Epiphytic Subaerial Algae as Biological Indicators of Air Pollutants in Bangkok, Thailand

1. Introduction

Epiphytic subaerial algae are a widespread subgroup of photosynthetic organisms living on aerial surfaces [1,2,3,4]. They are either single-celled or form colonies or filaments that, when sufficiently abundant to produce mats or crusts, are visible to the naked eye. Epiphytic subaerial algae differ in many aspects from algae that live in water or in conditions of high humidity. Many subaerial algae can tolerate drought and low atmospheric humidity, although they are metabolically active only when there is liquid water or a high atmospheric moisture [2,4,5,6]. Most subaerial algae are green algae (phyla Chlorophyta & Charophyta) and Cyanobacteria with a high tolerance to ultraviolet (UV) radiation and a reduced dependence on water [7,8,9]. Epiphytic subaerial algae grow on the surface of trunks, leaves, flowers, and fruits of vascular plants all over the world, but they are usually most abundant in humid tropical environments, from plains to highlands [1,2,3,10].

Epiphytic subaerial algae can grow on the surface of trees in many settings, from forests to city boulevards, although their diversity and abundance are influenced by the different climatic conditions of different regions [1,2,4] and specific conditions of local habitats. Species growing in urban environments can tolerate air pollution and can be used as biological indicators of air pollution in different areas and weather conditions [11,12,13]. Some studies have focused on the diversity and abundance of these organisms in urban areas with high levels of air pollutants due to emissions from automobiles, industrial plants, and households [14,15,16]. [16] and [17] argued that some of these organisms may be used as bioindicators of specific air pollutants such as sulfur dioxide (SO₂), nitrogen oxides (NO_x), carbon monoxide (CO), and fine particulate matter (PM_2.5).

Bangkok is the capital city of Thailand, and it is governed by the Bangkok Metropolitan Administration. The city is located in the tropical region, with a population of more than 10 million people [18,19,20]. Today, it suffers from severe air pollution [20,21]. The pollution derives from emissions by cars, factories, and various types of waste [22], such as hydrocarbons, particulate matter (PM₁₀), carbon monoxide (CO), sulfur dioxide (SO₂), nitrogen oxides (NO_x), benzene, and black smoke from diesel-powered vehicles [23,24,25]. Epiphytic subaerial algae that grow in green spaces such as parks and urban forests are exposed to high levels of such pollutants. It is therefore plausible that some species might be used as biological indicators of current climate conditions and pollution levels. Identifying species and assessing their distribution and possible correlation with amount and/or type of air pollutants may therefore guide researchers in recognizing tolerant species that can withstand city pollution. This information can then be used for further studies focusing on several aspects of the biology of these organisms.

The objectives of this study were to determine the diversity and abundance of epiphytic subaerial algae at several sites within the Metropolitan Region of Bangkok and identify species/taxa that might be used as indicators of air pollution in this area.

2. Materials and Methods

2.1. Study Sites

Extensive collections of epiphytic subaerial algae were made in May 2022 (the wet season, which in Bangkok spans from May to October) from five parks selected as sampling sites in the Bangkok Metropolitan Region (hereafter reported simply as Bangkok). They were located in the city’s northern area (Vachirabenjatas Park, or Rot Fai Park), eastern area (Suan Luang Rama IX), central area (Lumpini Park), western area (Thawi Wanarom Park), and southern area (Thonburirom Park). Distances among these sites ranged between 10 and 30 km (Figure 1 and Table 1).

2.2. Sample Collection

At each site, samples of epiphytic subaerial algae were collected from five trees with a circumference of more than 130 cm, at approximately 1.5 m height from the ground, by the method of line transect sampling by walk in the line of 5 trees in 4 corners and a center [26]. A sterile scraper or knife was used to scrape off a surface of 1 cm², and each sample was placed in a paper bag containing silica gel. Information concerning orientation of the sampled surface, species of tree, and characteristics of the surrounding environment was annotated for each sample. The geographical coordinates and altitude were recorded based on GPS data (Table 1). The paper bags containing the samples were sealed and transferred to the laboratory at the Department of Botany, Kasetsart University.

2.3. Measurement of Air Pollutants

The general atmospheric air quality standards for six types of air pollutants were used in this study. For ozone (O₃), nitrogen dioxide (NO₂), carbon monoxide (CO), and sulfur dioxide (SO₂), the measurements were recorded in units of parts per billion (ppb) or parts per million (ppm). For particulate matter less than 10 microns in size (PM₁₀) or less than 2.5 microns in size (PM_2.5) the measurements were recorded in units of micrograms per cubic meter (µg/m³). The measurements used were averages of the pollutant measurements taken in every hour over 24 hours in the algal collection dates. The measurements were taken in each site by the Air Quality and Noise Management Divisions of the Pollution Control Department and the Bureau of the Environment of the Bangkok Metropolitan Administration; the data are available from http://air4thai.pcd.go.th/webV2/.

2.4. Algal Identification

The species/taxa of epiphytic subaerial algae were identified using a metagenomic approach. The samples scraped from 5 trees at each site were mixed together and DNA was extracted using the OnePCR Kit (GeneDireX, USA) following the manufacturer’s recommendations. For the amplification of the 23S rDNA marker, the primers P23SrV_f1 (GGACAGAAAGACCCT) and P23SrV_r1 (TCAGCCTGTTATCCC) were used, following the amplification protocol of [27]. These primers amplify the chloroplast-encoded 23S rDNA marker from prokaryotic and eukaryotic algae [27,28]. PCR amplification was performed using the following cycling conditions: initial denaturation at 94˚C for 2 min, followed by 35 cycles of 94˚C for 20 s, 55˚C for 30 s, and 72˚C for 30 s, with a final extension of 72˚C for 10 min [27]. PCR products with the expected size and yield were selected after a check on 2% agarose gel electrophoresis. The same amount of PCR products from each sample was pooled, end-repaired, A-tailed, and further ligated with Illumina adapters. Libraries were sequenced on a paired-end Illumina platform to generate 450-bp paired-end raw reads. The library was checked with Qubit^TM 4.0 Fluorometer (Thermo Fisher Scientific, USA) following [29] and the real-time polymerase chain reaction (rtPCR) for quantification and a bioanalyzer (2100 Bioanalyzer Instrument, USA) for size distribution detection. Quantified libraries were pooled and sequenced on MGISEQ-2000 Sequencing Illumina platforms, according to effective library concentrations and the amount of data required.

2.4.1. Merging of Paired-End Reads and Quality Control

Paired-end reads were assigned to samples based on their unique barcodes and were truncated by cutting off the barcodes and primer sequences. Then, the paired-end reads were merged using FLASH (Version 1.2.11, http://ccb.jhu.edu/software/FLASH/) [30], a very fast and accurate analysis tool designed to merge paired-end reads when at least some of the reads overlap with the reads generated from the opposite end of the same DNA fragment; the splicing sequences are called raw tags. Quality filtering of the raw tags was performed using fastp (Version 0.20.0) software to obtain high-quality clean tags. Finally, the clean tags were compared with the reference database (SILVA database: https://www.arbsilva.de/ for 23S; UNITE database: https://unite.ut.ee/ for rDNA) using VSEARCH (Version 2.15.0) to detect the chimera sequences, and then the chimera sequences were removed to obtain the effective tags [31].

2.4.2. Amplicon Sequence Variants (ASVs) Denoising and Species Annotation

First, for the effective tags obtained as described above, denoising was performed with the DADA2 or deblurring module in QIIME2 software (Version QIIME2-202006) to obtain the initial ASVs (default: DADA2), and then ASVs with an abundance of less than 5 were filtered out [32]. Species annotation was performed using the QIIME2 software. For 23S, the annotation database was the SILVA database from LPSN and NCBI taxonomy [33], while for markers, primer, and protocols from [27], it was the UNITE database. To study the phylogenetic relationship of each ASV and the differences in dominant species in different samples, multiple sequencing alignment was performed using the QIIME2 software. Finally, the absolute abundance (individuals, meaning an individual cell, colony or filament of subaerial algae) of ASVs was normalized using a standard sequence number corresponding to the sample with the fewest sequences.

2.5. Algal Community Structure

The diversity index (Shannon–Weaver index (H')), richness index (Margalef index), equitability (evenness) index (Pielou index (J')), and similarity index or the percentage of overlap algae, were calculated from abundance data of epiphytic subaerial algae. They were used to compare the diversity and abundance of epiphytic subaerial algae at the various sites. Cluster analysis using the Bray–Curtis method was used to determine the similarities index for the various sites of collection of epiphytic subaerial algae by PC-ORDTM 6 program [34] and IBM SPSS Statistics version 21.0 (IBM Corporation, Armonk, New York, USA) [35].

2.6. Relationship Between Algal Diversity and Abundance and the Air Pollutants

The possible relationships between diversity and abundance of the epiphytic subaerial algae and the air pollutants were analyzed using a Principal Component Analysis (PCA) with a Bray‒Curtis distance in the PC-ORDTM 6 program [34]. The statistical values of the Pearson’s correlation coefficient (r) obtained with IBM SPSS Statistics version 21.0 (IBM Corporation, Armonk, New York, USA) also analyzed the relationships between the diversity and abundance of the epiphytic subaerial algae and the air pollutants [35,36].

3. Results

3.1. Diversity and Abundance of Algae

The overall number of taxa of epiphytic subaerial algae recorded in the urban area of Bangkok was 101. Overall, 101 taxa of algae belonging to the phyla Cyanophyta (70 taxa), Chlorophyta (21 taxa), Charophyta (5 taxa), Bacillariophyta (3 taxa) and Eustigmatophyta (2 taxa) were identified in the urban area of Bangkok from the algal samples (Figure 2; Table 2).

Concerning each site individually, 72 species were recovered in the western site (Thawi Wanarom Park), 55 in the eastern site (Suan Luang RAMA IX), 53 in the northern site (Vachirabenjatas Park), 49 in the southern site (Thonburirom Park) and 44 in the central site (Lumpini Park). Moreover, when comparing the abundance of the five phyla at each site, Cyanophyta had the highest diversity of taxa, followed by Chlorophyta. Charophyta, Bacillariophyta, and Eustigmatophyta were found in smaller numbers (Figure 3).

The overall abundance of epiphytic subaerial algae in Bangkok, expressed as number of individuals, was 17,031 individuals/cm². Cyanophyta had the highest abundance, followed by Charophyta, Chlorophyta, Eustigmatophyta, and Bacillariophyta (Figure 4 and Table A1).

Concerning the most abundant taxa, Chroococcidiopsis sp. 1 had the highest abundance, followed by Microcoleus sp. 1, Klebsormidium flaccidum, Oscillatoria sp., Scytonema mirabile, Tolypothrix sp. 1, Cyanothece sp., Trebouxia corticola and Gloeocapsa sp. (Figure 5 and Table A1).

For the epiphytic subaerial algae collected at the five sampling sites in Bangkok, in terms of numbers of individuals Cyanophyta were the most abundant group at four sites. At the fifth (the northern site), Charophyta had a slightly higher number of individuals than Cyanophyta. At the eastern, western, and southern sites, Chlorophyta was the most abundant phylum after the Cyanophyta. At the central site, Chlorophyta and Charophyta were recorded, but in small abundances (see Figure 6).

Figure 7 shows the relative abundance of different subaerial epiphytes at the five study sites within Bangkok. At the northern site and southern site, the most abundant taxa were the same, i.e., Klebsormidium flaccidum, Microcoleus sp. 1, and Chroococcidiopsis sp. 1. At the eastern site, Chroococcidiopsis sp. 1, Trebouxia corticola and Cyanothece sp. 1 had the highest abundance. At the central site, the most abundant taxa were Tolypothrix sp. 1, Cyanothece sp. 1 and Microcoleus sp.1; at the western site, Oscillatoria sp., Scytonema mirabile, and Microcoleus sp. 1. From these results it can be seen that in general Microcoleus sp. 1 and Chroococcidiopsis sp. 1 were the taxa with the highest abundance at the 5 sites sampled in Bangkok.

3.2. Types of Air Pollutants

The air pollutants affecting the general atmospheric air quality at the time of sampling in Bangkok are presented in Figure 8. The amounts of ozone (O₃) were at 16.34 to 23.84 ppb. Amounts of nitrogen dioxide (NO₂) ranged between 6.67 to 24.18 ppb; carbon monoxide between (CO) 29 and 233 ppb (a large amount), and sulfur dioxide (SO₂) between 1.34 and 2.41 ppb. Particulate matter less than 10 microns in size (PM₁₀) was present at high levels, from 26.8 to 53.85 µg/m³. Finally, particulate matter less than 2.5 microns (PM_2.5) was present at 10 to 15 µg/m³.

3.3. Index Values for Algal Communities

The diversity index (Shannon–Weaver index (H')), richness index (Margalef index), equitability, or evenness, index (Pielou index (J')), and similarity index were determined for the algal communities found at the sampling sites in Bangkok (Table 3 and Table 4). The diversity index ranged between 1.37 and 2.51, with the highest value for the southern site and the lowest value for the northern site. The western site had the highest richness index value (6.19), whereas the lowest was found for the central site (3.84). The equitability index values ranged between 0.30 and 0.54. Finally, the similarity index values ranged from 8.00% to 64.82%, with the highest similarity recorded between the central and southern sites, and the lowest similarity between the northern and eastern sites.

A cluster analysis showed that the composition of species and abundance of epiphytic subaerial algae separated the subaerial epiphytic communities into three groups of sampling sites (Figures 9), with 75% of the information remaining. For the Group 1, represented by the northern site, Klebsormidium flaccidum, Microcoleus sp. 1, and Chroococcidiopsis sp. 1 were the dominant taxa. In Group 2 (eastern site), Chroococcidiopsis sp. 1, Trebouxia corticola, and Cyanothece sp. were the dominant taxa. In Group 3 (which clustered the central, western, and southern sites), Microcoleus sp.1, and Tolypothrix sp. 1 were the dominant taxa.

3.4. Relationship between Diversity of Species and Air Pollutants

The PCA showed the influence of air pollutants on the presence of diversity and abundance of algal species (41.623% and 28.075% of variance explained for the first and second axis, respectively) (Figure 10). Only PM_2.5 was a major contributor to the first PCA axis (r = −0.468, r = 0.187), which was directly positively correlated for the western site but negatively correlated for the southern and central sites. CO, NO₂, and PM₁₀ were the major contributors to the second PCA axis (r = 0.423, r = 0.856; r = 0.646, r = 0.673; and r = 0.527, r = 0.621, respectively), which was directly positively correlated for the northern site. SO₂ also was a major contributor to the second PCA axis (r = 0.455, r = −0.634), which was directly positively correlated for the eastern site. Finally, O₃ also was a major contributor to the second PCA axis (r = −0.514, r = −0.611), which was negatively correlated for the northern site.

The relationship between the diversity of epiphytic subaerial algae found in Bangkok and air pollutants showed abundance of subaerial species and many air pollutants (see Figure 10). The abundance of subaerial species was broadly classified into 3 groups. The first group, based on PM_2.5, was the only major factor on the first PCA axis, meaning that areas with high PM_2.5 pollution had high abundance and composition of most subaerial algal communities. The second group, CO, NO₂ and PM₁₀, contributed significantly to the second PCA axis and had the same abundance and composition of most subaerial algal communities as PM_2.5, but a different species group. Whereas O₃ was a similar major contributor to the second PCA axis as CO, NO₂ and PM₁₀, but in the opposite direction; that is, when the area had high O₃, the abundance and composition of subaerial algal communities was low. Finally, the third group, following SO₂, contributed significantly to the second PCA axis in most epiphytic subaerial algae, as did CO, N₂, and PM₁₀, but had an impact on several species’ groups.

The possible relationship between abundance of each subaerial algal taxon and chemical pollutants was tested using the Pearson’s correlation coefficient (r). Pearson’s correlation coefficient (r) showed that all air pollutants had a significant correlation (* = statistically significant difference (p ≤ .05), 0.01, ** = high statistically significant difference (p ≤ .01)) with the abundance of several epiphytic subaerial algae (Table A2). Cyanothece sp. 2 had a correlation many air pollutants: it was negatively correlated with O₃ (−0.941**) and positively with NO₂ (0.988**) and CO (0.914*) (Table A2). Leptolyngbya sp. 1, Nostoc carneum, Phormidium tinctorium, Synechococcus sp., Symbiochloris irregularis, Stichococcus sp., Chlamydomonas zebra, and Nannochloropsis oculata were positively correlated with CO (0.986**) and PM₁₀ (0.964**). Significant correlations with these pollutants were also detected for Klebsormidium flaccidum (0.986**) (0.963**), Tolypothrix sp. 2 (0.925*) (0.999**), Nostoc sp. 1 (0.975*) (0.943*), Microcoleus sp. 1 (0.939*) (0.916*), Oscillatoria nigro-viridis (0.949*) (0.893*), Cylindrospermum sp. (0.949*) (0.994**), Diadesmis sp. (0.986**) (0.940*), and Parachlorella kessleri (0.957*) (0.980**).

Chroococcidiopsis thermalis and Watanabea reniformis were positively correlated with SO₂ (0.880* and 0.957*, respectively). Scytonema hofmannii (0.964**) was positively correlated with CO. Calothrix sp. 4 (0.917*) was positively correlated with O_3, whereas Chloromonas perforata (−0.962**) was negatively correlated with this pollutant. Symbiochloris reticulata (0.944*) was positively correlated with PM₁₀. Finally, Acutodesmus sp. (−0.896*) was negatively correlated with PM_2.5.

4. Discussion

The high diversity of epiphytic subaerial algae found in this study was not unexpected, due to the fact that Bangkok is located in a tropical region in Southeast Asia, with a humid climate characterized by high levels of rainfall, and therefore particularly suitable for growth of subaerial algae [13,16,38,39].

A noteworthy aspect of our study is the lack of sequences of Trentepohliales in our metagenomic sequencing results. The Trentepohliales is an order of subaerial green algae widespread in areas with humid climates, particularly in tropical regions [3,40,41]. These algae have been reported from urban habitats in the tropics [5,42,43], where they typically form bright red or orange patches on the colonized surfaces. At the time of sampling, growths with typical trentepohlialean habit were not visible on the tree trunks selected for this study. However, previous studies based on high-throughput sequencing carried out in the tropics recovered large numbers of sequences of Trentepohliales [10,44], revealing a great phylogenetic diversity of these algae in natural environments. Therefore, we expected to record some trentepohlialean sequences in our results. Further studies will be necessary to clarify if this reflects a real rarity of these chlorophytes in the urban area of Bangkok or is related to some technical problems for extraction of DNA from thalli of Trentepohliales. This result is even more striking considering that the sampling for this study was carried out in Bangkok’s wet season, which should be a favourable period for the growth of Trentepohliales. The high diversity of epiphytic subaerial algae recorded in this study was certainly related to this aspect. Obviously, the wet season’s high humidity and rainfall provide optimal conditions for the vegetative growth of subaerial algae [2]. Differences in microhabitat conditions related to microvariations in humidity may play a crucial role in determining the diversity and composition of tropical bark-growing algal assemblages [39].

Cyanobacteria (Cyanophyta) had the highest diversity and abundance among the epiphytic subaerial algae recorded in our study, followed by chlorophytan green algae and diatoms (Bacillariophyta). The success of cyanobacteria in subaerial environments is due to their adaptations to desiccation, favoured by the production of mucilaginous sheaths [2,45]. The tropics are well known to host a large diversity of Cyanobateria, which often grow in large amounts on many surfaces [46], especially artificial [3,47]. Chlorophyte green algae have a wide range of thallus and cellular organization compared to other groups of microalgae, and have developed several adaptations for survival and growth in subaerial habitats [3]. Diatoms are another algal group widely distributed in this type of environment [46,48,49]. Because they are limited by the availability of dissolved silica, their numbers are higher during periods of higher humidity and in habitats in which silica is more readily available [46,50,51,52], in a study similar to ours conducted in tropical South America and Hawaii, recorded environmental sequences inferred to be diatoms.

Chroococcidiopsis sp. 1, Microcoleus sp. 1, and Klebsormidium flaccidum were the most abundant subaerial algae found in Bangkok. Unicellular cyanobacteria belonging to the genus Chroococcidiopsis are extremely resistant to desiccation, UV irradiation, salt toxicity and high temperatures [53,54]. They require high light intensities for growth and are protected from high-energy UV light by pigments such as carotenoids and scytonemin [53,54]. Microcoleus is a well-known filamentous cyanobacterium that is resistant to desiccation thanks to several desiccation tolerance mechanisms, including accumulation of trehalose [55], stabilization of the photosynthetic apparatus [56] and accumulation of UV-protecting pigments [57]. A recent study reported the expression of genes involved in the oxidative and osmotic stress response, the desaturation of membrane lipids, and the production of EPS at the onset of desiccation [58]. Klebsormidium flaccidum was one of the most common subaerial algae recorded in this study. It is generally reported as one of the most widespread subaerial algae in the world, with cosmopolitan distribution according to morphology-based identifications [59,60,61]. The taxonomic circumscription of this species has been reassessed in recent years based on molecular data. In general, species of Klebsorbidium are known as fast-evolving organisms, which may rapidly develop physiological adaptations enabling them to colonize new habitats, including some with extreme conditions [62,63].

The wet season in Bangkok has an AQI of 28, or green, which indicates that a “good quality of air” is present and that residents “can participate in outdoor activities and travel as usual.” The AQI is a universal index with five levels from 0 to 201 or more [64,65]. It is widely used in many countries, such as the United States, Australia, Singapore, Malaysia, and Thailand, and measures levels of O₃, NO₂, CO, SO₂, PM₁₀, and PM_2.5 [64,65,66].

The diversity index in the wet season in Bangkok was 1.37 to 2.51 for taxa of epiphytic subaerial algae. The richness index ranged between 3.84 and 4.75, indicating that in the sites studied there were generally high abundances of epiphytic subaerial algae [67]. The equitability index had values ranging from 0.30 to 0.54, showing that there was a similar representation of taxa in terms of abundances. [68] stated that if the equitability index approaches 1, this indicates that the study site/area hosts taxa with generally similar abundances. The similarity index had values ranging from 8.00% to 64.82%; these values were consistent with the results of the cluster analysis.

Cluster analysis showed that the subaerial algal communities could be subdivided in three separate groups distributed in different sampling sites. Group 1 was represented by the northern site, Group 2 by the eastern site, and Group 3 by the central, western, and southern sites. Each group had different abundances of taxa and ecological index values (i.e., diversity index, richness index, and equitability index), in agreement with the similarity index results. In all groups cyanobacteria were the dominant group, whereas differences in abundance of the other algal groups were detected. In Group 1 Charophyta were well represented, and this group had the lowest value for the ecological index values. In Group 2 (eastern site) Chlorophyta were well represented; this group had a low-value ecological index. Group 3, which comprised the central, western, and southern sites, consisted largely of Cyanobacteria and had the highest ecological index values. The abundance of cyanobacteria in urban areas is well documented, with particular abundance in tropical regions [3,45].

A significant relationship between air pollutants and some epiphytic subaerial algae was detected, in particular PM₁₀. Cyanothece sp. 2 corresponds to Cyanothece sp. ATCC 51142 in the genomic database from NCBI (National Center for Biotechnology Information of USA) [69,70,71,72]. In this study it showed a negative correlation with O₃ and a positive correlation with NO₂ and CO. Species of Cyanothece are widely distributed in various environments worldwide, usually at a pH lower than 7 [73]. Typically, this genus is associated with water in benthic marine environments, rice fields, acidic marshes, peaty bogs, intertidal zones, moors, and clear lakes, but it is sometimes found in mountain soils [69,74], and records of it are also available from rocks and trees. The taxa Leptolyngbya sp. 1, Nostoc carneum, Phormidium tinctorium, Synechococcus sp., Symbiochloris irregularis, Stichococcus sp., Chlamydomonas zebra, Nannochloropsis oculata, and Klebsormidium flaccidum were also positively correlated with CO and PM₁₀ and were found only at some sites. These correlations indicate their potential value as bioindicators of these pollutants, which should be further explored in future studies.

5. Conclusions

This study unraveled the presence of 101 taxa of epiphytic subaerial algae on tree barks in Bangkok, consisting mainly of Cyanophyta. In terms of abundance, the three most abundant taxa were Chroococcidiopsis sp. 1, Microcoleus sp. 1 and Klebsormidium flaccidum. All environmental pollutants had correlations with the abundance of epiphytic subaerial algae. Cyanothece sp. 2 was the taxon that showed the strongest correlation and has therefore the best potential to be used as bioindicator, being negatively correlated to O₃ and positively correlated to NO₂ and CO.