Aliinostoc bakau sp. nov. (Cyanobacteria, Nostocaceae), a New Microcystin Producer from Mangroves in Malaysia

: A new microcystin-producing mangrove cyanobacterium, Aliinostoc bakau sp. nov., was isolated from a tropical mangrove in Penang, Malaysia, and characterized using combined morphological and phylogenetic approaches. Cultures were established in liquid media of different salinities (0, 7, 14, 21, 28, and 35 ppt). Optimal growth observed at both 7 and 14 ppt was consistent with the origin of the strain from an estuarine mangrove environment. Phylogenetic analysis based on the 16S rRNA gene strongly indicated that the strain is a member of the genus Aliinostoc and is distinct from other currently sequenced species in the genus. The sequences and secondary structure of the 16S–23S ITS region D1–D1’ and Box–B helices provided further confirmation that the new species is clearly distinct from previously described Aliinostoc species. Amplification of the mcyE gene fragment associated with the production of microcystin in A. bakau revealed that it is identical to that in other known microcystin-producing cyanobacteria. Analysis of the extracts obtained from this strain by HPLC-MS/MS confirmed the presence of microcystin variants (MC-LR and -YR) at concentrations of 0.60 µ g/L and MC-RR at a concentration of 0.30 µ g/L. This is the first record of microcystin production from Aliinostoc species in tropical mangrove habitats.


Introduction
Mangroves are coastal ecosystems that constitute a transitional forest between the sea and land.These ecosystems are extremely productive and provide niches for a diversity of microbes, plants, and animals, as well as breeding grounds for various benthic and pelagic species [1][2][3].Cyanobacteria are key species adapted to the generally very changeable circumstances among the highly varied microbial communities observed in mangrove habitats [1,3].They are also one of the main primary producers supporting marine fisheries that rely on mangrove ecosystems [4].The organic material produced by these organisms at the base of the mangrove ecosystem cascades through multiple levels in the food web [5].
The majority of the mangroves in the world (50.5%) are found in Malaysia, along with Indonesia, Australia, Brazil, Mexico, and Nigeria [6].Mangroves are a common feature in the coastal areas of Malaysia, reaching their greatest extent on the north-east coast of Sabah, where about 60% of the country's coverage occurs.In Sarawak, a further 23% is located in the deltas of the Sarawak, Rejang, and Trusan-Lawas rivers, and the remaining 17% is found along the east and west coasts of peninsular Malaysia [7].Most published studies of mangroves in Malaysia have focused on faunal diversity, primarily on fish diversity and larval abundance [8,9], mollusks ( [10][11][12]), horseshoe crabs [13], mud crabs [14], and food web interactions [15].Studies of micro-organisms have been conducted only on selected groups, for instance on the diversity and features of Actinobacteria [16,17], Proteobacteria [18], and Firmicutes [19].The diversity of macroalgae [20][21][22] and diatoms [23] has also been studied.
The genus Aliinostoc Bagchi, Dubey, & P. Singh was established with the type species A. morphoplasticum Bagchi, Dubey, & P. Singh [47], based on molecular assessment confirming that it was distinct from other Nostoc-like genera.Although morphological characteristics alone do not distinguish members of Aliinostoc from Nostoc sensu stricto, the loosely arranged filaments and motile hormogonia with gas vesicles are considered diacritical features of the genus [47,48].The genus originally included six species, all originating from tropical regions.Three of these, A. constrictum Kabirnataj & al. [48] A. alkaliphilum Christodoulou, Economou-Amilli, M. Fiore, & Sivonen [51], and A. vietnamicum S. Maltseva, E. Kezlya, & Y. Maltsev [52].All are characterized based on 16S rRNA gene sequences, as well as morphological and ecological features.
Several studies have reported toxin production by mangrove cyanobacteria.These include one record from the island of Guadeloupe [53], 18 records from two islands in Brazil [30], two records from the Red Sea coast of Saudi Arabia [54,55], and one record from the northern coast of the Persian Gulf, Iran [56].All these records originated from subtropical mangroves.The present study aims to identify a novel cyanobacterial strain capable of producing MCs obtained from a tropical estuarine mangrove forest in Pulau Betong, Malaysia.Phylogenetic analysis shows that the new strain is included in a distinct clade with previously identified Aliinostoc strains.Based on morphological and molecular assessments, we establish the new species Aliinostoc bakau sp.nov., following the procedures of the International Code of Nomenclature for algae, fungi, and plants [57].

Site Description and Sampling
The microbial mat sample used in this study was obtained from a rotting tree trunk in the intertidal zone of an estuarine mangrove area in Kampung (Kg.) Pulau Betung, Penang (5 • 18 ′ 23.3 ′′ N, 100 • 12 ′ 02.4 ′′ E) (Figure 1), located close to a residential area and shrimp aquaculture ponds.Field samples were collected as part of a broad study of mangrove cyanobacteria, based on the visual presence of macroscopic mats, gelatinous colonies, and the crusts of cyanobacteria on both natural and artificial substrates.Each sample was stored in a separate 60 mL sterile polycarbonate screw top container and immediately transported to the laboratory in Universiti Sains Malaysia, Penang, for further analysis.

Site Description and Sampling
The microbial mat sample used in this study was obtained from a rotting tree trunk in the intertidal zone of an estuarine mangrove area in Kampung (Kg.) Pulau Betung, Penang (5°18′23.3″N, 100°12′02.4″E) (Figure 1), located close to a residential area and shrimp aquaculture ponds.Field samples were collected as part of a broad study of mangrove cyanobacteria, based on the visual presence of macroscopic mats, gelatinous colonies, and the crusts of cyanobacteria on both natural and artificial substrates.Each sample was stored in a separate 60 mL sterile polycarbonate screw top container and immediately transported to the laboratory in Universiti Sains Malaysia, Penang, for further analysis.

Culture Establishment
The mat, from which the strain reported here was obtained, was dominated by Oscillatoriales.A small sub-sample of the field material was cultured in both full-strength BG 11 and BG110 (lacking nitrogen with 1% agarised medium) media [58].The culture media were supplemented with 0.1 µg/L vitamin B12, artificial seawater (Instant Ocean) that was adjusted to 6 ppt salinity, and 100 µg/mL cycloheximide to eliminate eukaryotes [59].Isolation was carried out after a 2-week incubation by removing single colonies from the original enrichment culture and transferring them onto new culture media.All cultures were incubated at 25 °C and a 12:12 h light:dark cycle under a white, fluorescent lamp (±27.03µmol photon m −2 s −1 ) for 2 weeks before microscopic examination.The present strain was only observed growing in culture and was not detected in the initial observation of the field specimen.After isolation, the strain was grown in 50 mL BG110 liquid cultures in a range of salinity conditions (0, 7, 14, 21, 28, and 35 ppt), with all other

Culture Establishment
The mat, from which the strain reported here was obtained, was dominated by Oscillatoriales.A small sub-sample of the field material was cultured in both full-strength BG 11 and BG11 0 (lacking nitrogen with 1% agarised medium) media [58].The culture media were supplemented with 0.1 µg/L vitamin B12, artificial seawater (Instant Ocean) that was adjusted to 6 ppt salinity, and 100 µg/mL cycloheximide to eliminate eukaryotes [59].Isolation was carried out after a 2-week incubation by removing single colonies from the original enrichment culture and transferring them onto new culture media.All cultures were incubated at 25 • C and a 12:12 h light:dark cycle under a white, fluorescent lamp (±27.03µmol photon m −2 s −1 ) for 2 weeks before microscopic examination.The present strain was only observed growing in culture and was not detected in the initial observation of the field specimen.After isolation, the strain was grown in 50 mL BG11 0 liquid cultures in a range of salinity conditions (0, 7, 14, 21, 28, and 35 ppt), with all other conditions as described above.Good growth was observed at a salinity of 7 ppt, and the cultures were maintained at this salinity to achieve the biomass required for further analysis.

Morphological Characterisation
Morphological examination was carried out on the strain in culture using an Olympus BX-53 (Olympus America Inc., Center Valley, PA, USA) bright field microscope at 100-2000× magnification.Detailed descriptions of morphological characteristics were recorded, including trichome color and shape, vegetative cell length and width, shape, and dimensions of heterocyst and akinetes, apical cell shape, and the presence or absence of a sheath.Measurements were made on 30 replicates of randomly chosen specimens.Illustrations were made with the aid of a camera lucida.Initial morphological identification followed [49][50][51][52]60,61].

Molecular Analyses
Colonies were aseptically transferred from liquid media into 1.5 mL microcentrifuge tubes containing 1 mL BG11 0 (lacking nitrogen) liquid culture medium.DNA was then extracted using the Intron G-spin™ Total DNA Extraction Mini Kit for bacteria (iNtRON Biotechnology, Inc., Seongnam, Kyonggi-do, Republic of Korea), following the manufacturer's instructions.The extracted DNA was stored frozen at −20 • C. The quality of the extracted DNA was determined using a Nanodrop Quawell UV Spectrophotometer Q3000 (Thermo Fisher Scientific, Loughborough, UK).The extracted DNA was used to amplify an approximately 1300 bp product of the 16S rRNA gene using the combination of primers 2 (5 ′ -GGG GGA TTT TCC GCA ATG GG-3 ′ ) and 3 (5 ′ -CGC TCT ACC AAC TGA GCT A-3 ′ ) [62].The 16S-23S ITS region was amplified using primers 1 (5 ′ -CTC TGT GTG CCT AGG TAT CC-3 ′ ) and 5 (5 ′ -TGT AGC TCA GGT GGT TAG-3 ′ ) [62].Thermal cycling conditions were 94 • C for 4 min for pre-denaturation, followed by 35 cycles of denaturation at 95 • C for 15 s, annealing at 55 • C for 15 s, extension at 72 • C for 20 s, and a final extension at 72 • C for 7 min.The genes were amplified using the polymerase chain reaction (PCR) with 25 µL of MyTaq™ Red Mix (Bioline, Little Clacton, UK), 2 µL of each 20 µM forward and reverse primer, 2 µL of approximately 5-10 ng of genomic DNA template, and 19 µL of milli-Q, giving a final volume of 50 µL.An extra reaction tube with all the chemicals except for the DNA template was used as a negative control.
PCR products were loaded onto a 1% agarose electrophoresis gel stained with 1 µL ready-to-load RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Inc., Seongnam, Kyonggi-do, Republic of Korea) and inserted into a gel tank with 0.5 × TBE running buffer (iNtRON Biotechnology, Inc., Seongnam, Kyonggi-do, Republic of Korea).Amplified DNA was separated by electrophoresis and visualised with the power system supplied by Major Science MP-300V (100 V, 500 mA) for 30 min.A Bioneer 100 bp DNA ladder was also loaded to determine the size of the DNA extracted.The gel was viewed using Syngene GeneFlash Bio Imaging Gel Documentation UV/VVIS.The PCR product was then purified using the MEGAquick-spinTM Total Fragment DNA Purification Kit (iNtRON Biotechnology, Inc., Seongnam, Kyonggi-do, Republic of Korea).The purified PCR product was sequenced commercially by the Bioneer Corporation (Daejeon, Republic of Korea).

Screening for Cyanotoxin Genes
The extracted DNA was screened for five cyanotoxin genes using PCR: the mcyE gene coding for microcystin (MCY) production in microcystin producers was amplified using the primer pair mcyE-F2/R4 [63]; the ndaF gene in nodularin (NOD) producers was amplified using the primer pair HEP-F/R [64]; the anaC gene for anatoxin (ATX) production was amplified using primers anxgen-F/R [65]; the cyrJ gene-encoding cynlindrospermopsis (CYN) was amplified with cynsulf-F and cynlnam-R [66]; and the gene encoding for saxitoxin (SXT) was amplified with sxtAF/R [67].Extra reaction tubes with these chemicals and DNA template strains, which were previously confirmed to be from microcystin (Microcystis aeruginosa FSS-164), nodularin (Iningainema pulvinus ES0614), saxitoxin (Dolichospermum circinale FSS-124), anatoxin (Anabaena UHC0054), and cylindrospermopsin (Raphidiopsis raciborski FSS-127) producers, were used as positive controls.A positive result in a specific PCR would indicate that the strain contained the respective gene and, if so, the PCR product was purified and sequenced as described above.Only the mcyE gene was identified by the BLAST (Basic Local Alignment Search Tool) and, hence, we tested the strain for microcystin production specifically.A 20 mL sample was sent to the Cawthron Institute, New Zealand, for quantifying microcystin concentration using HPLC-MS/MS analysis [68,69].

Phylogenetic Analysis and ITS Folding
All sequences were edited and assembled using the Geneious Prime software package (Biomatters, http://www.geneious.com(accessed on 25 December 2023)).Sequences that shared more than 97% sequence identity with our strains were considered as the same operational taxonomic unit (OTU) [70].Multiple sequence alignments were built with the MUSCLE algorithm in Geneious Prime (Biomatters Ltd., Auckland, New Zealand) and then manually checked.Phylogenetic trees were constructed using two different methods: maximum likelihood (ML) and Bayesian inference (BI).ML analyses were performed using RaxML v8 [71] in Geneious using the general time-reversible invariant-sites (GTRI) nucleotide substitution model with 1000 bootstrap replicates.BI analyses were carried out using MrBayes v3.1.2[72] as implemented in Geneious under the GTR + I + Γ model of sequence evolution.Two independent analyses, each consisting of four simultaneous Markov chains, were run for 3,000,000 generations, and trees were sampled every 100 generations.Log likelihood and parameter values were determined with the Tracer program v1.5 [73].The first 25% of trees were discarded as burn-in, and the remaining trees were used to compute the Bayesian posterior probability values.ML and BI trees were edited in FigTree v1.3.1 [74].Uncorrected pairwise genetic distances (p-distance) were calculated in MEGA X [75].
The 16S-23S ITS region was used for the modelling of secondary structure folding.The tRNA genes were identified using tRNAscan-SE 2 [76].The secondary structure of the D1-D1' and Box-B helices was modelled using the Mfold WebServer with default conditions.

Results
The new strain was isolated from an oscillatorialean-dominated mat and showed the diagnostic traits of the genus Aliinostoc Bagchi, Dubey, & P. Singh; however, it has morphological characteristics that are distinct from those of previously described species in the genus.Phylogenetic analysis based on the 16S rRNA gene also placed this strain within the clade of the genus Aliinostoc species (see below), but as phylogenetically distinct from other Aliinostoc species.On this basis, we propose establishing the new species of Aliinostoc.
Holotype.Strain USMNA collected by Faradina M. and Nur Afiqah A. R. Strain deposited in the School of Biological Sciences Herbarium, Universiti Sains Malaysia, Malaysia, and the Faculty of Fisheries, Kasetsart University, Thailand.

GenBank accession number. 16S rRNA (MH182619)
Colonies with trichomes irregularly twisted and embedded within a common mucilage conform with the genus Nostoc.This genus is widespread with over 200 species recorded to date, making it difficult to distinguish morphospecies confidently using morphological evaluation [77,78].Comparison with previously described Nostoc species showed a resemblance in the size of vegetative cells, heterocytes, and akinetes to Nostoc passerinianum Bornet et Thuret ex Bornet et Flahault [78]; however, colony morphology, heterocyst and akinete shapes, and habitat differ from the present specimen.

Phylogenetic Analysis
The partial 16S rRNA gene sequences of 1316 bp were successfully amplified from A. bakau sp.nov.USMNA (MH182619) and trimmed to 1143 bp to provide fully bidirectional contiguous sequences.The trees constructed using ML and BI analyses were largely congruent and, therefore, only the ML tree is presented (Figure 3).Sequences of Aliinostoc species, including A. bakau, and four strains labelled as 'Nostoc' sp.formed a well-supported clade (ML = 99%, BI = 1.00).Our new species was grouped with 'Nostoc' sp.(MN864652) with high bootstrap support (ML = 100%, BI = 1.00) and sister to all other Aliinostoc species (Figure 3).
Genetic distances within the Aliinostoc clade were 0-4.4%, and A. bakau and other Aliinostoc species were at least 2.5% divergent.The sequences of A. bakau obtained here were identical to that of 'Nostoc' sp.(MN864652) from a mangrove in Iran and, therefore, the latter strain should be re-examined with the consideration of re-naming it A. bakau.contiguous sequences.The trees constructed using ML and BI analyses were largely congruent and, therefore, only the ML tree is presented (Figure 3).Sequences of Aliinostoc species, including A. bakau, and four strains labelled as 'Nostoc' sp.formed a well-supported clade (ML = 99%, BI = 1.00).Our new species was grouped with 'Nostoc' sp.(MN864652) with high bootstrap support (ML = 100%, BI = 1.00) and sister to all other Aliinostoc species (Figure 3).

ITS Secondary Structure
The ITS region sequence of A. bakau was 641 bp long, containing the sequences coding for two tRNA molecules (tRNA Ala and tRNA Ile ).The D1-D1' and Box-B helices, a semiconserved region of the 16S-23S ITS region, were analyzed.The putative secondary structures of A. bakau, together with other Aliinostoc species, are presented in Figures 4 and 5.
The comparison of secondary structure provides additional information in confirming the identity of the new species in comparison with previous records.

Microcystin Production from A. bakau
A partial mcyE gene sequence (733 bp) associated with the microcystin biosynthetic pathway was successfully amplified from the strain.Phylogenetic analysis of this sequence with comparable sequences from other microcystin-producing cyanobacteria revealed that the sequence of A. bakau sp.Nov. USMNA (MT982365) was similar to those obtained from 'Nostoc' sp., Anabaena sp., N. linckia, and Anabaena lemmermannii, and formed a clade with other mcyE-producing strains, with 0.99/100 bootstrap values for ML and BI analyses (Figure 6).
bakau were clearly distinct from A. catenatum and A. magnakinetifex by the length of the basal stem and sequences of internal and terminal loop (Figure 5).On the other hand, the pattern in the Box-B secondary structure of A. bakau was identical to A. alkaliphilum, A. morphoplasticum, and A. vietnamicum by having a 5 bp long basal stem (CAGCA-UGCUG), a 3-residue asymmetrical internal loop, and a 4-residue terminal loop (Figure 5).The differences among these four species were mainly based on the nucleotide sequence at position 10 (A. bakau vs. A. alkaliphilum and A. vietnamicum), and 18 (A.bakau vs. A. vietnamicum), and terminal loop (AAUU for A. morphoplaticum, GAAA for A. alkaliphilum, GAGA for A. vietnamicum, and GCUA for A. bakau) (Figure 5).
The comparison of secondary structure provides additional information in confirming the identity of the new species in comparison with previous records.

Microcystin Production from A. bakau
A partial mcyE gene sequence (733 bp) associated with the microcystin biosynthetic pathway was successfully amplified from the strain.Phylogenetic analysis of this sequence with comparable sequences from other microcystin-producing cyanobacteria revealed that the sequence of A. bakau sp.Nov. USMNA (MT982365) was similar to those obtained from 'Nostoc' sp., Anabaena sp., N. linckia, and Anabaena lemmermannii, and formed a clade with other mcyE-producing strains, with 0.99/100 bootstrap values for ML and BI analyses (Figure 6).

Discussion
This study establishes a new microcystin-producing species of cyanobacteria, Aliinostoc bakau sp.nov., isolated from a tropical estuarine mangrove environment in Malaysia.The genus Aliinostoc is morphologically indistinguishable from Nostoc except for the loosely arranged filaments and formation of motile hormogonia with gas vesicles [47].The new species differs from the other five currently recognised species of Aliinostoc in the absence of hormogonia (Table 1).Although the formation of hormogonia with gas vesicles has been proposed to be diagnostic for the genus [47], the current study suggests otherwise.
The capacity to produce motile hormogonia has been reported to be inconsistently present or entirely lacking in Nostoc-like strains [58,79].Environmental conditions, including exposure to various light wavelengths, have been shown to either induce differentiation into hormogonia or suppress such development [80][81][82].Furthermore, under specific conditions, hormogones may be retained within the colony, undergoing developmental processes without subsequent release, resulting in an increased number of trichomes within the colony [83].While the formation of hormogonia has been previously described in both A. magnakinetifex and A. catenatum, the motility of this structure has not been explicitly mentioned [48].
The genus was erected based on the type species, A. morphoplasticum, which originated from a stagnant, eutrophic, polluted pond.The specimen was collected amongst benthic rocks and other submerged substrates [47].Gas vesicles are common in planktonic cyanobacteria and they provide buoyancy [84]; however, non-planktonic species have been reported to form them only under certain conditions [85].In A. alkaliphilum, A. morphoplasticum, and A. vietnamicum, the formation of hormogonia with gas vesicles observed in culture suggests that these species have a planktonic phase in their life cycle.The gas vesicles may play an important role in facilitating hormogonia dispersal by increasing their time in suspension in the water column before sedimentation [86].The hormogonia of A. magnakinetifex and A. catenatum, both collected from garden soil, lacked gas vesicles.The absence of hormogonia in A. bakau, and the absence of gas vesicles together with unknown motility in the hormogonia of A. magnakinetifex and A. catenatum, indicate that the possession of motile hormogonia with gas vesicles is not diagnostic for the genus.In contrast to akinetes, hormogonia represent a transient life stage lasting for only 1-2 days [87].Hence, we propose the shape and dimensions of both heterocytes and akinetes as diagnostic morphological features for the identification of the genus.
Genetically, A. bakau is well separated from the previously described species of Aliinostoc.Based on available GenBank 16S rRNA sequences, the current strain is identical to 'Nostoc' sp.(MN864652; deposited in December 2019), which was isolated from a mangrove environment in Iran.Although 'Nostoc' sp.(MN864652) lacks morphological characterization to enable a reliable comparison with our strain, the genetic evidence presented here strongly supports the correct identity for that strain to be A. bakau, highlighting the need for caution in the use of species names assigned in such publicly accessible databases.Similarly, based on the analyses conducted here, we suggest that three further strains, 'Nostoc' elgonese TH3S05 (AM 711548), 'Nostoc' sp.CENA543 (CP023278), and 'Nostoc' sp.SK6A-PS (OQ247923) should also be reclassified into the genus Aliinostoc.The names applied in GenBank for these previously deposited strains were assigned before the genus Aliinostoc was established [47].
The modelling of the secondary structure of the ITS region indicated that the structure of the D1-D1' and Box-B regions was distinct in both sequence and folding patterns from other Aliinostoc species.Previous studies have shown that the analysis of 16S gene phylogeny, coupled with the secondary structure of the ITS region, provides a better tool for the separation of cyanobacteria species [49][50][51][52]62,77,88,89].
The amplification of a fragment of the mcyE gene involved in MC biosynthesis from the A. bakau strain USMNA confirmed that the species has a genetic determinant vital for MC production, consistent with the result of HPLC-MS/MS analysis.This is the first member of the genus Aliinostoc shown to be capable of toxin production and only the fifth species of toxin-producing cyanobacteria identified from mangrove ecosystems worldwide.MC production has been detected in benthic mats in Red Sea mangrove swamps, with the presence of variants MC-YR, MC-LR, and MC-RR at higher concentrations of 4.89-9.74µg/L [54] than recorded in the current study (0.0-0.60 µg/L).World Health Organization provisional guidelines [90] for microcystin consumption stipulate a maximum concentration of 1 µg/L.
The role of toxin-producing cyanobacteria in mangrove ecosystems is currently unknown.Previous studies have reported that known major producers of cyanotoxins are planktonic and able to form toxic blooms [91]; however, in mangrove habitats, blooms of benthic cyanobacteria are common [30,[54][55][56].Where they are present in algal mats, cyanotoxin producers may pose ecological risks to grazers if the toxins are produced at sufficiently high concentrations.Invertebrates that feed on toxic cyanobacteria can serve as vectors transferring cyanotoxins to higher trophic levels and even into the human food chain, for instance by the consumption of contaminated seafood [56].Studies conducted in tropical and temperate lakes have reported MC accumulations ranging from 0.5 to 1917 and 4.5-215.2µg/kg wet mass in muscle tissue and whole fish, respectively [92].These levels are generally higher than health guideline values (24 µg/kg of whole organism) for MC levels in seafood and, therefore, pose a significant risk for human consumption [93].
In previous investigations of sub-tropical mangroves, the prevalence of toxin-producing cyanobacteria was primarily attributed to non-heterocytous species [30,[54][55][56].A study from Brazil reported the highest number of microcystin (MC) producers, with 18 out of 55 identified species originating from two sampling localities [54].The current study unveils a novel finding in the identification of a heterocytous cyanobacteria with toxin-producing abilities identified from a total of 33 species recorded from a tropical mangrove ecosystem [94]. A. bakau's ability to differentiate specialized cells allows exceptional adaptation to environmental stressors, including osmotic stress, desiccation, increasing temperatures, limited nutrient availability, and prolonged darkness [95,96].The ability to form heterocyst allows A. bakau to survive in a nitrogen deprived environment, while akinetes increase resilience to fluctuating environmental conditions [95].The formation of specialised cells highlights the potential long-term impact of A. bakau's survival mechanisms in a changing environment.
Escalating human population pressures in South-east Asia, including in Malaysia, have led to the extensive destruction of mangrove forest habitats, primarily attributable to anthropogenic activities including land reclamation, aquaculture expansion, oil palm expansion, the development of human settlements, and industrial expansion [97].These human-induced alterations pose a substantial threat to the microbial communities inhabiting tropical mangrove environments.While the response of toxin-producing cyanobacteria to environmental stimuli remains unknown within mangrove ecosystems, an earlier study on phytoplankton diversity and community composition in a Malaysian mangrove revealed the dominance of a single species in a disturbed habitat [98].Further research is needed to understand both the survival strategies of A. bakau and the physical stimuli that induce toxin production in order to fully assess potential future threats.An environmental monitoring programme should be established, particularly when animals or plants intended for human consumption are harvested from this habitat.
The general lack of microbiological studies, and specifically of cyanobacteria, in mangrove habitats, is exemplified by our identification of the first strain capable of MC production in Malaysia, a country with some of the most important remaining expanses of mangrove forest globally.Further studies across other mangroves in these regions are required to facilitate identification of other toxin-producing cyanobacteria and to clarify their functional roles in the mangrove environment.

Figure 1 .
Figure 1.Location map of study site.(A) Peninsular Malaysia, and location of Penang (arrow).(B) Penang, and location of Kampung (Kg.) Pulau Betung (red circle).(C) Study site shown by red circle.

Figure 1 .
Figure 1.Location map of study site.(A) Peninsular Malaysia, and location of Penang (arrow).(B) Penang, and location of Kampung (Kg.) Pulau Betung (red circle).(C) Study site shown by red circle.

Figure 3 .
Figure 3. ML tree based on the partial 16S rRNA gene sequence showing the phylogenetic position of Aliinostoc bakau sp.nov.(MH182619).ML bootstrap values (left) and Bayesian posterior probabilities (right) are indicated at the nodes.Bootstrap values of >90% for ML and >0.90 for BI are presented, and full support is indicated by asterisk (*).

Figure 3 .
Figure 3. ML tree based on the partial 16S rRNA gene sequence showing the phylogenetic position of Aliinostoc bakau sp.nov.(MH182619).ML bootstrap values (left) and Bayesian posterior probabilities (right) are indicated at the nodes.Bootstrap values of >90% for ML and >0.90 for BI are presented, and full support is indicated by asterisk (*).

Figure 6 .
Figure 6.Phylogenetic tree based on the partial mcyE gene sequence showing the phylogenetic position of Aliinostoc bakau USMNA (MT982365).ML bootstrap values (left) and Bayesian posterior probabilities (right) are indicated at the nodes.Asterisk (*) indicates full support (100%, 1.0) in both analyses.

Figure 6 .
Figure 6.Phylogenetic tree based on the partial mcyE gene sequence showing the phylogenetic position of Aliinostoc bakau USMNA (MT982365).ML bootstrap values (left) and Bayesian posterior probabilities (right) are indicated at the nodes.Asterisk (*) indicates full support (100%, 1.0) in both analyses.Analysis of the extract from strain USMNA by HPLC-MS/MS confirmed the presence of microcystin variants MC-LR and MC-YR at concentrations of 0.60 µg/L and MC-RR at a concentration of 0.30 µg/L.