Unveiling the Diversity of Periphytic Cyanobacteria (Cyanophyceae) from Tropical Mangroves in Penang, Malaysia

Cyanobacteria are one of the most important groups of photoautotrophic organisms, contributing to carbon and nitrogen fixation in mangroves worldwide. They also play an important role in soil retention and stabilisation and contribute to high plant productivity through their secretion of plant growth-promoting substances. However, their diversity and distribution in Malaysian mangrove ecosystems have yet to be studied in detail, despite Malaysia hosting a significant element of remaining mangroves globally. In a floristic survey conducted in Penang, peninsular Malaysia, 33 morphospecies of periphytic cyanobacteria were identified and described for the first time from a mangrove ecosystem in Malaysia. Sixteen genera, comprising Aphanocapsa, Chroococcus, Chroococcidiopsis, Cyanobacterium, Desmonostoc, Geitlerinema, Leptolyngbya, Lyngbya, Microcystis, Myxosarcina, Oscillatoria, Phormidium, Pseudanabaena, Spirulina, Trichocoleus and Xenococcus, were obtained from field material growing on diverse natural and artificial substrata. Oscillatoriales was the dominant order with Phormidium the dominant genus at nine of the 15 sampling sites examined. Three of the morphospecies, Aphanocapsa cf. concharum, Xenococcus cf. pallidus and Oscillatoria pseudocurviceps, are rare and poorly known morphospecies worldwide. Chroococcus minutus, Phormidium uncinatum, P. amphigranulata, and some species of Oscillatoriales are considered as pollution indicator species. This study provides important baseline information for further investigation of the cyanobacterial microflora present in other mangrove areas around Malaysia. A complete checklist will enhance understanding of their ecological role and the potential for benefits arising from useful secondary metabolites or threats via toxin production to the ecosystem.


INTRODUCTION
Mangroves are marine coastal ecosystems that constitute a transitional forest between the coast and the mainland.Mangroves are highly productive ecosystems and create unique niches for a diversity of plants and animals as well as providing nursery grounds for many benthic and pelagic marine organisms (Yabuki 2004;Alongi 2005;Sundararaman et al. 2007;Rigonato et al. 2013).Malaysia is one of six countries (the others being Indonesia, Australia, Brazil, Mexico and Nigeria) which together host 50.5% of the world's mangroves (Alvarenga et al. 2015).Mangroves are a common feature in coastal areas of Malaysia, with the largest areal contribution particularly along the north-east coast of Sabah.In Sarawak, most mangroves are located in the deltas of the Sarawak, Rejang and Trusan-Lawas Rivers [Food and Agriculture Organisation (FAO) 2014].Mangroves sustain and support production, income and employment in coastal fisheries.
Cyanobacteria are one of the earliest organisms to have evolved on earth and may have existed for 3.5 billion years (Komárek 2016).They play a major role in oxygen production and nutrient cycling, with some members of the group also capable of fixing atmospheric nitrogen (Gehringer & Wannicke 2014).Among the highly diverse microbial communities in mangrove ecosystems, cyanobacteria are one of the most commonly noted organisms, adapted to highly unstable environmental conditions (Alongi 2005;Nedumaran et al. 2008;Rigonato et al. 2013).They are one of the main primary producer groups that support marine fauna and fisheries in mangrove ecosystems (Essien et al. 2008), carrying out the same photosynthetic function as do eukaryotic algae.The organic material produced by these organisms is the foundation of the entire food web in these ecosystems (Dadheech et al. 2013).Cyanobacterial communities can be observed developing on a variety of surfaces in mangroves, such as sediments, roots, leaves and branches (Rigonato et al. 2013).
Cyanobacteria vary morphologically and physiologically in response to different environmental conditions, making them reliable environmental indicators in the mangrove ecosystem (Chaurasia 2015).Morphological characteristics can provide valuable information about the nutrient status of a site.The presence of well-developed multicellular hyaline hairs in many filamentous forms is a response to phosphorus limitation (Chaurasia 2015).A higher number of heterocytes in trichomes is an indicator of water lacking combined nitrogen compared to other nutrients, especially phosphate (Chaurasia 2015).Changes have also been demonstrated in the composition of the cyanobacterial community as a function of water quality.An increase in abundance of heterocytic cyanobacteria such as Calothrix, Scytonema, Nostoc and Rivularia has been observed in response to low nitrate concentrations, while mass growth of Oscillatoriales species can be associated with eutrophication (Mateo et al. 2015).Shifts in species diversity, along with changes in morphological characteristics and photosynthetic behaviour, are reliable indicators that can be utilised to assess environmental changes (Wan Jusoh et al. 2020).
Some cyanobacteria also have proven ability to produce useful bioactive natural compounds, which may have antifungal, antibacterial, antiviral and protease inhibition activities (Shishido et al. 2015;Briand et al. 2016).However, some species can also have strongly detrimental effects on the environment, humans, animals and other organisms through their ability to produce toxins under certain environmental condition (Lopes & Vasconcelos 2011;Neilan et al. 2013;Silva et al. 2014;Briand et al. 2016).Non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes (Silva et al. 2014) are involved in the synthesis of most compounds produced by cyanobacteria.However, to further develop the potential of applied research on these organisms, their diversity and distribution must first be understood.
Amongst the countries hosting the world's mangroves, a significant amount of research on cyanobacteria has been conducted in Brazil alone (Nogueira & Ferreira-Correia 2001;Rigonato et al. 2012;Alvarenga et al. 2015).Although India makes a relatively small contribution to global mangrove extent, it has been an active source of research on mangrove cyanobacteria (Sakthivel & Kathiresan 2013;Diana & Papiya 2014;Ray et al. 2014).
The study of cyanobacterial biodiversity and distribution in brackish environments has been neglected worldwide (Ram & Paul 2021).To our knowledge, studies of cyanobacterial diversity in Malaysia have been conducted solely in freshwater environments and aquaculture ponds, and then only with identification to the genus level (Mohd.Nasarudin & Ruhana 2007;2011a;2011b;Sinang et al. 2015;Sinden & Sinang 2015).The existence and diversity of cyanobacteria colonising Malaysian mangroves requires recognition, identification of their potential as bioindicators or as producers of useful bioactive compounds, and of threats to the ecosystem (including associated and dependent human populations).Here we present the first extensive study of cyanobacterial diversity in a Malaysian mangrove ecosystem.

Sample Collection
A floristic survey was conducted at all accessible mangrove locations along Balik Pulau, located in the south-west of Penang Island, and Gurney, located in the north-east of Penang Island (Fig. 1) between December 2014 and October 2015.Fifteen mangrove sampling sites, including 13 from Balik Pulau and two from Gurney, were examined.Summary descriptions of these sampling sites are presented in Table 1.All visible cyanobacterial growths were sampled from different natural and artificial substrates (Fig. 2).The natural substrates included pneumatophores of mangrove trees, rotting tree branches, rock and sediment, while the artificial substrates included plastic bottles, plastic food containers, plastic bags, PVC pipe and linen rope.Each site was examined and samples of visible mats, gelatinous colonies or crust were collected.Crusts from the substrate were removed by scraping the surface using a spatula while mats and gelatinous colonies were removed by hand.Samples were kept in 60 mL polycarbonate screw-top containers and transported to the laboratory for further analysis.

Microscopic Evaluation
Slides containing fresh material mounted in habitat water were observed under a compound microscope (Olympus BX53) at 100-2,000× magnification.Diacritical characteristics for morphological identification included: vegetative cell width and length, and those of other specialised cells if present; heterocytes and akinetes were recorded and 30 random measurements were taken.Other characteristics of the samples including cell shape and colour, the presence of granules, constriction of cross walls and the structure of apical cells were recorded.Illustrations were made with the aid of a camera lucida.Identifications were made to the lowest taxonomic level possible following Komárek and Anagnostidis (1998;1999;2005) and Komárek (2013a;2013b).The abbreviation "cf." (Latin confertim: to compare with) was used when uncertainty existed, indicating ambiguity between the identified specimen and closely similar morphospecies in the identification key.The high dependence on temperate region keys in identifying specimens can potentially lead to the loss of information on the possibility of a new variety of a given species or even of a novel species.We have taken a conservative approach in listing all the differences between the strains to ensure information on morphospecies that might be useful in confirming variant forms or novel species will be available to future researchers.
Morphospecies was previously recorded from marine environment epiphytic on seaweeds (Komárek & Anagnostidis 1998).Description: Field specimens epiphytic on rotting tree branches (Fig. 2b).Cells densely arranged, bright blue-green, older cells yellowish, and spherical to almost cylindrical with homogeneous content, 4.0 μm-5.5 µm diameter.Reproduction by baeocyte division.Baeocytes spherical to irregular in shape, 1.0 μm-2.0 µm diameter.Sheath communal, thin, hyaline.Ecology: Sites BP-KPB2, BP-KP and G-KM Notes: Cell dimensions consistent with the description of Komárek and Anagnostidis (1998), although this did not include baeocytes.The morphospecies has been recorded as epiphytic on seaweeds in marine habitats (Komárek & Anagnostidis 1998), and from Northern Cyprus in the marine benthos (Ulcay et al. 2015).Trichomes in mat forming flat macroscopic, entangled, colonies suggest placement in Anabaena sp.Akinete morphology is the main characteristic to identify this species (Komárek 2013a).This feature serves as a dormant cell in unfavourable conditions and their absence in the cultured strain has been recorded previously (Komárek 2013b), but hinders the identification of this strain.Description: Colonies on agarised and in liquid media forming dark olive-green to brownish-green crust.Trichome bright blue-green to dark olive-green, isopolar.Cells barrel-shaped to cylindrical, longer than wide, 2.0 μm-2.5 μm wide, 3.8-7.5 μm long.Apical cell rounded.Heterocytes develop in both terminal and intercalary positions, spherical to cylindrical shaped, 3.8-5.0μm × 5.0-6.4μm.Akinetes oval, slightly larger than vegetative cells, 3.8-5.0μm × 5.0-7.5 μm.Ecology: Specimen did not form macroscopic growth in the field a nd w as only observed in cultures.Notes: Specimens recorded only from cultures in agar plate and liquid media.
Ecology: Sites BP-KBAH and G-KRB Notes: Previously recorded as a cosmopolitan species from rocks, stones, wood and other substrata, and rarely from moist soils and brackish swamps (Komárek & Anagnostidis 2005).Description: Field specimens epipelic inside PVC water pipe (Fig. 2h) and periphytic on rotting tree branches (Fig. 2b).Trichomes pale blue-green or bright bluegreen, straight, motile with slowly oscillation movement, isopolar.Cells cylindrical, distinctly longer than wide, 3.0 μm-3.8 μm wide, 5.0 μm-7.5 μm long, slightly constricted at cross walls.Apical cells conical or pointed, slightly bent, attenuated with no calyptra.Sheath absent.Ecology: Sites BP-KPB, BP-KBAH and G-KM Notes: The motility of the trichome and mat forming characteristics suggest placement in Geitlerinema rather than Jaaginema, which has immotile trichomes and occurs mainly in clusters (Komárek & Anagnostidis 2005).This morphospecies recorded as halophilic and epiphytic on other algae and cyanoprokaryotes in saline lakes (Komárek & Anagnostidis 2005).Description: Field specimens epipelic inside plastic bottle (Fig. 2e).Trichomes bright blue-green, straight and entangled, isopolar.Cells cylindrical, slightly longer than wide, 1.8 μm-2.5 μm wide, 3.2 μm-6.2 μm long, slightly constricted at cross walls.Apical cells conically rounded or pointed, not attenuated with no calyptra.Sheath absent.Ecology: Sites BP-KPB4 and BP-KSB1 Notes: Specimens differ from G. tenuius described by Komárek and Anagnostidis (2005) in having the morphology of the cross wall not constricted.G. tenuius is previously recorded from moist soils, shallow ditches and paddy fields (Komárek & Anagnostidis 2005).However, the presence of this morphospecies in brackish or saline water is not well documented.

Leptolyngbya subuliforme (Kützing ex
Previously recorded as widely distributed, occurring in marine ecosystem and on coastal rocks as well as from brackish waters.

Cyanobacteria in Balik Pulau and Gurney Mangrove Ecosystems
The 33 morphospecies identified in this study provide new records of periphytic cyanobacteria occurring in Malaysian mangrove ecosystems.The morphospecies are representatives of the groups Chroococcales, Chroococcidiopsidales, Nostocales, Oscillatoriales, Pleurocapsales, Spirulinales and Synechococcales.
Oscillatoriales was the most widespread group recorded.The dominance of the group has also been noted in previous studies in tropical mangroves (Ram & Paul 2021;Ram & Shamina 2017;Branco et al. 2003;Nogueira & Ferreira-Correia 2001).The ability of members of this group to tolerate considerable fluctuation in salinity, temperature, water volume, light intensity and UV radiation enables their successful colonization in this extreme ecosystem (Mandal & Rath 2015).Members of this group, for example in the genera Phormidium and Oscillatoria, are able to survive under osmotic stress and tolerate a wide range of salinity fluctuation (Mandal & Rath 2015).Mangrove salinity is a major environmental factor that changes rapidly through the tidal cycle, along with temperature changes.Salinity generally increases during the flooding tide and decreases during the ebbing tide, although desiccation of exposed surfaces can conversely lead to increased osmotic stress locally.High temperature will increase evaporation rate thus further increasing salinity.The presence of organic osmoregulatory solutes in members of the group enables them to maintain their intracellular ionic concentration at low levels despite constant inwards diffusion of K + and Cl -ions from the environment (Mandal & Rath 2015).
Seven of the 33 morphospecies recorded in this study were motile.Motility rates differed between them, with six morphospecies (O.pseudocurviceps, P. cf. janthiphorum, P. uncinatum, S. cf.labyrinthiformis, S. meneghiniana and S. cf.robusta) having rapid gliding movement, while one morphospecies (G.attenuatum) showed a slow oscillation movement.Downwards and upwards movements of motile Oscillatoriales allow migration from microbial mat surfaces into soft sediments, to avoid long-term exposure to high levels of ultra-violet radiation (Bagmi et al. 2007).High exposure to solar radiation is common in areas within tropical mangroves and can have an adverse impact on growth, survival, pigmentation, orientation, metabolism and photosynthesis in cyanobacteria (Xue et al. 2005).Motility could also enable these morphospecies to reposition buried trichomes to the surface of the sediment following disturbance during the tidal cycle.
Heterocytous cyanobacteria were the group least recorded in this study compared to unicellular/colonial or non-heterocytous filamentous types.Only three genera were recorded, Anabaena, Desmonostoc and Nostoc, all representing the order Nostocales.Among these, Anabaena sp. and Nostoc sp. were only recorded in culture.There may be two possible explanations for this observation -both morphospecies may have been present in very low abundance, hindering their identification in fresh sample material, or they could derive from wind-dispersed spores from the nearby terrestrial environment.
Most studies of cyanobacteria in tropical mangroves have reported low occurrence of heterocytous species (Ram & Paul 2021;Ram & Shamina 2017;Alvarenga et al. 2015).This may be attributed to the likely high levels of nitrogen and the instability of their macroscopic growth form in this harsh environment.Low nitrogen levels in the environment favour the occurrence of nitrogen-fixing cyanobacteria and this can be reflected in the number of heterocytous groups present (Sakthivel & Kathiresan 2013).Poor adaptation and weak resistance of heterocytous cyanobacteria towards water disturbance may break the weaker connections between the heterocyte and vegetative cells (Stal & Krumbein 1985;Stal 1995).
The most common macroscopic growth form noted in this study was that of mats.Cyanobacterial mats collected from both sampling areas were usually dominated by more than one morphospecies, contradicting the conclusion of Stal (1995) who noted that many cyanobacterial mat types are dominated by a single species.In the present study, the co-occurrence of various species in the mat may relate to the instability of the environment.We suggest that the occurrence of different morphospecies together allows interaction and enables sharing of resources, thereby enhancing survival of the community.The presence of different morphospecies with similar functions, or morphospecies with different functions, could improve overall tolerance of the rapidly changing environment.
Among the cyanobacteria collected, three morphospecies-Aphanocapsa cf.concharum, Xenococcus cf.pallidus and O. pseudocurviceps-are rare and poorly-known morphospecies worldwide.Two of these morphospecies, A. cf.concharum and O. pseudocurviceps, were found both in Balik Pulau and Gurney.X. pallidus has previously been collected from the marine ecosystem (Komárek & Anagnostidis 1998), but here was only recorded in the mangrove area, and only at the Balik Pulau sampling site.
Chroococcus minutus in this study was epiphytic on rotting tree branches from Balik Pulau and on a pneumatophore in Gurney, while X. schousboei was recorded from rotting tree branches in both areas.Submerged parts of mangrove trees, branches and pneumatophores potentially serve as habitat for epifloral and faunal communities (Sundararaman et al. 2007;Mohamed & Al-Shehri 2015).However, previous records of both C. minutus and X. schousboei are from sediment (Hussain & Khoja 1993;Branco et al. 2003;Sakthivel & Kathiresan 2013;Mohamed & Al-Shehri 2015).The occurrence of these two morphospecies appears not to be entirely dependent on attachment structure, allowing them to be more widespread on a wider range of available substrata.
Lyngbya cf.aestuarii was found growing on diverse substrates in this study.Similar observations have been reported from mangroves in India (Sakthivel & Kathiresan 2013).Members of the genus Lyngbya, together with Oscillatoria, Phormidium and Microcoleus, are widespread in mangrove areas (Sundararaman et al. 2007).Lyngbya aestuarii was previously recorded as a dominant mat-forming cyanobacteria in hypersaline Laguna Guerrero Negro, Mexico (Bagmi et al. 2007).The widespread occurrence of this species may be attributed to the presence of the sheath, which helps in binding cells to support their structure and attachment to sand grains (Rajeev et al. 2013;Rossi & De Philippis 2015).The sheath pigment, scytonemin, also gives effective protection from excessive ultra-violet exposure (Rastogi & Incharoensakdi 2014), reduces dehydration (Rossi & De Philippis 2015) and acts as a structural defence against predators (Camacho & Thacker 2006).
Phormidium cf.nigroviride was found mainly on sediment inside plastic food containers.Similar morphospecies have been recorded previously but differ in their type of attachment, occurring on pneumatophores and on sediment (Sakthivel & Kathiresan 2013;Mohamed & Al-Shehri 2015).In this study, P. cf. nigroviridis was collected at exposed sampling sites and near to aquaculture farms and residential areas, which had high abundance of plastic containers and debris.This species may not require a specific substrate for attachment but rather display a more opportunistic behaviour that allows it to thrive at such disturbed sites.Spirulina cf.labyrinthiformis and S. meneghiniana were found epipelic on sediments.S. labyrinthiformis was previously recorded as ubiquitous in mangrove areas (Hussain & Khoja 1993;Lugomela et al. 2001;Sakthivel & Kathiresan 2013;Ahmad et al. 2016).Sakthivel and Kathiresan (2013) reported this morphospecies from all three sampling sites examined in a study on the south-east coast of India, Pichavaram, Porto Novo and Mudasal Odai.A similar pattern observed in the present study further supports the ubiquity of these species.
Comparison of the morphospecies recorded in this study with those from other mangrove ecosystems worldwide revealed only seven morphospecies to be common.The present study raises the possibility of the cyanobacterial microflora of Penang mangroves being distinct from other assemblages around the world.Limitations in the identification keys currently available, and particularly their reliance on material derived from temperate regions, could provide a significant source of confusion as well as the loss of important information on endemic and rare species occurring in tropical regions.There is clearly a need to further develop taxonomic knowledge and keys to fully assess the diversity of this group.Robust identification of many tropical cyanobacteria requires further integrated study of their morphological, ecological and molecular characteristics in Malaysian and other mangrove ecosystems.
Land conversion is already extensive in both mangrove areas surveyed in this study.Hundreds of hectares in the mangrove forest area of Pulau Betung have been developed to provide shrimp ponds and residential areas.Large parts of the Gurney Drive mangrove area have also undergone reclamation and with the development of roads and buildings.These disturbances are reflected by the dominance of the Oscillatorialles in our data.Members of the Oscillatorialles are excellent indicators of eutrophication related to alterations in land use (Chaurasia 2015), as observed at this study site.The highly enriched wastewater from commercial aquaculture is likely to have triggered a rapid response by these cyanobacteria, leading to their dominance over heterocytous species.Floristic studies can provide important baseline information not only on the diversity but also on the potential use of cyanobacteria in monitoring the health of mangroves in other mangrove areas around Malaysia and more widely.

Figure 1 .
Figure 1.Map showing the study sites at Penang Island, Malaysia (centre).(a) Balik Pulau and (b) Gurney.Source: Derived from ArcGis.

Figure 2 .
Figure 2. A variety of periphytic cyanobacteria growing on different substrates.(a) bluegreen mats on a pneumatophore (arrow); (b) brown mats on a rotting tree branch (arrow); (c) olive-green crust on rocks (arrow); (d) blue-green mats on sediments (arrow); (e) bluegreen crust inside a plastic bottle (arrow); (f) blue-green crust inside a plastic food container (arrow); (g) dark blue-green crust inside a plastic bag (arrow); (h) dark blue-green mats on sediments inside PVC pipe (arrow); (i) dark blue-green mats on linen rope (arrow).

Table 2 .
List of cyanobacteria identified from mangroves sampled Balik Pulau and Gurney.
Khusnul et al. (2014)r cell diameter, 4.0 μm-8.0 μm.The morphospecies was previously recorded from a salt marsh in California and is known to occur on coastal rocks in the Mediterranean(Komárek & Anagnostidis  1998).TikaKhusnul et al. (2014)recorded Myxosarcina sp. as epiphyte on an Avicennia marina pneumatophore in a mangrove ecosystem in Indonesia.