Abstract:
Cyanobacteria, previously referred to as blue-green algae, are a diverse group of
microorganisms that inhabit a wide range of ecological niches and are well known for their
toxic secondary metabolite production. Knowledge of the evolution and dispersal of these
microorganisms is still limited, and further research to understand such topics is vital. In Sri
Lanka, a number of studies have been conducted for identifying fresh water cyanobacteria
based on morphology, but molecular studies on cyanobacterial phylogeny and on cyanotoxin
production have been scarce.
The objectives of the present study were to isolate and identify cyanobacteria using their
morphological characters from different geographical locations of Sri Lanka, to determine the
cyanobacterial phylogeny using 16S rRNA gene partial sequences, and to identify potential
microcystin producing cyanobacteria from environmental samples and cultured isolates using
molecular techniques. To determine the cyanobacterial diversity, samples were collected from
water reservoirs representing wet, intermediate, dry and arid climatic zones. Sampling was
also carried out from thermal springs, paddy fields, brackish water and tsunami affected areas
(fresh water mixed with seawater).
Considering the distribution of cyanobacterial morphotypes, 37 genera were recorded from
studied areas. Among them, 25 genera were observed in environmental samples representing
the orders of Chroococcales, Pleurocapsales, Oscillatoriales and Nostocales. In cultured
isolates, 35 genera were observed for all five cyanobacterial orders, including Stigonematales.
Scytonema and Anabaenopsis species were only observed in environmental samples but could
not get them into cultures. Cyanobacterial isolates belonging to genus Gloeothece,
Aphanocapsa, Planktolyngbya, Aphanizomenon, Tolypothrix, Hapalosiphon, Westiellopsis,
Nodularia, Schizothrix, Cylindrosperm um, Chlorogloeopsis and Fischerella were only
observed in cultures. According to the results obtained from present study, the highest
diversity of cyanobacteria was recorded from dry zone reservoirs while the lowest
cyanobacterial diversity was recorded from arid zone reservoirs. According to observations
the genus Planktolyngbya and Nodularia were recorded from wet zone, genus Anabaenopsis,
Scytonema, Cylindrospermopsis, Tolypothrix, Aphanizomenon and Chlorogloeopsis
recorded only from dry zone. In addition, genus Coelosphaerium was recorded only from
intermediate zone. Microscopic examination of samples collected from hot spring showed
dominance of cyanobacterial species belonging to Synechococcus, Chroococcus,
were
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Chroococcidiopsis, Xenococcus, Phormidium, Leptolyngbya, Calothrix and Chlorogloeopsis.
In this study, the genus Chroococcidiopsis was identified for the first time in Sri Lanka.
Ninety eight cyanobacterial isolates were identified using 16S rRNA sequences and were
deposited in NCBI database. Eighteen isolates belonged to order Chroococcales, 49 to
Oscillatoriales, ten to Pleurocapsales, six to Nostocales and three to Stigonematales. Order
Oscillatoriales cyanobacteria were most abundant throughout the studied areas. Based on 16S
rRNA gene sequences and their morphology, two isolates were identified up to species level,
72 to genus level, one isolate up to family and 11 up to order level. Twelve isolates could not
be assigned to any taxonomic level. Three unclassified cyanobacteria did not show any
sequence similarity to previously recorded sequences of known isolates recorded either from
Sri Lanka or from other parts of the world. Therefore sequences of such species were
deposited either as filamentous cyanobacterial species or unicellular cyanobacterial species
according to their morphology. These cyanobacterial isolates could be considered as novel
cyanobacterial genera. Nine other unclassified cyanobacterial isolates had sequence
similarities ranging from 94%- 100% to previously recorded cyanobacteria, however
considering their morphology or 16S rRNA gene sequences they could not be assigned to any
taxonomic level.
According to the positions of the cyanobacteria in the phylogenetic tree, morphology could be
correlated to the 16S rRNA sequences of unknown isolates. The 16S rRNA sequences of nine
unclassified isolates were compared with known cyanobacterial sequences in GenBank and
uncultured cyanobacterium Pal was identified to genus level as Synechococcus, uncultured
cyanobacterium PFB-A2 to genus Leptolyngbya, unicellular cyanobacterium N2a to order
level as Chroococcales cyanobacterium. These results confirmed the usefulness of 16S rRNA
gene as a valuable tool for identification of cyanobacteria up to order or genus level.
Determination of potential microcystin producing ability of cyanobacteria was carried out to
both cultured isolates and environmental samples using mcyA and mcyE genes in the
microcystin synthetase pathway. Results confirmed the genetic potential of microcystin
production of cultured isolates and environmental water samples collected from water
reservoirs using both primers for mcyA and mcyE genes. Based on PCR results and analysis
of nucleotide sequences of mcy gene confirmed the utility of molecular markers for early
detection of potential microcystin producers in both environmental and cultured isolates