BALIDO DIVERSITY AND DISTRIBUTION OF CORTICOLOUS LICHENS AND THEIR RELATIONSHIP TO LEVELS OF NITROGEN DIOXIDE IN CHIANG MAI PROVINCE, THAILAND BACHELOR THESIS Study Mode: Full-time
INTRODUCTION
Research rationale
Thailand's economic growth has led to significant challenges, particularly in terms of air pollution, which is increasingly harming both the population and the environment This worsening issue has resulted in a rise in respiratory-related health problems, contributing to approximately 50,000 deaths annually (Pacific Prime Thailand).
Chiang Mai Province in Northern Thailand has experienced significant economic growth since 2017, driven by an increase in tourist attractions, accommodations, factories, restaurants, road construction, and business buildings This growth is reflected in the rising number of tourists visiting the area Additionally, the industry and service sectors have emerged as the two largest employers in the province in recent years.
The economic growth in the province has led to significant trade-offs, including traffic congestion, inadequate waste management, and increased water and air pollution, all of which negatively impact the natural environment and resources Among these issues, air pollution stands out as the most critical, severely affecting public health The Pollution Control Department reported that PM 2.5 levels on March 18, 2018, ranged from 48 to 86 micrograms per cubic meter, significantly exceeding safe levels.
50 micrograms per cubic meter of air
Each year, residents and tourists experience health issues and even fatalities due to pollution, primarily from hazardous pollutants like sulfur dioxide, benzene, lead, carbon monoxide, and nitrogen oxide Sulfur dioxide can lead to shortness of breath, while benzene is linked to leukemia Lead negatively impacts the central nervous system and brain function Additionally, excessive exposure to carbon monoxide can be fatal, and high levels of nitrogen oxide can cause respiratory problems.
The establishment of standard levels for nitrogen dioxide is primarily intended to safeguard human health In Thailand, the Pollution Control Department, part of the Ministry of Natural Resources and Management, has set these standards for hazardous pollutants to assess current levels over hourly, monthly, and yearly intervals.
Effective air pollution monitoring can be achieved through various methods, including the use of lichens as a biomonitoring technique (Blasco et al., 2008) and passive samplers that measure nitrogen dioxide levels Lichens serve as indicators of pollution's impact on organisms, while passive samplers provide crucial data on the concentration of hazardous pollutants Both approaches are widely recognized for their effectiveness in assessing air quality.
Research objectives
The study aims to explore the diversity and distribution of lichens in Chiang Mai Province, while also monitoring environmental factors and nitrogen dioxide levels Additionally, it seeks to establish the correlation between nitrogen dioxide, environmental conditions, species richness, and lichen diversity.
Research Questions and Hypotheses
The research questions are based on the objectives of the study which are the following:
1 What is lichen diversity in Chiang Mai Province?
2 What is the similarity of the species communities and lichen distribution within the study sites?
3 What is the light intensity, temperature, relative humidity, bark pH, elevation, species richness and nitrogen dioxide levels in each site?
4 What is the correlation between nitrogen dioxide levels, environmental factors (light intensity, temperature, relative humidity, elevation and bark pH), species richness and lichen diversity in Chiang Mai Province?
The study comprises two hypotheses such as
1 Null Hypotheses: No significant correlation between nitrogen dioxide levels, environmental factors, species richness and lichen diversity
2 Alternative Hypotheses: Significant correlation between nitrogen dioxide levels, environmental factors, species richness and lichen diversity.
Limitations
The study faced limitations due to the restricted time available for research, leading to a brief observation of lichen diversity and distribution The use of efficient equipment, such as passive samplers, was essential for obtaining results within this short timeframe, as passive samplers are recognized for their simplicity and effectiveness in measuring nitrogen dioxide levels.
Definitions
Lichens – organism made up from the combination of fungi and algae and/or cyanobacteria
Passive sampling - physico- chemical method which can access the amount of pollutants
Pearson correlation coefficient- statistical measure of the strength of the association of two variables
LITERATURE REVIEW
Lichens
Lichens are unique composite organisms formed through a symbiotic relationship between fungi, algae, and/or cyanobacteria The fungal component, known as the mycobiont, provides structure and protection, while the photobiont, which can be either an algal (phycobiont) or cyanobacterial (cyanobiont) partner, supplies essential food through photosynthesis This intricate relationship places lichens within the Fungi Kingdom, highlighting the crucial role of fungi in determining their overall appearance.
Lichens are composite organisms primarily composed of mycobionts from two fungal groups: the common Ascomycetes and the rarer Basidiomycetes Their photobionts include the green algae Trentepohlia and Trebouxia, while cyanobionts consist of Stigonema, Nostoc, and Scytonema (Wolseley et al., 2015) These organisms thrive on various substrates, including soil (terricolous lichens), bark (corticolous lichens), and rocks (saxicolous lichens), and are found in diverse habitats such as mountains, seashores, and rainforests Lichens play a crucial role in ecological processes, including slowing soil formation and providing food and shelter for various animal species.
Morphology
Lichens are structured with a body known as the thallus, which consists of filaments of fungal cells There are two main types of thalli based on their internal structure: homoiomerous thalli, which are simple and contain only fungal filaments and algae, and heteromerous thalli, which are layered and consist of four distinct parts: the upper cortex, algae layer, medulla, and lower cortex (Constantine et al., 2018).
The upper cortex serves as the protective outer layer of the thallus, while the algae layer functions as the photosynthetic region, where algae coexist with fungal hyphae The medulla acts as the central support structure of the thallus, featuring robust walls that facilitate gas exchange in lichens In the medulla, fungal hyphae are loosely arranged, whereas the lower cortex contains densely packed hyphae Additionally, some hyphae at the base, known as rhizines, anchor the thallus to substrates; in the absence of a lower cortex, a hypothallus forms from a thin layer of hyphae to fulfill this role.
Lichens possess various structures, including hapter, holdfast, cephalodia, cilia, pruina, cyphellae, and pseudocyphellae Hapter and holdfast play a crucial role in anchoring lichens to substrates, complementing the function of rhizines Cephalodia, which are small structures, appear exclusively in lichens that contain both cyanobacteria and green algae, typically located in the upper or lower regions of the lichen.
7 of the thallus and their important function is to execute nitrogen fixation in the environment
Cilia are tiny hair-like structures, while pruina is a white, icy coating typically composed of calcium oxalate, both of which are present on the margins of lichens Cyphellae are pores located on the lower surface of the thallus, whereas pseudocyphellae are small pores created by the expansion of the medulla layer on the lower cortex, appearing as a line along the margins of the upper cortex (Lepp, 2011a).
Growth forms and reproduction
Lichens exhibit three primary growth forms: crustose, foliose, and fruticose Crustose lichens are the most prevalent, characterized by their crust-like appearance and strong attachment to substrates In contrast, foliose lichens, often referred to as leaf-like lichens, have a flattened shape and are loosely attached to their surfaces Fruticose lichens, which are also distinct in their structure, complete the trio of common lichen growth forms.
Lichens, such as those described by Sharnoff (2018), exhibit a shrub-like or hair-like appearance and grow by attaching their lower parts to substrates, allowing for multidimensional forms (Lepp, 2011b) Typically, these crustose, foliose, and fruticose lichens grow at a rate of approximately 0.5 to 5 mm per year.
Lichens reproduce through both sexual and asexual (vegetative) methods In sexual reproduction, only the fungal component is involved, producing fruiting bodies with structures known as ascus and basidium, which serve as containers for spores These spores are generated by two fungal partners: Ascomycetes, which produce ascospores, and Basidiomycetes, which produce basidiospores.
Fruiting bodies are categorized into two main forms: apothecia and perithecia Apothecia, which are cup-like structures located on the upper surface, can be further divided into three types: lecanorine, lecideine, and lirellae Lecanorine apothecia contain algae and have margins that match the color of the thallus, while lecideine apothecia lack margins and algae, displaying different colors Lirellae apothecia are elongated and feature distinct black margins In contrast, perithecia are spherical fruiting bodies characterized by a small opening, differentiating them from apothecia.
Figure 1 Three types of lichens (a) crustose (b) foliose (c) fruticose
Asexual or vegetative reproduction in lichens involves three key structures: isidia, soredia, and lobules Unlike sexual reproduction, these structures enable the reproduction of both algal and fungal components Isidia are barrel-shaped or coral-like formations, while soredia consist of small, powdery clusters of hyphae Lobules are flat lobes found in the thallus These structures detach from the thallus and reproduce through environmental conditions and physical interactions, such as wind and animal disturbances (Smith, 1921).
Factors affecting the lichens
Lichens are composite organisms that thrive in various habitats, depending on suitable substrate and environmental conditions They are commonly found on rough, flaky, and persistent barks, while their presence is less frequent on smooth barks Additionally, lichens can adapt to different surface components, such as pH levels and water content, allowing them to survive in conditions with minimal water and moderate acidity in the substrates (Lam et al., 2013).
Environmental conditions such as temperature, relative humidity, light intensity, and elevation significantly impact lichen growth Extreme temperatures, whether high or low, can limit lichen richness by affecting their reproduction and food sources Additionally, humidity plays a crucial role in lichen development, with growth being particularly pronounced in areas with high humidity.
Lichens thrive in environments with low humidity, as noted by Čabrajić (2009) Sunlight intensity is crucial for their existence, leading to their prevalence in areas with ample sunlight exposure (Lam et al., 2013) Additionally, the growth and diversity of lichens are significantly affected by elevation, with greater diversity and species richness observed at higher elevations (Kumar et al., 2014).
2.5 Lichens as biomonitor of air pollution
Lichens play a crucial role in monitoring environmental issues, especially air pollution, due to their sensitivity and adaptive capabilities Their unique structure, lacking an outer layer such as an epidermis or cuticle and roots, makes them particularly sensitive to atmospheric conditions Furthermore, their adaptive capability allows for structural variations based on their growing environment, reinforcing their significance as indicators of air quality.
Crustose, foliose, and fruticose lichens exhibit varying levels of environmental tolerance Crustose lichens are the most resilient, thriving even in polluted air, while foliose lichens can only survive in moderately polluted areas In contrast, fruticose lichens require completely clean air and cannot thrive in environments with even minimal pollution Understanding the presence of sensitive and tolerant lichen species is crucial for assessing environmental health.
11 within the three common lichens specifically helps in distinguishing the possible indicator species as well as the changes in the environment
Recent studies have assessed the impact of climate change on global environments by examining lichen diversity and abundance For instance, a sixteen-year study in the US Pacific Northwest (1993-2009) found that high elevation lichens were significantly affected, with a decrease in species numbers and distribution compared to lower elevations, indicating that warming has a pronounced effect on cooler environments (Glavich et al., 2018) Additionally, research in northwestern Alaska reported a low abundance of lichens in the Arctic tundra (Joly et al., 2009).
A study by Shukla et al (2018) indicates a decline in cold temperature species of lichens in both highland regions, particularly in the mountains of Western and Central Europe, as well as in lowland areas However, some research suggests that lichens may be increasing in cold climates For instance, a study conducted in Swedish forests examined specific lichen species, including Bryoria spp., Usnea spp., and Alectoria sarmentosa, revealing notable findings regarding the Usnea spp and Alectoria sarmentosa.
Ach shows a greater prevalence in colder regions compared to warmer areas, while Bryoria spp is absent in both temperature conditions (Esseen et al., 2016) Additionally, climate change, characterized by extreme heat and increased rainfall, will continue to impact the growth and diversity of lichens.
Therefore, lichens in local climate scale were considered to be influenced by the varying levels of microclimatic factors from each environment it grows In
A study conducted in the Badrinath Valley of the Western Himalayas revealed that lichens are influenced by temperature, altitude, and relative humidity Additionally, research in the White Mountains of California indicated that light intensity also affects lichen distribution (Gupta et al., 2014).
Lichens, known for their sensitivity and tolerance to environmental changes, serve as effective bioindicators for assessing air pollution, a significant environmental issue caused by excessive pollutants from human activities Key contributors to air pollution include vehicle emissions, power plants, and agricultural and industrial operations, which release harmful substances such as sulfur dioxide and nitrogen dioxide into the atmosphere.
Lichens have been utilized to monitor atmospheric pollutants, specifically nitrogen dioxide and sulfur dioxide, due to their sensitivity to these harmful substances (Richardson, 1988) High concentrations of these pollutants adversely affect chlorophyll, ultimately leading to the death of lichens Consequently, research has focused on assessing pollutant levels by examining both sensitive and tolerant lichen species in relation to each type of pollutant.
A study revealed the discovery of new lichen species and the reappearance of some in London, indicating improved air quality free from sulfur dioxide (Hawksworth & McManus, 1989) In the United Kingdom, five terricolous alpine lichen species, known to be sensitive to nitrogen dioxide pollution, were adversely affected (Nash & Gries, 2002).
A study conducted in Colombo and its suburbs assessed the levels of sulfur dioxide and nitrogen dioxide pollution by evaluating lichens and disturbances in the area The findings revealed high pollution levels, evidenced by a significant decline in lichen populations and the complete absence of tropical lichens, which are recognized as pollution-sensitive species (Attanayaka et al 2007).
Muhammad et al (2018) identified the D picta lichen species as being associated with vehicular emissions in Malaysia, particularly in the Bahut Pahut area This species was found in all locations, regardless of traffic levels, and is considered a tolerant lichen among those studied Additionally, research in Chiang Mai, Thailand, revealed that P cocoes also exhibits tolerance to air pollution, highlighting the varying sensitivity of different lichen species to environmental stressors.
D picta which may be considered as a bioindicator of air pollution within the area and the tolerance of P.cocoes (Pimwong, 2002)
The passive sampler is a device that utilizes molecular diffusion to gather pollutants from the environment This method allows for the free flow of analytes, including heavy metals and various inorganic and organic compounds, from the atmosphere to the collecting media These compounds can be found in different matrices such as soil, water, and air.
2012) Furthermore, the average amount from the collected pollutants were estimated by using the Fick’s law of diffusion
Passive Sampling
The passive sampler is a device that utilizes molecular diffusion to gather pollutants from the environment This method allows for the free flow of analytes, including heavy metals and various inorganic and organic compounds, from the atmosphere to the collecting media These pollutants can be found in different matrices such as soil, water, and air.
2012) Furthermore, the average amount from the collected pollutants were estimated by using the Fick’s law of diffusion
Passive sampling is a cost-effective and flexible physico-chemical method widely utilized for air pollution monitoring This approach is advantageous as it requires no maintenance or electricity to function, making it an efficient tool for assessing pollutant levels in various study areas Additionally, its lightweight and simple design enhances its usability in diverse environments.
Recent studies utilizing passive sampling methods have emerged in various countries to monitor air pollutants and their impact on air quality In Malaysia, findings indicated that human activities, particularly from construction sites and vehicle emissions, significantly affect air quality Among three selected areas, the one furthest from roads and construction exhibited the lowest nitrogen dioxide level at 14.0 ppbv, while the area closest to these activities recorded a peak of 36.8 ppbv Similarly, a monitoring study in Abeokuta, Nigeria, identified high concentrations of nitrogen dioxide, sulfur dioxide, and carbon dioxide, particularly during the dry season The Iyana Mortuary site showed the highest levels of nitrogen dioxide and sulfur dioxide, attributed to domestic cooking, road expansion, and traffic congestion, with Kuto also experiencing elevated carbon dioxide levels.
Vehicular emissions have led to pollutant concentrations exceeding the WHO's 2005 threshold limit value (TLV) of 500 ppm, indicating a significant decline in air quality in the area This deterioration poses serious health risks to the local population (Olaynika et al., 2015).
A study conducted in Chiang Mai, Thailand, revealed that nitrogen dioxide (NO2) concentrations were significantly higher in urban areas compared to suburban and rural regions Notably, the highest levels of NO2 were found near areas experiencing severe traffic congestion.
Recent studies utilizing passive sampling and lichens to assess air quality have been conducted, including one in South Korea that measured sulfur dioxide (SO2) and nitrogen dioxide (NO2) across various forest types: Quercus, coniferous, and deciduous The deciduous forest, situated on the Korea Peninsula, was more affected by human activities, while the coniferous and Quercus forests on Jeju Island experienced higher humidity levels Consequently, the coniferous forest exhibited the highest nitrogen dioxide levels, while the deciduous forest recorded the highest sulfur dioxide concentrations, with the Quercus forest showing the lowest levels of both pollutants The study revealed a negative correlation between SO2 and NO2 concentrations and lichen diversity (Udeni et al., 2016) In contrast, research by Pomphueak (2005) in Chiang Mai, Thailand, indicated a positive correlation between lichen diversity and SO2 levels.
NO2 concentrations collected by passive sampling method
Figure 2 The process of gas diffusion in the passive sampling procedure
MATERIALS AND METHODS
Time frame and description of the study areas
Lichens were collected over the period of April 27 to May 18, 2018 within the six study areas in Chiang Mai Province
Chiang Mai, the second-largest province in Thailand, spans approximately 20,170.1 square kilometers With an annual average temperature of 25.6°C and yearly rainfall of 1,184 mm, the province experiences a tropical monsoon climate The region observes three distinct seasons, including a cold season that starts in November.
February and the hot season during March – May Wet season begins in the month of May- October (World Weather and Climate Information, 2016)
In particular, the six study areas were selected The study areas investigated are Mae Tang, Mae Rim, Doi Saket, Mae On, San Pa Tong, and Chiang Mai City
In particular,these areas chosen for the study are based on the degree of human activities present within these places
Table 1 Location of the six study sites Site number (No.), Study sites, longitude and latitude and elevation
No Study sites Abb Location
6 Chiang Mai City CM city 18°47'43"N 98° 59' 55"E 310
Figure 3 Location of the six selected areas within Chiang Mai Province
Materials
The study utilized various materials for collecting and analyzing lichens and nitrogen dioxide through passive sampling methods Data collection for lichens involved tools such as a compass, pens, recording forms, a pocketknife, a grid frame, a basket, a camera, small paper envelopes, a dropper, and measuring tape For nitrogen dioxide, a cool box and plastic zip lock bags were employed In the laboratory, essential materials included slide and cover glass, a hand lens, beakers ranging from 25-600 ml, a 10 ml graduated cylinder, an analytical balance, a pH meter, compound and stereo microscopes, an ultraviolet lamp, GPS, a Lux meter, and wet and dry thermometers The passive sampling method required an oven, ultrasonic bath, shelter, 10 ml polypropylene tubes, parafilm, Whatman (GF/A), a 10 ml syringe, a 50 ml beaker, 10 ml micropipettes, volumetric flasks ranging from 25-250 ml, glass stirring rods, and a pipet bulb.
The study of lichens involved the use of several chemicals, including deionized water, sodium hypochlorite, potassium hydroxide at a concentration of 10%, and 5% Lugol’s iodine For the passive sampling method, the chemicals utilized were deionized water, phosphoric acid, Triethanolamine (TEA), N-(1-Napthyl)ethylenediamine dihydrochloride (NEDA), and Sulfanilamide (C6H8N2O2S).
Methods
The study examined 10 mango trees (Mangifera indica L.) within a 2 km x 2 km area, utilizing a grid frame to assess lichen frequency on the selected trees The grid frame, measuring 20 x 50 cm² and divided into 10 subsquares of 10 x 10 cm², was positioned 150 m above ground on the tree trunk where lichen coverage was most abundant Lichen species and their frequencies within the grid frame were documented using a hand lens, while lichens smaller than 3 mm in diameter were excluded to prevent misidentification Additionally, lichens outside the grid frame were identified, with their frequency recorded as one.
Lichens that were not identified in the field were collected in paper envelopes for further analysis using ultraviolet lamps, compound microscopes, and stereo microscopes Their morphology, growth, reproduction, and dispersal were assessed using keys from Sipman (2003), Wolseley and Aquire-Hudson (1997), and Awasthi (1991) Additionally, data on the tree's circumference, bark type, bark pH, grid frame placement on the trunk, sun exposure, and the surrounding environment, including nearby activities and establishments, were recorded, along with the distance between trees at each site.
22 b) Measurement of some environmental factors
Data was collected using various instruments, including a Lux meter to measure light intensity, a Global Positioning System (GPS) for altitude, and both dry and wet thermometers to determine temperature and relative humidity, respectively, positioned at 1.5 meters above the ground Elevation data for each site was obtained through Google Earth Additionally, bark pH analysis was conducted.
Bark samples devoid of lichens were collected at a height of 1 meter above the ground and stored in paper envelopes within a freezer The samples were then weighed using an analytical balance with a precision of 0.5 grams and placed in plastic cups Each sample was soaked in 5 ml of distilled water for 8 hours The pH of the bark from each of the 10 trees across six different sites was measured using an electrochemical analyzer equipped with a pH meter.
Figure 4 The position of a grid frame on selected tree
The selection of sites was based on one of the ten investigated trees from each location To prevent the loss of passive samplers during the study, careful consideration was given to their safety and necessary exposure The procedures included the preparation of the solutions.
The 20 ml of Triethanolamine solution was adjusted to volume with deionized water in 100 ml volumetric flask by using micropipette and a pipet bulb Saltzman reagent was prepared by mixing the reagent A and B The reagent A was made up of 5.375 g of Sulfanilamide solution and 14 ml of phosphoric which was adjusted to volume with deionized water in a 250 ml beaker
The preparation of the reagent involved dissolving 0.038 g of N-(1-Naphthyl)ethylenediamine dihydrochloride in a 25 ml beaker to achieve the desired volume The Saltzman reagent was then stored in the freezer until the day of collection Additionally, the diffusion tube was prepared for subsequent use.
The GF/A filter paper, with a diameter of 110 mm, was cut into small pieces to fit into polypropylene tubes These pieces were soaked in deionized water three times and then placed in an oven to prevent stretching and ensure they dried properly.
Forty-eight polypropylene tubes were arranged on six passive samplers, with each sampler containing eight tubes Among these, five diffusion tubes served as replicates of the samples, while three blank tubes acted as control samples under identical conditions.
Triethanolamine mixed with deionized water was added on the filter paper placed in the bottom of all tubes c) Exposure of the diffusion tube
The passive sampler's tubes were installed vertically at a height of 150 meters in trees at each site The exposure period for the sampling took place from May 25 to June 1, 2018.
The passive samplers were sealed with paraffin film and stored in plastic zip lock bags, then kept in a freezer until the samples were extracted.
Figure 5 Configuration of passive sampler
Samples were extracted by adding 2 ml of deionized water to the tubes with a syringe, followed by shaking the tubes for approximately 10 minutes prior to analysis.
The samples were placed in small bottles and treated with 2 ml of Saltzman reagent The resulting colors were then compared to a standard color chart for NO2, allowing for the determination of NO2 levels.
Figure 6 Analysis of the sample using NO 2 test kit
Figure 7 The NO 2 standard color chart
3.3.3 Data Analysis a) Lichens Diversity: Shannon Index
The study on lichen diversity employs the Shannon Diversity Index (H'), a widely recognized metric for assessing species diversity within a community This index effectively captures both the abundance and evenness of the species present, providing a comprehensive analysis of lichen diversity.
The resulting product is summed across species and multiplied by -1 Below is the following formula (Kerkhoff, 2010); Η′ = − ∑(𝑃𝑖 𝐼𝑛𝑃𝑖)
𝑃 𝑖 = proportion of the individuals found in species i
𝑛 𝑖 = the number of individuals in species i
N = total number of individuals in a community
∑ = sum of the calculations ln= natural logarithm b) Similarity of lichen communities and distribution of lichens
Cluster analysis was employed to assess the similarity of lichen communities across all sites, utilizing the Multivariate Package Program (MVSP) The Bray-Curtis Similarity Index was applied within MVSP, and the results were presented in a dendrogram Additionally, Detrended Correspondence Analysis (DCA) was conducted to visualize the distribution of lichens in the study, using Past3 software.
28 c) Correlation between nitrogen dioxide, environmental factors, species richness and lichens diversity
The correlation between nitrogen dioxide, environmental factors, species richness and lichens diversity were done by using Pearson CorrelationCoefficient, Past3 software, Excel 2016
RESULTS AND DISCUSSION
Results and Discussion
A total of 14 families and 22 genera, comprising 38 species of lichens, were identified, including 24 crustose lichens and 14 foliose lichens The complete list of families, genera, and species is provided in Table 2, while Table 3 presents the species along with their frequencies across all study areas.
Table 2 List of lichens families, genera and species of lichens found in the study areas
Arthoniaceae Arthonia Arthonia cinnabarina Crustose
Arthonia white fruiting body group Crustose
Arthonia black fruiting body group Crustose
Caliciaceae Buellia Buellia stillingiana Crustose
Chrysotrichaece Chrysotrix Chrysothrix sp Crustose
Laureraceae Laurera Laurera sp Crustose
Lecanoraceae Lecanora Lecanora group.1 Crustose
Table 2 List of lichens families, genera and species of lichens found in the study areas (continued)
Lecanoraceae Lecanora Lecanora group 2 Crustose
Letrouitiaceae Letrouitia Letrouitia sp Crustose
Malmideaceae Malmidea Malmidea piae (Kalb)Kalb Crustose
Parmeliaceae Parmotrema Parmotrema praesorediosum (Nyl.) Hale, Foliose
Parmotrema saccatilobum (Taylor) Hale, Foliose
Parmotrema tinctorum (Despr ex Nyl.) Hale, Foliose
Peltulaceae Phyllopeltula Phyllopeltula cf corticola
Physiaceae Dirinaria Dirinaria applanta Foliose
Table 2 List of lichens families, genera and species of lichensfound in the study areas (continued)
Physiaceae Dirinaria Dirinaria confluens Foliose
Physcia Physcia cf dilatata Foliose
Table 2 List of lichens families, genera and species of lichens found in the study areas (continued)
Physiaceae Rinodina Rinodina roboris Crustose
Pyrenulaceae Pyrenula Pyrenula sp Crustose
Trypathelieae Nigrovothelium Nigrovothelium tropicum Crustose
Table 3 List of lichen species and their frequencies in all study areas
Family Taxa No of Sites Sum of total frequency
MR MT DSK SPT MO CM
Arthonia white fruiting body group
Arthonia black fruiting body group
Table 3 List of lichen species and their frequencies in all study areas (continued)
Family Taxa No of Sites Sum of total frequency
MR MT DSK SPT MO CM
Table 3 List of lichen species and their frequencies in all study areas (continued)
Family Taxa No of sites
MR MT DSK MO SPT CM
Malmideaceae Malmidea piae (Kalb)Kalb 7 7
Parmeliaceae Parmotrema praesorediosum(Nyl.) Hale, 1 1 2
Parmotrema tinctorum (Despr ex Nyl.)
Table 3 List of lichen species and their frequencies in all study areas (continued)
Family Taxa No of sites Sum of total frequency
MR MT DSK MO SPT CM
Table 3 List of lichen species and their frequencies in all study areas (continued)
Family Taxa No of sites Sum of total frequency
MR MT DSK MO SPT CM
Note: MR: Mae Rim, MT: Mae Tang, DSK: Doi Saket, MO: Mae On,
SPT: San Pa Tong, CM: Chiang Mai City
The Mae Tang area exhibited the highest diversity index, while both Mae Tang and Mae On demonstrated the greatest richness In contrast, Chiang Mai City recorded the lowest diversity index, and Doi Saket had the least richness Detailed findings on richness and diversity indices across the study areas are presented in Table 4.
Table 4 List of richness and diversity index in the study areas
No site Study Site Abb Richness H'
The study showed that the number of lichen species in the study areas was high in suburban areas in comparison to urban areas which corresponded on Pomphueak
In a study comparing crustose and foliose lichens, crustose lichens were found to be more abundant, particularly at altitudes between 250-400 m, consistent with findings by Saipunkaew et al (2007) The highest lichen species richness and dominance were observed in the Family Physiaceae, while the Families Caliciaceae and Malmideaceae exhibited the lowest richness and dominance Notably, Pyxine cocoes, Hyperphyscia adglutinata, and Dirinaria picta were the most frequently encountered species, with Pyxine cocoes having the highest frequency and Dirinaria picta the lowest Additionally, certain species like Hyperphyscia adglutinata, Phyllopeltula cf corticola, and Rinodina roboris were exclusively found in urban areas with significant anthropogenic impacts, aligning with the observations of Sransupphasirigul (2012).
In this study, Dirinaria picta were found only in suburban areas which had less anthropogenic impacts and implied the sensitivity of Dirinaria picta on air pollution
In suburban areas, several lichen species were identified, including Physcia poncisii, Parmotrema praesoredium, Hyperphyscia pruinosa, Trypatheliem eluteriae, and Nigrovothelium tropicum Notably, Letroutia transgressa and Letroutia aureola were exclusively found in Mae On, a region characterized by moist conditions typical of moist forests The proximity of Mae On to mountainous areas with minimal human activity likely accounts for the presence of these lichens, as suggested by Wolseley and Aguirre-Hudson (1997).
4.1.2 Similarity of lichen communities and distribution of lichens
The study employed cluster analysis using the Bray-Curtis similarity index to assess the similarity of lichen communities across six sites, as illustrated in the dendrogram (Figure 8) The unweighted pair-group method with arithmetic averaging (UPGMA) revealed two distinct groups with a 30% similarity between them The first group included San Pa Tong, Mae Rim, Mae Tang, Doi Saket, and Mae On, while the second group was represented by Chiang Mai City Notably, the lichen communities in Chiang Mai City were significantly different from those in the first group, as they were predominantly found in areas with high human activity.
The distribution of lichens across the sites was analyzed using Detrended Correspondence Analysis (DCA), which aligned with the findings from the Bray-Curtis analysis, as illustrated in Figure 9 Notably, certain species prevalent in the first group were frequently observed.
In 41 suburban areas, including San Pa Tong, Mae Rim, Mae Tang, Doi Saket, and Mae On, notable species such as Chrysothrix sp., Pyxine cocoes, Dirinaria picta, and Hyperphyscia adglutinata were identified Each site also featured distinct species like Trypathelium eluteriae, Bactrospora dryina, and Pyrenula sp Additionally, species commonly found in urban areas, particularly in Chiang Mai City, included Rinodina roboris and Phyllopeltula cf corticola.
,Dirinaria picta and Pyxine cocoes
Hyperphyscia adglutinata was widely distributed across all sites, with a notably high frequency in urban areas In contrast, Phyllopeltula cf corticola and Rinodina roboris were exclusively found in urban environments Both Hyperphyscia adglutinata and Phyllopeltula cf corticola were identified as species tolerant to air pollution.
(Sransupphasirigul, 2012) Saipunkaew et al (2005) found that Dirinaria picta was identified in suburban areas and as a sensitive species on air pollution as same as the results found in the study
Figure 8 Dendrogram of similarity of lichen communities
Figure 9 Distribution of lichens in all study sites
4.1.3 Environmental factors in the study sites
Elevation, light intensity, temperature, relative humidity and bark pH acquired in the study were illustrated in Table 5
Table 5 List of environmental factors collected in study sites
Lichens are influenced by various environmental factors, including elevation, light intensity, temperature, relative humidity, and bark pH In a recent study, these factors showed no significant differences across sites with similar land use patterns and human activities However, the data on light intensity, relative humidity, and temperature from different sites were collected at varying times and days, making direct comparisons challenging; consistent daily measurements are recommended for more accurate analysis.
4.1.4 NO 2 concentration in the study sites
The determination of NO2 concentration was done by passive sampling method which diffusion tubes were installed in each study sites one week during May – June
2018 The NO2 concentrations ranges from 9.5 -19.1 ppbv The collected NO2 concentrations from all sites were shown in Table 6
Table 6.List of the NO 2 concentrations in study sites
Note: *, data were excluded as the results were affected by some factors
The passive sampling method involves five key procedures: preparing solutions such as 20% Triethanolamine (TEA), N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDA), sulfanilamide, ortho-phosphoric acid, and Saltzman reagent; preparing diffusion tubes by absorbing TEA solution in GF/A filter paper; exposing the diffusion tubes; and analyzing NO2 concentrations using a standard NO2 color chart (Bootdee, 2009) This method was implemented at six different sites, where passive samplers were deployed to evaluate NO2 concentration ranges across all locations.
46 by using standard NO2 color chart The color of the samples was specified in two colors namely light misty rose (9.5 ppbv) and misty rose (19.1 ppbv)
The study found NO2 concentrations ranging from 9.5 to 19.1 ppbv, which aligns with the lower range of 0.21 to 13.09 ppbv reported by Sransupphasirigul (2012) Both studies measured NO2 levels during the rainy season, with rainfall identified as a significant factor contributing to the observed low concentrations (Thammapanya, 2012) To ensure unbiased results, samples affected by ants and soil were excluded, as detailed in Table 5.
4.1.5 Correlation between nitrogen dioxide, environmental factors, species richness and lichens diversity
The Pearson correlation test was conducted to analyze the relationship between eight parameters: relative humidity, temperature, light intensity, bark pH, elevation, NO2, species richness, and lichen diversity, with results presented in Table 7 The analysis revealed no significant correlation between lichen diversity and temperature (r = -0.13, p < 0.05), relative humidity (r = -0.81, p < 0.05), light intensity (r = 0.93, p < 0.05), bark pH (r = -0.58, p < 0.05), and NO2 (r = -0.40, p < 0.05).
10 m
No Lichens present Frequency Remarks
No of Crustose Lichens: No of Foliose Lichens: Total F:
Crustose Lichens ( Bacidia sp and Phyllopeltula cf corticola – lack of picture of the specimen)
Arthonia black fruiting body Arthonia cinnabarina Arthonia white fruiting body
Arthothelium Cryptothecia sp.1 Cryptothecia sp.2
Bacidia cf medalis Buellia stillingiana Chrysothrix sp
Laurera sp Lecanora group.1 Lecanora group 2
Letroutitia aureola Letrouitia transgressa Malmidea piae
Rinodina roboris Pyrenula sp Bactrospora dryina
Lecanographa sp Nigrovothelium tropicum Trypethelium eluteriae
Foliose Lichens (Dirinaria complicata -lack of picture of the specimen)
Parmotrema praesorediosum Parmotrema saccatilobum Parmotrema tinctorum
Dirinaria applanta Dirinaria confluens Dirinaria picta
Hyperphyscia adglutinata Hyperphyscia pruinosa Hyperphyscia cf tuckermanii
Physcia cf.dilatata Physcia undalata Physcia cf poncinsii
Mae Rim (Temple) Mae Tang (Household)
Doi Saket (Household) Mae On- (Household)
San Pa Tong (Household) Chiang Mai City (Donation Center)