2002); thus, for each of the considered taxa, the structure tures were constrained in the basal region (GCCCG-CGUAAC) to
with the lowest free energy was constrained to bring it in line with resemble the structures proposed for some Leptolyngbyaceae by
the crystallographic structure of H22–H23 helix. Two structural Řeháková et al. (2014), but they were left as generated by the pro-
types were found among all of the considered taxa (Fig. 4A). Struc- gram RNAstructure in the remaining part. Six structural types of
tural type 1 of H22–H23 helix was common to the genus Leptolyn- H44 helix were found among the considered taxa (Fig. 5B). Lep-
gbya sensu stricto, to the genus Oculatella, to strain Leptolyngbya sp. tolyngbya sensu stricto had structural type 1 of H44 helix, Leptolyn-
PltLPT2, and to all but one of the Thermoleptolyngbya strains; struc- gbya sp. PltLPT2 showed structural type 2, Oculatella had structural
tural type 2 was found in strain Thermoleptolyngbya sp. tBTRCCn types 3 and 4, and Thermoleptolyngbya showed structural types 5
408 (accession number DQ471446) (Table 3). and 6 (Table 3).
Structural type 1 was longer (57 residues) than structural type 2
(56 residues) and had a 3 residue asymmetrical interior loop after 3.3. 16S–23S ITS secondary structures
the basal stem and the single base right bulge, instead of the 4 resi-
due asymmetrical interior loop found in structural type 2 (Fig. 4A). Hypothetical secondary structures of the 16S–23S ITS (contain-
Several non-canonical base pairings were necessary to make struc- ing both tRNA genes) were estimated for strain ETS-08, strain PCC
tural type 2 matching the crystallography proofed structure of 8501, and other representative strains of focus clades in the 16S–
H22–H23 helix (Fig. 4A). At the primary structure level, H22–H23 23S ITS tree. Only one hypothetical structure was produced for
domain showed variable nucleotide regions that were conserved each of the considered domains except the V2 hypervariable region
inside each of the analyzed focus taxa (Fig. 4B). of strain ETS-08 for which two hypothetical structures were gener-
For H41 domain, RNAstructure produced one structure for each ated. The obtained hypothetical structures are shown in Figs. 6–9.
of the considered sequences, and seven structural types were The percent identities of each single 16S–23S ITS domain were
found among the investigated taxa (Fig. 5A). Leptolyngbya sensu also calculated between strains belonging to Thermoleptolyngbya
stricto had structural types 1 and 2 of H41 helix, Leptolyngbya sp. and the focal strains for which hypothetical secondary structures
PltLPT2 showed structural type 3, Oculatella had structural types had been estimated (Table 4). Interspecific percent identities of
1 and 6, and Thermoleptolyngbya showed structural types 4, 5, the single spacer domains inside the genera Thermoleptolyngbya,
and 7 (Table 3). Several compensatory base changes were observed Leptolyngbya sensu stricto and Oculatella are also reported (Table 5).
G
A (A)
U
C C
A
G
G
G
G C
U G
H41 - type 1 H41 - type 2 H41 - type 3 H41 - type 4 H41 - type 5 H41 - type 6 H41 - type 7
U (B)
A
H44 - type 1 H44 - type 2 H44 - type 3 H44 - type 4 H44 - type 5 H44 - type 6
Fig. 5. Hypothetical secondary structures of the 16S rRNA H41 (A) and H44 (B) helices. Structures of a same helix are inscribed in a box and the structural type is reported
under each structure. In paired regions, A-U pairings are represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by
a circle. Nucleotide substitutions for a given structure are illustrated by double-headed black arrows plus circles inscribing nucleotide symbols near the interested nucleotide
positions. Compensatory base changes are highlighted by yellow circles with red border. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Table 3
Structural types of each 16S rRNA helix for the genus Leptolyngbya sensu stricto, strain Leptolyngbya sp. PltLPT2, the genus Oculatella, and the new genus Thermoleptolyngbya. The
structural types are depicted in Figs. 3–5.
Leptolyngbya sensu stricto Leptolyngbya sp. PltLPT2 Oculatella Thermoleptolyngbya

Helices
H15 Type 1 Type 1 Type 1 Type 1
H17 Type 1 Type 2 Type 1, type 2 Type 1, type 3
H21 Type 1 Type 1 Type 2, type 3 Type 1, type 4, type 5, type 6
H22–H23 Type 1 Type 1 Type 1 Type 1, type 2
H41 Type 1, type 2 Type 3 Type 1, type 6 Type 4, type 5, type 7
H44 Type 1 Type 2 Type 3, type 4 Type 5, type 6
The obtained D1-D10 helices are depicted in Fig. 6. All helices D1-D10 helix (95.31% nucleotide identity) that was 64 nucleotides
were characterized by the same basal stem structure (GACCU- in length with only three nucleotide differences (Fig. 6A and B);
AGGUC). Strains ETS-08 and PCC 8501 showed an almost identical two of these nucleotide differences were located inside interior
D1-D1' HELIX
(A) strain ETS-08 (B) strain PCC 8501
(C) L. boryana and

(E) Leptolyngbya
L. tenerrima sp. PltLPT2
(D) L. corticola
(F) O. subterranea (G) O. cataractarum
(H) O. atacamensis (I) O. neakameniensis
Fig. 6. Hypothetical secondary structures of the 16S–23S ITS region D1-D10 helix for strain ETS-08 (A), strain PCC 8501 (B), and the following focal strains: Leptolyngbya
boryana PCC 73110 and Leptolyngbya tenerrima UTCC 77 (C); Leptolyngbya corticola CCALA 085 (D); Leptolyngbya sp. PltLPT2 (E); Oculatella subterranea VRUC135/Albertano
1985 (F); Oculatella cataractarum GSE-PSE-MK49-07D (G); Oculatella atacamensis ATA3-4Q-KO2 (H); Oculatella neakameniensis Kovacik 1990/54 (I). In paired regions, A-U
pairings are represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by a circle. The new genus Thermoleptolyngbya
is inscribed in a box. Black arrows indicate differences between strain ETS-08 and strain PCC 8501.
loops and the remaining one was inside a 5 residue stem towards stem was found in Leptolyngbya sp. PltLPT2 (Fig. 6E) compared to
the terminal part of the helix. This last nucleotide difference the single base left bulge followed by a 5 residue stem in Ther-
caused the replacement of a canonical G-C base pairing in strain moleptolyngbya (Fig. 6A and B), a 7 residue asymmetrical internal
ETS-08 with a G-U non-canonical pairing in strain PCC 8501. The loop was present in Leptolyngbya sp. PltLPT2 (Fig. 6E) with respect
D1-D10 helix of Thermoleptolyngbya was clearly distinct from the to the 9 residue loop in Thermoleptolyngbya (Fig. 6A and B), and a 5
helices of strains belonging to Leptolyngbya sensu stricto residue hairpin loop was found in Leptolyngbya sp. PltLPT2 (Fig. 6E)
(Fig. 6C and D), while some similarities were present with respect compared to the 3 residue hairpin loop of Thermoleptolyngbya
to the helices of Leptolyngbya sp. PltLPT2 (Fig. 6E) and the strains (Fig. 6A and B). Numerous nucleotide differences were observed
belonging to genus Oculatella (Fig. 6F–I). However, the D1-D10 helix between the D1-D10 primary structures of Thermoleptolyngbya
of Leptolyngbya sp. PltLPT2 (Fig. 6E) was 65 nucleotides in length (Fig. 6A and B) and those of Oculatella (Fig. 6F–I), causing
and had several nucleotide differences that caused divergence in consequent differences in the secondary structures. In detail, Ocu-
the secondary structure. From the basal stem onwards, a first sym- latella subterranea (Fig. 6F) and O. cataractarum (Fig. 6G) had a 6
metrical internal loop of 4 residues was present in Leptolyngbya sp. residue stem after the first symmetrical internal loop, whereas
PltLPT2 (Fig. 6E) with respect to the 2 residue loop in Thermolep- the corresponding Thermoleptolyngbya stem was 5 residues
tolyngbya (Fig. 6A and B), a 2 base left bulge followed by a 4 residue (Fig. 6A and B). O. subterranea (Fig. 6F) and O. cataractarum (Fig. 6G)
V2 HELIX
(A) strain ETS-08 (C) strain PCC 8501

(structure I)
(B) strain ETS-08

(structure II)
(F) Leptolyngbya
(D) L. boryana and sp. PltLPT2
L. tenerrima (E) L. corticola
(G) O. subterranea
(I) O. atacamensis and

O. neakameniensis
(H) O. cataractarum
Fig. 7. Hypothetical secondary structures of the 16S–23S ITS region V2 helix for strain ETS-08 (structure I (A) and structure II (B)), strain PCC 8501 (C), and the following focal
strains: Leptolyngbya boryana PCC 73110 and Leptolyngbya tenerrima UTCC 77 (D); Leptolyngbya corticola CCALA 085 (E); Leptolyngbya sp. PltLPT2 (F); Oculatella subterranea
VRUC135/Albertano 1985 (G); Oculatella cataractarum GSE-PSE-MK49-07D (H); Oculatella atacamensis ATA3-4Q-KO2 and Oculatella neakameniensis Kovacik 1990/54 (I). In
paired regions, A-U pairings are represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by a circle. The new genus
Thermoleptolyngbya is inscribed in a box.
had another symmetrical internal loop of 4 residues followed by a structure I was 152 residues in length and had the lowest free
4 residue stem instead of the single base left bulge followed by a 5 energy, whereas structure II was 146 residues in length. Strain
residue stem found in Thermoleptolyngbya (Fig. 6A and B). The 9 ETS-08 had the longest V2 helix, followed by strain PCC 8501
residue asymmetrical internal loop of Thermoleptolyngbya (Fig. 7C) whose helix was 71 residues in length, O. subterranea
(Fig. 6A and B) was replaced by a 6 residue symmetrical internal (Fig. 7G) with a helix of 70 residues, and Leptolyngbya sp. PltLPT2
loop in O. subterranea (Fig. 6F) and O. cataractarum (Fig. 6G). The (Fig. 7F) with a helix of 65 residues. The remaining helices were
D1-D10 secondary structures of O. atacamensis (Fig. 6H) and O. considerably shorter: 17 residues in O. cataractarum (Fig. 7H), 10
neakameniensis (Fig. 6I) were the most similar to those of Ther- residues in all the strains belonging to Leptolyngbya sensu stricto
moleptolyngbya (Fig. 6A and B); the only difference was that the (Fig. 7D and E), and 8 residues in O. atacamensis and O. neakame-
stems upstream and downstream of the single base left bulge were niensis (Fig. 7I).
6 and 4 residues in length, respectively, compared with the 5 and 5 Fig. 8 illustrates the obtained boxB helices, which all shared the
residues found in Thermoleptolyngbya. same basal stem structure (AGCA-UGCU). The major length of the
Fig. 7 shows the obtained hypothetical V2 helices. The helices boxB helix of Thermoleptolyngbya (Fig. 8A and B) clearly distin-
were variable among all of the considered strains even inside gen- guished this group from the other taxa, which showed a very con-
era, and a common basal primary structure was not found. In strain sistent boxB helix among them. The helix was 47 residues in length
ETS-08, two alternative structures were obtained (Fig. 7A and B); in strain ETS-08 (Fig. 8A) and 60 residues in length in strain PCC
boxB HELIX
(A) strain ETS-08

(C) L. boryana and (D) L. corticola
L. tenerrima
(B) strain PCC 8501
(E) Leptolyngbya (F) O. subterranea (G) O. cataractarum (H) O. atacamensis and

sp. PltLPT2 O. neakameniensis
Fig. 8. Hypothetical secondary structures of the 16S–23S ITS region boxB helix for strain ETS-08 (A), strain PCC 8501 (B), and the following focal strains: Leptolyngbya boryana
PCC 73110 and Leptolyngbya tenerrima UTCC 77 (C); Leptolyngbya corticola CCALA 085 (D); Leptolyngbya sp. PltLPT2 (E); Oculatella subterranea VRUC135/Albertano 1985 (F);
Oculatella cataractarum GSE-PSE-MK49-07D (G); Oculatella atacamensis ATA3-4Q-KO2 and Oculatella neakameniensis Kovacik 1990/54 (H). In paired regions, A-U pairings are
represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by a circle. The new genus Thermoleptolyngbya is inscribed
in a box. Double-headed dotted line arrows indicate insertions in the primary structures of ETS-08 and PCC 8501 causing longer helixes in these strains.
8501 (Fig. 8B) compared to the 33–34 residue length found in the sp. PltLPT2 (Fig. 8E) had a terminal hairpin loop of 6 residues
other considered strains (Fig. 8C–H). All of the compared strains (GUUUDG in Oculatella and GUAAGA in Leptolyngbya sp. PltLPT2).
had similar boxB helices as follows: a stem structure fragmented The major length of the boxB helices of Thermoleptolyngbya, due
by an asymmetrical internal loop (in L. boryana, L. tenerrima, O. to primary sequence insertions, also affected the secondary struc-
cataractarum, O. acatamensis, and O. neakameniensis)/single base ture. In both strains belonging to this new genus, 4 unpaired
right bulge (in L. corticola, Leptolyngbya sp. PltLPT2 and O. subter- regions fragmented the stem structures as follows: from the basal
ranea) and a following single base left bulge (in all Leptolyngbya stem onwards, strain ETS-08 (Fig. 8A) showed a single base right
and Oculatella strains)/asymmetrical internal loop (in Leptolyngbya bulge, a single base left bulge and another single base right bulge
sp. PltLPT2). All Leptolyngbya sensu stricto box B helices contiguous to an asymmetrical internal loop. Strain PCC 8501
(Fig. 8C and D) terminated with a 5 residue hairpin loop (GAGAU), (Fig. 8B) had an asymmetrical internal loop, a single base left bulge,
whereas the boxB helices of Oculatella (Fig. 8F–H) and Leptolyngbya a single base right bulge, and another asymmetrical internal loop.
V3 HELIX
(C) Leptolyngbya
sensu stricto
(D) Leptolyngbya
sp. PltLPT2
(A) strain ETS-08 (B) strain PCC 8501
(G) O. atacamensis
(F) O. cataractarum
(E) O. subterranea (H) O. neakameniensis
Fig. 9. Hypothetical secondary structures of the 16S–23S ITS region V3 helix for strain ETS-08 (A), strain PCC 8501 (B), and the following focal strains: Leptolyngbya boryana
PCC 73110, Leptolyngbya tenerrima UTCC 77 and Leptolyngbya corticola CCALA 085 ( Leptolyngbya sensu stricto (C)); Leptolyngbya sp. PltLPT2 (D); Oculatella subterranea
VRUC135/Albertano 1985 (E); Oculatella cataractarum GSE-PSE-MK49-07D (F); Oculatella atacamensis ATA3-4Q-KO2 (G); Oculatella neakameniensis Kovacik 1990/54 (H). In
paired regions, A-U pairings are represented with a single line, G-C pairings with a double line, and unconventional pairings with a line interrupted by a circle. The new genus
Thermoleptolyngbya is inscribed in a box. Black arrows indicate differences between strain ETS-08 and strain PCC 8501.
Table 4
Percent identities of each single 16S–23S ITS domain among the strains belonging to the new genus Thermoleptolyngbya (ETS-08 and PCC 8501) and the following focal strains:
Leptolyngbya boryana PCC 73110, Leptolyngbya tenerrima UTCC 77, Leptolyngbya corticola CCALA 085, Leptolyngbya sp. PltLPT2, Oculatella subterranea VRUC135/Albertano 1985,
Oculatella cataractarum GSE-PSE-MK49-07D, Oculatella atacamensis ATA3-4Q-KO2, Oculatella neakameniensis Kovacik 1990/54.
L. boryana L. tenerrima L. corticola Leptolyngbya O. subterranea O. cataractarum O. atacamenis O. neakameniensi

sp. PltLPT2
D1-D10 strain ETS-08 54.90% 54.90% 56.86% 75.00% 79.69% 76.56% 75.00% 75.00%
strain PCC 8501 52.94% 52.94% 54.90% 75.00% 76.56% 73.44% 73.44% 71.88%
boxB strain ETS-08 75.76% 75.76% 69.70% 70.59% 73.53% 85.29% 82.35% 82.35%
strain PCC 8501 75.76% 75.76% 66.67% 52.94% 64.71% 76.47% 73.53% 73. 53%
V2 strain ETS-08 35.71% 50.00% 35.71% 46.58% 55.70% 55.00% 69.23% 69.23%
strain PCC 8501 35.71% 40.00% 35.71% 44.07% 70.97% 62.50% 50.00% 50.00%
V3 strain ETS-08 90.48% 90.48% 90.48% 52.78% 73.58% 65.38% 66.00% 64.00%
strain PCC 8501 90.48% 90.48% 90.48% 55.56% 71.70% 67.31% 68.00% 66.00%
Table 5
Percent identities of the single 16S–23S ITS domains inside the genera Thermoleptolyngbya, Leptolyngbya and Oculatella, based on the analyzed strains.
D1-D10 boxB V2 V3
Thermoleptolyngbya 95.31% 59.57% 52.11% 91.89%
Leptolyngbya 84.31–100% 90.91–100% 90–100% 100%
Oculatella 84.38–92.19% 88.24–100% 45.45–100% 62.18–85.11%
The terminal hairpin loop was 4 residues long in strain ETS-08 The densely packed trichomes of young cultures of strain ETS-
(GGAC) and 3 residues long in strain PCC 8501 (CAA). 08 (Fig. 11A) became large membranaceous floating aggregates in
Fig. 9 shows the inferred hypothetical V3 helices, which all had older liquid cultures (Fig. 11B). This behavior was probably due
the same basal stem structure (GUC-GAC). This 16S–23S ITS to the production of a large amount of exocellular mucilaginous
domain showed different characteristics for each of the considered material, exopolysaccharides, able to embed many trichomes.
taxa. The longest V3 helix was that of Leptolyngbya sp. PltLPT2 Further investigations carried out on strain ETS-08 by a light
(Fig. 9D) with 94 residues, followed in order by Thermoleptolyngbya confocal laser scanning microscope highlighted that allophyco-
(Fig. 9A and B) with 74 residues and Oculatella (Fig. 9E–H) with 50– cyanin and C-phycocyanin were the main phycobiliproteins
53 residues. The shortest V3 helix was that of Leptolyngbya sensu (Fig. 11C and D).
stricto (Fig. 9C) with only 21 residues. While all the considered Lep- Based on 16S rRNA gene data obtained from the literature, it
tolyngbya sensu stricto strains had identical V3 helices, strains was possible to trace the distribution of strains belonging to Ther-
belonging to the genus Oculatella showed some differences among moleptolyngbya (Table 1, Fig. 12). Ecological information was also
their V3 helices (nucleotide identity of 62.18–85.11%). retrieved from the literature. Organisms belonging to this taxon
Strain ETS-08 and strain PCC 8501 had almost identical V3 were found at and could be cultivated in a wide temperature range
helices (91.89% nucleotide identity) with only 6 nucleotide differ- (25–71 °C, Fig. 12) and in many cases were also sampled from alka-
ences. In detail, the first nucleotide difference was at residue 10 line environments (pH range 5–10.3, Fig. 12) (Castenholz, 1970;
and caused the replacement of the first single base left bulge of Boomer et al., 2009; Coman et al., 2013; Peng et al., 2013;
strain ETS-08 (Fig. 9A) with a 2 residue asymmetrical internal loop Andreote et al., 2014; Gaisin et al., 2015).
in strain PCC 8501 (Fig. 9B). Other 3 nucleotide differences fell in
an 8 residue symmetrical internal loop of strain ETS-08 (Fig. 9A)
and led to a smaller internal loop (4 residues) followed by a longer 4. Discussion
stem region in strain PCC 8501 (Fig. 9B). The 2 remaining nucleo-
tide differences entailed 2 non-canonical base pairings G-U in the 4.1. The new genus Thermoleptolyngbya
terminal stem region of strain ETS-08 (Fig. 9A) compared to the 2
corresponding classical A-U pairings found in strain PCC 8501 The new genus Thermoleptolyngbya represents a monophyletic
(Fig. 9B). The basal region and the terminal region of the V3 helix lineage in both the 16S rRNA and 16S–23S ITS phylogenetic recon-
were identical in both strains belonging to Thermoleptolyngbya: a structions with good statistical support in all of the performed
4 residue asymmetrical interior loop was present after the basal analyses (NJ, MP, ML, and BI). Although fewer strains were
stem and a 2 residue symmetrical interior loop was found included, this monophyletic lineage was also indirectly found in
upstream and close to the terminal hairpin loop of 9 residues. several previous investigations, most of which were ecological sur-
veys of microorganisms living in thermal environments (Moro
3.4. Morphological and ecological information et al., 2010; Coman et al., 2013; Mackenzie et al., 2013; Peng
et al., 2013; Andreote et al., 2014; Gaisin et al., 2015). The strict
After a bibliographic search, we found and compared the mor- evolutionary relationship between strain ETS-08 and strain PCC
phological descriptions available for some of the cyanobacteria 8501 was also formerly demonstrated by Moro et al. (2010) with
belonging to genus Thermoleptolyngbya: strains ETS-08 and PCC the rbcL and rpoC1 genes. Unfortunately, no further new sequences
8501 (Moro et al., 2010) and the organism Leptolyngbya sp. O-77 of strains belonging to genus Thermoleptolyngbya were available at
(Nakamori et al., 2014). For the uncultured cyanobacterial clone the INSD for these two loci.
ST7S_1CY and for Leptolyngbya sp. CENA538, we observed and The analysis of the 16S–23S ITS secondary structures of strains
compared LM pictures [Figs. S2H–L in Amarouche-Yala et al. ETS-08 and PCC 8501, representing the genus Thermoleptolyngbya,
(2014), Figs. 2C and 2D in Andreote et al. (2014)]. compared with those of other related clades confirms the exis-
Further morphological and ultrastructural investigations were tence of this new cryptic taxon. In fact, all of the considered
carried out on strains ETS-08 and PCC 8501 by light and electron domains (especially the D1-D10 helix, boxB helix, and V3 helix)
microscopy (SEM and TEM) analyses. Both strains were composed clearly distinguished Thermoleptolyngbya strains from the other
of flexuous and curved, sometimes intertwined, unbranched tri- analyzed organisms. The V2 helix was the most variable and was
chomes (Fig. 10A, B, E, and F). With TEM and SEM, trichomes of less taxonomic-informative; this finding was in agreement with
strain ETS-08 were made up of elongated cells, 1.5–3 lm in length the observations by Iteman et al. (2000) who suggest that the high
and up to 1 lm in width. Cells exhibited constrictions at the cross variability of this region and its absence in some cyanobacteria
walls (Fig. 10C and E). With TEM strain ETS-08 cells showed 3–4 indicate that it ‘‘serves no essential function”. Inside the genus
parietal thylakoids and trichomes were surrounded by a multilay- Thermoleptolyngbya, the D1-D10 helix was the most conserved,
ered sheath (Fig. 10C). As regards strain PCC 8501, TEM and SEM whereas the two remaining helices (boxB and V3) showed more
observations showed trichomes made up of cells, 2–6.5 lm in variation, compatible with the existence of two distinct species.
length and 0.8–1.8 lm in width, with distinct constrictions at the Differences in the boxB helix suggesting the presence of more than
cross walls (Fig. 10D and F). TEM analyses revealed that cells were one species have been reported in other cyanobacterial genera
characterized by 4–6 thylakoids arranged at the periphery of the (e.g., Perkerson et al., 2011; Dvořák et al., 2015; Vaz et al.,
cells (Fig. 10D). Moreover, each trichome was individually sur- 2015). Thus, the D1-D10 helix seems to be more important for dis-
rounded by an unlayered sheath (Fig. 10D). tinguishing between organisms belonging to a given genus,
A B
C D
t s
c
t
s
E F
Fig. 10. Microscope micrographs of strains ETS-08 and PCC 8501. (A–B) Light microscope images showing unbranched trichomes of strains ETS-08 (A) and PCC 8501 (B).
Bar = 5 lm. (C–D) Transmission electron micrographs showing elongated cells of strains ETS-08 (C) and PCC 8501 (D). Note the different number of thylakoids (t) and the
multilayered sheath (s) in ETS-08 compared with the unlayered sheath of PCC 8501. c = carboxysome. Bar = 0.5 lm. (E–F) Scanning electron micrographs of trichomes with
constrictions at the cell wall of strains ETS-08 (E) and PCC 8501 (F). Bar = 5 lm.
whereas boxB and V3 are also informative at the species level. The genera of the family Leptolyngbyaceae; however it is useful to
question of whether strains ETS-08 and PCC 8501 belong to the check the presence of sequencing errors in the sequences used
same species or should be considered as separate species was for phylogenetic analyses, as suggested by Řeháková et al. (2014).
already addressed by Moro et al. (2010), who reported some mor- Based on this, the sequence DQ471446 of the thermophilic strain
phological and biochemical differences between the two strains. In tBTRCCn 408 very probably contains sequencing errors, but they
particular, strain ETS-08 had a multilayered sheath that was were not so significant to justify its exclusion from the phyloge-
thicker than the sheath observed in strain PCC 8501 and two myx- netic analyses.
oxanthophylls (sensu Karsten and Garcia-Pichel, 1996) compared H15 domain of 16S rRNA was the most conserved both in
to the one mxyoxanthopyll found in strain PCC 8501. Moreover, sequence and structure. It is worth noticing that at the primary
the cell size was different between the two strains kept in the structure level a nucleotide substitution distinguished most mem-
same culture conditions, with strain PCC 8501 showing cells gen- bers of the genus Thermoleptolyngbya from the other analyzed taxa
erally 2-fold longer and wider than the cells of strain ETS-08. Here, of Leptolyngbyaceae and inside Thermoleptolyngbya the same
we confirm the morphological differences between the two strains nucleotide substitution distinguished the South American strains
and we show that these organisms represent two distinct species: from the others.
Thermoleptolyngbya albertanoae Sciuto & Moro and T. oregonensis The second more conserved 16S rRNA domain at the structure
Sciuto & Moro. level was H22–H23 domain; this was also reported by Řeháková
Based on the 16S rRNA phylogenetic reconstruction, at least 14 et al. (2014) and its probably due to the crucial function of H22–
other cyanobacterial strains are attributable to genus Thermolep- H23 helix for the core of the ribosome (Meares et al., 2002). We
tolyngbya besides strains ETS-08 and PCC 8501, including uncul- cannot exclude that the differences observed in sequence
tured and cultivated microorganisms (Fig. 1, Table 1); and more DQ471446, leading to several non-canonical base pairings in struc-
organisms very probably belong to this taxon (see for example tural type 2, can be due to sequencing errors; nevertheless non-
Fig. 12). In addition to their phylogenetic position in the tree, the canonical base pairings are accepted to occur in prokaryotic rRNA
16S rRNA percent identities among the strains inside this clade molecules (Gutell et al., 2002). At the primary structure level,
are above 95% (Table S1), which is the threshold value proposed H22–H23 domain was the only one that allowed distinction among
by Stackebrandt and Goebel (1994) to assign two bacterial strains the investigated taxa.
to the same genus. H17 was the third more conserved 16S rRNA domain, with only
The analysis of 16S rRNA secondary structures does not allow a three structural types found among the investigated taxa, of which
clear distinction of the genus Thermoleptolyngbya from the other structural type 3 was found only in the genus Thermoleptolyngbya.
A B
C D
Fig. 11. Morphological, ecological, and biochemical features of strain ETS-08. (A) Flexuous and curved trichomes. Bar = 10 lm. (B) Large floating aggregates (arrow) in older
liquid cultures. (C) Light confocal laser scanning micrograph showing the predominance of autofluorescence of allophycocyanin and C-phycocyanin (light red). Bar = 10 lm.
(D) Light confocal laser scanning micrograph of trichomes made up by cylindrical cells with distinct constrictions at the cross walls. Bar = 5 lm. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Also in the case of H21 and H41 domains, some structural types sampling temperature was approximately 45 °C and the strain
were present solely in specific taxa. In detail, H21-structural types 2 could be kept in culture at the same temperature (Castenholz,
and 3 distinguished the genus Oculatella and H21-types 4, 5, and 6 1970); however, we maintained this strain at a temperature range
were found only in the genus Thermoleptolyngbya; structural types identical to that used for strain ETS-08 (data not shown). Strain O-
4 and 6 are unique since they lack the AA-U residue motif typical of 77, that is the only other Thermoleptolyngbya strain for which a
this helix (Powers and Noller, 1995; Řeháková et al., 2014). H41- morphological description is available and that here we re-name
structural type 2 was present only in Leptolyngbya sensu stricto, Thermoleptolyngbya sp. O-77, has a growth range between 25 °C
H41-type 3 was found only in strain Leptolyngbya sp. PltLPT2, and and 60 °C, with its optimum at 55 °C and no growth above 65 °C
H41-type 6 was present only in Oculatella; structural types 4, 5, (Nakamori et al., 2014). Strain ST7S_1CY was isolated from the
and 7 of H41 helix distinguished the genus Thermoleptolyngbya Meskoutine hot spring in Algeria and was one of the most abun-
from the other investigated taxa. Nevertheless for each of these dant cyanobacteria in the investigated Algerian hot springs, found
genomic regions (H17, H21, and H41), one structural type was gen- also in the hottest sites at 70 °C (Amarouche-Yala et al., 2014).
erally present in different lineages (Table 3), suggesting that it was Strain AE1b_A12, strictly related with the species T. oregonensis
analogous, rather than homologous, in different taxa. (99% identity between its 16S rRNA sequence and strain PCC
H44 domain was the longest one (101–103 residues) and differ- 8501), and strain AE1b_F12, showing a 16S rRNA sequence identity
ent H44 structural types were found for the considered lineages, of 98% with Thermoleptolyngbya sp. O-77, were among the most
allowing distinction among the investigated taxa; however H44 abundant taxa in the Ady Endre geothermal spring, Romania, at
domain could be analyzed for less cyanobacterial strains since 55 °C.
many 16S rRNA sequences were too short to include it. One strain belonging to the Thermoleptolyngbya clade (Leptolyn-
The new genus Thermoleptolyngbya has a worldwide distribu- gbya sp. CENA538) appeared to be an exception since it was not
tion in thermal environments based on both the reports from the isolated from a typical thermal environment. The taxonomic posi-
16S rRNA gene (Weller et al., 1992; Lacap et al., 2007; Oren tion of this organism, whose morphology observed with LM resem-
et al., 2009; Boomer et al., 2009; Moro et al., 2010; Coman et al., bled the morphology described here for other strains of
2013; Mackenzie et al., 2013; Peng et al., 2013; Amarouche-Yala Thermoleptolyngbya, was also uncertain based on the phylogenetic
et al., 2014; Andreote et al., 2014; Nakamori et al., 2014; Gaisin reconstruction because it could be attributed to this new genus or
et al., 2015) (Fig. 12) and classical investigations (Castenholz, be a strictly related taxon. However, based on the 16S rRNA per-
1970; Roy et al., 2015). cent identity it more plausibly represents a species of Thermolep-
Organisms belonging to this taxon seem to be thermotolerant tolyngbya distinct from both T. albertanoae and T. oregonensis.
rather than thermophilic because they have been found at and Moreover, its strong phylogenetic relationship with the type
can be cultivated in a wide temperature range (Fig. 12). For exam- strains of the latter two species (ETS-08 and PCC 8501) as well as
ple, strain ETS-08 grows well between 25 °C and 40 °C (data not with strain O-77 was previously reported by Andreote et al.
shown) and is one of only two taxa able to thrive above 50–55 °C (2014) based on 16S rRNA phylogenetic analyses. Unfortunately,
in its natural environment (Moro et al., 2010). Strain PCC 8501’s the 16S–23S ITS sequence was not available for this strain. The
Fig. 12. Geographical and ecological information of the genus Thermoleptolyngbya. The phylogenetic reconstruction is based on the 16S rRNA gene ‘‘short” dataset analyses.
The NJ topology is depicted and the numbers associated with nodes indicate support values for NJ, MP, ML with MEGA, respectively. Only bootstrap supports P50% are
reported. Values for nodes that obtained support in only one of the performed phylogenetic analyses were omitted. The new genus Thermoleptolyngbya is highlighted by a
blue box, whereas the other genera and clades A, B, C are indicated by vertical bars. Horizontal bar represents expected number of nucleotide substitutions per site. For the
genus Thermoleptolyngbya, also collection sites are reported and each OTU is evidenced by a color referring to the continent where the OTU was sampled. OTU distribution is
illustrated also by the colored circles on the world map. For most Thermoleptolyngbya OTUs, also temperature and pH available data are reported in the table. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
water temperature of the saline-alkaline lake where strain isodiametric and by the presence of necridia, whereas the second
CENA538 was found was not reported. However, we cannot by cells distinctly longer than wide and by the absence of necridia.
exclude that the temperature can reach high values during the Only two species from thermal environments are listed under the
Brazilian dry season (the period when this organism was sampled) subgenus Leptolyngbya: L. gelatinosa (Voronichin) Anagnostidis
based on the limited dimensions of this water pond (Andreote and Komárek and L. thermobia Anagnostidis. Most of the described
et al., 2014). Indeed, water ponds, which often have high salinity, Leptolyngbya species belong to the subgenus Protolyngbya, which
can be considered a sort of thermal environment (Castenholz, includes also many species from thermal environments. Under this
1969). Further analyses are required to better characterize this subgenus, Komárek and Anagnostidis (2005) list ten thermal spe-
strain that here we re-name Thermoleptolyngbya sp. CENA538. cies (corresponding to the ‘‘ecological group VIII”), which are
Another feature observed in many members of genus Ther- divided into two further groups based on the trichome width: spe-
moleptolyngbya is their tolerance to alkaline conditions (Fig. 12). cies with trichomes wider than 2 lm (L. subuliformis (Gomont)
Many of the strains were sampled from alkaline environments Anagnostidis and L. phormidioides (Anagnostidis) Anagnostidis
(Castenholz, 1970; Boomer et al., 2009; Coman et al., 2013; Peng and Komárek) and species with trichomes narrower than 2 lm
et al., 2013; Andreote et al., 2014; Gaisin et al., 2015) and we (L. granulifera (Copeland) Anagnostidis, L. copelandii Anagnostidis,
observed that old cultures of strain ETS-08 could survive for sev- L. laminosa (Gomont) Anagnostidis and Komárek, L. treleasii
eral days, maintaining their blue-green color, even after treatment (Gomont) Anagnostidis and Komárek, L. ramosa (Boye-Petersen)
with sodium hypochlorite, probably due to protection by the thick Anagnostidis and Komárek, L. orientalis (G.S. West) Anagnostidis
sheath surrounding the filaments (Fig. 11B). and Komárek, L. thermalis Anagnostidis in Anagnostidis and Komá-
rek, and L. thermarum (Voronichin) Anagnostidis and Komárek).
4.2. Comparison of Thermoleptolyngbya taxa with other described The new genus Thermoleptolyngbya very probably corresponds
thermal species of Leptolyngbya to this second group of the subgenus Protolyngbya, ecological group
VIII, even if the two species described here, T. albertanoae and T.
Komárek and Anagnostidis (2005) divided the heterogeneous oregonensis, differ from all the other species reported by Komárek
genus Leptolyngbya into the two subgenera Leptolyngbya and and Anagnostidis (2005). In detail, L. ramosa is the first to be
Protolyngbya. The first is characterized by cells more or less excluded because of its main, distinctive feature, that is a thallus
forming macroscopic mucilaginous strands up to 15 cm long. Also ter represents a new genus separated from Leptolyngbya sensu
L. treleasii, with its unconstricted trichomes and cells up to 11-fold stricto. This finding is supported by the 16S–23S phylogenetic
longer than wide, does not correspond to T. albertanoae and T. ore- reconstruction. Another cyanobacterium called Leptolyngbya sp.
gonensis. L. copelandii is excluded because of its unconstricted tri- PltLPT2, isolated from the Platistomo spring in Greece (collection
chomes, for the slightly broadened apical cells, and for its cell temperature 26 °C), was a sister taxon to clade A with high support
content, which usually presents several large granules. Also L. ori- values in the 16S rRNA tree. The 16S rRNA sequence identities
entalis, L. thermalis, and L. thermarum cannot match T. albertanoae among the strains forming clade A and strain PltLPT2 (95.75–
and T. oregonensis because of their trichomes not constricted at 96.44%, Table S1) were compatible with their belonging to the
the cross walls. Moreover, L. orientalis has a very thin, often imper- same genus; however, the 16S–23S ITS tree did not agree with this
ceptible, sheath and L. thermalis has generally a single granule on hypothesis.
either side of the cross walls. L. granulifera and L. laminosa are Strains grouping into clade B (ANT.L67.1, ANT.LG2.5, and ANT.
the most similar to T. albertanoae and T. oregonensis for their L18.1) were all isolated from Antarctica and were attributed to
trichomes slightly constricted at the cross walls and for the the species Leptolyngbya antarctica. Their strong evolutionary
capability to form conspicuous, membranaceous thalli. However, relationship was previously observed (Taton et al., 2006). The
L. granulifera trichomes are usually slightly tapering at the ends, phylogenetic position of clade B and the 16S rRNA identities among
with long-conical apical cells, and the cells generally present a the strains inside this group (Table S1) indicate that it may repre-
granule on either side of the cross walls. Similarly, L. laminosa sent a new, separate genus.
has most cells with a single granule on either side of the cross walls Finally, clade C was composed of two strains (Greenland_7 and
and the apical cells usually are acute-conical or rounded-conical. tBTRCCn 407) isolated from thermal environments with identical
Based on our data, we could describe only two species inside 16S rRNA gene sequences. This cluster possibly represents a new
the genus Thermoleptolyngbya, but we think that more species genus and has been detected by previous 16S rRNA phylogenetic
are present inside the genus and that some of these species can reconstructions (e.g., Moro et al., 2010). The organism Leptolyngbya
match the already described species of the genus Leptolyngbya, sp. IkmLPT16 isolated from the Moustafa hot spring in Greece was
subgenus Protolyngbya, ecological group VIII, with trichomes nar- strictly related to clade C; however the analysis of 16S rRNA iden-
rower than 2 lm (Komárek and Anagnostidis, 2005). tity between this strain and the clade C strains (Table S1) as well as
Amarouche-Yala et al. (2014) identified the uncultured the branch length in the tree led us to not to include it in this
cyanobacterial clone ST7S_1CY as L. laminosa; this is one of the cluster.
most widespread Leptolyngbya species in thermal environments, Further investigations are needed to establish whether clades A,
reported with this taxonomic name or with its basionym Phormid- B, and C are actually new genera and to describe them if verified.
ium laminosum Gomont ex Gomont (Guiry and Guiry, 2016). As dis-
cussed above, the species L. laminosa (Gomont) Anagnostidis and 4.4. Identity values of genomic regions and taxonomic inferences
Komárek likely belongs to the genus Thermoleptolyngbya; however
the isolation of cyanobacterial strains morphologically ascribable Strain Leptolyngbya sp. PltLPT2 is an organism that particularly
to this species and their accurate description following a polypha- attracted our attention, leading us to analyze its 16S–23S ITS sec-
sic method, as it was done in this paper, are needed to re-assign it ondary structures. Indeed, as reported above, the phylogenetic
under this new genus. reconstruction based on the 16S rRNA gene combined with the
Based on the LM pictures reported in Andreote et al. (2014), percent identity values of this marker would have led to include
Thermoleptolyngbya sp. CENA538 has trichomes not constricted at this strain in clade A, but the 16S–23S ITS tree rejected this
the cross walls, cells longer than wide (even if we cannot establish hypothesis. Based on the percent identities of the 16S rRNA gene
the exact cell size), rounded apical cells, in some cases slightly sequence alone, strain PltLPT2 could have been included in the
tapered, and apparently most cells present a single granule on Thermoleptolyngbya clade because it had a percent identity just
either side of the cross walls. Thus the Brazilian strain CENA538 below 95% with many strains of this new genus and just above
can match the species L. thermalis Anagnostidis in Anagnostidis 95% with five cyanobacterial clones sampled from Mongolian and
and Komárek and it is worth underlining that this is the only ther- Russian hot springs (Alla11otu1-1, Alla11otu1-4, Alla11otu1-5,
mal species of the subgenus Protolyngbya with trichomes narrower Tsenher12otu4-1, and Tsenher12otu4-2; Table S1). However, both
than 2 lm reported from South America (Algeria, Argentina) the phylogenetic reconstructions and the 16S–23S ITS secondary
(Komárek and Anagnostidis, 2005). Further and more accurate structure analysis were against this conclusion.
morphological observations are required to re-name strain Information on Leptolyngbya sp. PltLPT2 is scarce, and we hope
CENA538 as Thermoleptolyngbya thermalis and thus to transfer L. that further studies will be performed on this cyanobacterium,
thermalis to the genus Thermoleptolyngbya. which may represent a new taxon. Nevertheless, its case lead us
to emphasize how caution should be taken when drawing taxo-
4.3. Other lineages requiring further investigation nomic conclusions based solely on percent identity values of the
16S rRNA locus as frequently occurs during ecological surveys
In addition to the monophyletic lineage coinciding with genus where cyanobacterial strains are attributed to taxa based solely
Thermoleptolyngbya, three highly supported clades were found on BLAST searches using their 16S rRNA sequences. 16S rRNA gene
with the phylogenetic reconstruction based on the 16S rRNA gene: data have been and are a precious tool, particularly for taxonomists
clade A, clade B, and clade C. Clade A comprised three strains, two involved in cyanobacterial systematics. However, as previously
of which (ANT.LMA.1 and ANT.L70.1) were identified as Leptolyng- demonstrated by several authors (e.g., Sciuto et al., 2012; Sciuto
bya frigida and collected from Antarctica and the third strain and Moro, 2015; Eckert et al., 2015), these data should be consid-
named Leptolyngbya sp. 1T12c was isolated from a green biofilm ered together with other evidence, such as data obtained with a
growing on the marble pedestal of a fountain in Firenze, Italy. polyphasic method, including a multilocus approach, and possibly
The strong evolutionary relationship among at least two of these with secondary structure analysis.
cited strains was previously reported in phylogenetic investiga- The same consideration is also valid for other genomic regions
tions using the 16S rRNA gene (e.g., Taton et al., 2006; Cuzman as shown here through the analysis of the 16S–23S ITS primary
et al., 2010; Mackenzie et al., 2013). The 16S rRNA gene percent structure. The most emblematic example of this issue is that the
identities among the three strains strongly suggests that this clus- percent identity value for the boxB domain between the two
strains representing the genus Thermoleptolyngbya is 59.57% Diagnosis: circumscribed by molecular and phylogenetic analy-
(Table 5), whereas the percent identity values for this 16S–23S ses based on the 16S rRNA gene and the 16S–23S ITS region and
ITS portion between strain ETS-08 and PCC 8501 and each of the previously (Moro et al., 2010) with the rbcL and rpoC1 genes. The
selected focus strains belonging to other taxa are above 64.71% 16S–23S ITS region with distinctive D1-D10 , boxB, and V3 helices.
(Table 4). Without the secondary structure domain analysis, the Etymology: the generic name ‘‘Thermoleptolyngbya” (fem. noun.)
percent identity values would have been misleading. This finding refers to the fact that members of this new cryptic genus are gen-
is particularly true for the 16S–23S ITS marker, which shows highly erally found in thermal environments and that the genus was sep-
variable and more conserved domains, and is the reason why align- arated from Leptolyngbya sensu stricto.
ing the 16S–23S ITS sequences of distantly related taxa is so diffi- Type species: Thermoleptolyngbya albertanoae Sciuto & Moro.
cult and why many authors suggest taking care when considering
Thermoleptolyngbya albertanoae Sciuto & Moro sp. nov.
this locus for phylogenetic reconstructions (e.g., Johansen et al.,
[Figs. 10A, 10C, 10E, 11A–11D in this paper and Figs. 1–3 in Moro
2011). However, with the support of secondary structure analysis
et al. (2010)].
and by limiting the investigation to related taxa, reliable
Description: Filaments forming expanded blue-green mats on
phylogenies can be obtained with this molecular marker (e.g.,
the substratum and large floating membranaceous aggregates in
Johansen et al., 2014; Bohunická et al., 2015).
older liquid cultures. With LM, variously curved, flexuous, densely
packed, isopolar trichomes, with rounded apical cells. Each tri-
4.5. Conclusions
chome individually surrounded by a thin colorless sheath. Cells
longer than wide (1.2–3 lm long and 0.8–1 lm wide) with distinct
The molecular, structural, and phylogenetic surveys based on
constrictions at the cross walls. Homogeneous cell content and lack
the 16S rRNA gene and the 16S–23S ITS region allowed us to cir-
of gas vesicles. Reproduction by trichome fragmentation into short
cumscribe a new taxon of filamentous non-heterocytous cyanobac- hormogonia without necridia. Cell division always symmetrical,
teria, the genus Thermoleptolyngbya, and to describe two of the perpendicular to the longitudinal axis of the trichome, and daugh-
species belonging to this new group, T. albertanoae and T. oregonen- ter cells dividing and growing to the size of the mother cell before
sis. This is one of the most widespread cyanobacterial genera in the next division. Slow gliding motility on solid medium. With
thermal environments and has been repeatedly encountered by TEM, 3–4 parietal thylakoids and a multilayered sheath approxi-
researchers during ecological surveys, but, up to now, it was erro- mately 0.15 lm thick. Water-soluble pigments: allophycocyanin,
neously and simplistically attributed to the genus Leptolyngbya. In C-phycocyanin (the most abundant), and phycoerythrin. Lipid-
fact, Thermoleptolyngbya gen. nov. is a cryptogenus, i.e., ‘‘a genus soluble pigments: two different myxoxanthophylls (sensu Karsten
that can be only detected based on molecular and phylogenetic and Garcia-Pichel, 1996), isozeaxanthin, zeaxanthin, chlorophyll
analyses, not showing distinctive morphological and/or ecological a, echinenone and b-carotene. Typical of thermal environments,
features” (Komárek et al., 2014). This concept was first explicitly usually growing on thermal muds.
introduced by Komárek and collaborators in 2014 and has been Diagnosis: detected by molecular and phylogenetic analyses
already applied for a number of taxa (e.g., Hrouzek et al., 2013; based on the 16S rRNA gene and 16S–23S ITS region. 16S–23S
Dvořák et al., 2015; Vaz et al., 2015). If not abused and based on ITS region with distinctive boxB and V3 helices.
strong phylogenetic evidence, it can be very useful in cyanobacte- Type locality: Euganean Thermal District, Montegrotto Terme,
rial systematics where cryptic taxa can be more abundant than Padova, Italy (45°190 N, 11°460 E).
thought. Importantly, the taxonomic recognition of cryptogenera Etymology: the specific epithet ‘‘albertanoae” (fem. gen.) was
can help provide a systematic basis to ecological surveys, which given in the honor of the phycologist Patrizia Albertano, who
are often based on modern molecular techniques able to deeply devoted a great part of her research career to the study of Leptolyn-
screen cyanobacterial diversity (e.g., high throughput sequencing), gbyaceae and who gave a precious contribution to the first paper
but which are equally often performed by non-taxonomists. on strain ETS-08 (i.e., Moro et al., 2010).
Holotype: resin-embedded sample of strain ETS-08 deposited at
5. Taxonomic treatment PAD (A000626).
Living cultures: active cultures of the type strain, from which the
The classification system used is that reported by Komárek et al. holotype was derived, are available at the Culture Collection of
(2014), and the taxon description follows the prescriptions of the Autotrophic Organisms (CCALA), under code CCALA 10257, and at
International Code of Nomenclature for Algae, Fungi and Plants the Culture Collection of Algae at Göttingen University (SAG),
(Melbourne code) (McNeill et al., 2012). under code SAG 2016.001.
DNA sequences available: 16S rRNA gene and 16S–23S region:
Thermoleptolynbgya Sciuto & Moro gen. nov.
FM210757; rbcL gene: FM955229; rpoC1 gene: FM955230.
Phylum: Cyanobacteria Thermoleptolyngbya oregonensis Sciuto & Moro sp. nov.

Order: Synechococcales [Figs. 10B, 10D, 10F in this paper and Figs. 4–6 in Moro et al.
Family: Leptolyngbyaceae (2010)]
Description: Filaments forming expanded blue-green mats on
Description: Filaments forming expanded blue-green mats on the substratum and large floating membranaceous aggregates in
the substratum and large floating membranaceous aggregates in older liquid cultures. With LM, variously curved, flexuous, isopolar
older liquid cultures. Under natural conditions, thalli live solitarily trichomes, with rounded apical cells. Each trichome individually
or mixed with other cyanobacteria and diatoms. Slow gliding surrounded by a thin colorless sheath. Cells longer than wide (2–
motility on solid medium. Flexuous, indefinite length long, isopo- 6.5 lm long and 0.8–1.8 lm wide) with distinct constrictions at
lar, unbranched trichomes. Each trichome individually surrounded the cross walls. Homogeneous cell content. Reproduction by tri-
by a thin colorless sheath. Inside the trichomes, cells 1–3.6-fold chome fragmentation into short hormogonia without necridia. Cell
longer than wide. Cells usually 1.2–6.5 lm long and 0.8–2 lm division always symmetrical, perpendicular to the longitudinal
wide. Parietal thylakoids. Reproduction by trichome fragmentation axis of the trichome, and daughter cells dividing and growing to
into short hormogonia without necridia. Allophycocyanin and C- the size of the mother cell before the next division. Slow gliding
phycocyanin rich. Typical from thermal environments. motility on solid medium. With TEM, 4–6 parietal thylakoids and
unlayered sheath. Water-soluble pigments: allophycocyanin, Cuzman, O.A., Ventura, S., Sili, C., Mascalchi, C., Turchetti, T., D’Acqui, L.P., Tiano, P.,
2010. Biodiversity of phototrophic biofilms dwelling on monumental fountains.
C-phycocyanin, and phycoerythrin. Lipid-soluble pigments: one
Microb. Ecol. 60, 81–95.
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anthin, zeaxanthin, chlorophyll a, echinenone and b-carotene. RNA secondary structure. Bioinformatics 25, 1974–1975.
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cyanobacterium Pinocchia polymorpha gen. et sp. nov. derived from the genus
Diagnosis: detected by molecular and phylogenetic analyses Pseudanabaena. Fottea, Olomouc 15, 113–120.
based on the 16S rRNA gene and 16S–23S ITS region. 16S–23S Eckert, E.M., Fontaneto, D., Coci, M., Callieri, C., 2015. Does a barcoding gap exist in
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Molecular Phylogenetics and Evolution 105 (2016) 15–35

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

Detection of the new cosmopolitan genus Thermoleptolyngbya

1. Introduction Antarctica and thermal springs (e.g., Anagnostidis and Komárek,

Strain Sequence identity with Isolated from References

H15 H17 - type 1 H17 - type 2 H17 - type 3

H22-H23 - type 1 H22-H23 - type 2

Leptolyngbya sensu stricto Leptolyngbya sp. PltLPT2 Oculatella Thermoleptolyngbya

(A) strain ETS-08 (B) strain PCC 8501

(C) L. boryana and

(F) O. subterranea (G) O. cataractarum

(H) O. atacamensis (I) O. neakameniensis

(A) strain ETS-08 (C) strain PCC 8501

(B) strain ETS-08

(I) O. atacamensis and

(A) strain ETS-08

(B) strain PCC 8501

(E) Leptolyngbya (F) O. subterranea (G) O. cataractarum (H) O. atacamensis and

(A) strain ETS-08 (B) strain PCC 8501

(E) O. subterranea (H) O. neakameniensis

L. boryana L. tenerrima L. corticola Leptolyngbya O. subterranea O. cataractarum O. atacamenis O. neakameniensi

Phylum: Cyanobacteria Thermoleptolyngbya oregonensis Sciuto & Moro sp. nov.

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