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
The economic growth of Thailand causes several issues which has come to a point where it is negatively affecting its people and environment Air pollution is one of the issues that beset the country as it is significantly worsening problem in recent times Several health implications concerning on respiratory- related conditions increased and caused 50, 000 deaths per year (Pacific Prime Thailand,
2017) In which particularly, Chiang Mai Province located in Northern Thailand greatly showed rapid growth of its economy since the number of tourist attractions, accommodations, factories, restaurants, road constructions and business buildings have increased as well as the number of tourists visited Chiang Mai Province Moreover, the industry and service sectors were considered to be the top two largest laborers in the province over the past few years (Kitirianglarp, 2015)
By these gains of the economic sector, there were trade-offs observed in the province, these are the traffic congestion, poor waste management, water and air pollution which generally affect the natural environment and resources of the province And out of these problems, air pollution is the biggest and greatly affects most of the people in terms of health According to the Pollution Control Department, PM 2.5 levels recorded on March 18, 2018 ranged from 48 – 86 micrograms per cubic meter of air which is high compared on the safety level of
50 micrograms per cubic meter of air
Annually, there are many cases wherein the residents and tourists in the hospital complain some illnesses and even caused several deaths because of the pollution These deaths caused by the excessive exposure to hazardous pollutants such as sulphur dioxide, benzene, lead, carbon monoxide and nitrogen oxide Particularly, sulphur dioxide causes shortness of breath, benzene can cause leukemia (ATDSR, 2007) and lead affects the central nervous system and the brain (WHO, 2018) On the other hand, too much exposure to carbon monoxide can lead to death (NCEH, 2017) and respiratory problems when someone intake nitrogen oxide in extremely high amount (US EPA, 2016)
In general, conception of imposing standard levels of nitrogen dioxide aimed to protect human health Pollution Control Department under the Ministry of Natural Sources and Management in Thailand set the standard levels for the hazardous pollutants in Thailand to compare to the present levels in an hour, month and a year
Likewise, other ways of monitoring the air pollution is considered to be useful and helpful These ways, for example is using lichens as biomonitoring method (Blasco et al., 2008) and by the use passive sampler which collects the data of nitrogen dioxide levels which is physicochemical monitoring method Both methods are known and commonly used in determining air pollution as the biomonitoring method determine the effects of the pollution to organisms and the physicochemical method identifies the concentration of the hazardous pollutants
Research objectives
With that, the objectives of the study are to investigate lichens diversity and their distribution of the lichens within the basin of Chiang Mai Province, to monitor the environmental factors and nitrogen dioxide levels and to determine the correlation between nitrogen dioxide, environmental factors, 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 limitation of the study was the lack amount of time in conducting the research which results on a short observation on lichens diversity and distribution Usage of only capable equipment on producing results within short study period was considered such as the passive sampler Passive sampler is known as a simple and easy device on collecting 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 composite organisms made up by the symbiotic relationship of fungus, algae and/or cyanobacteria The fungus is called mycobiont and algae and/or cyanobacteria is called photobiont (De Santis, 1999) There are two specific names for photobiont, it is phycobiont (algal photobiont) and cyanobiont (cyanobacterial photobiont) The mycobiont provides the structure and protection while the photobiont supplies food since it has chlorophyll Therefore, this composite organism is situated in the Fungi Kingdom as the function of fungus is associated with overall appearance of the lichen
The mycobiont components in lichens are from the two groups of fungi which are Ascomycetes (common) and Basidiomycetes (rare) group whereas, the common photobionts for phycobiont are Trentepohlia and Trebouxia and for cyanobiont are Stigonema, Nostoc, and Scytonema (Wolseley et al., 2015) Also, it is a composite organism that is found in many substrates such as soil (terricolous lichens), bark (corticolous lichens), rocks (saxicolous lichens) and habitats (mountains, seashores and rainforests) which contributes in some important process such as the process of slowing down the soil formation and providing food and shelter for animals
Morphology
The structure of the lichens is comprised of its body which is made up of filaments of fungal cells and this lichen body is called thallus (singular thalli) It has two types based on the internal structure namely homoiomerous thalli that is a simple type of thallus having only the filaments of fungal cells and algae in it while heteromerous thalli is a layered type wherein it is composed of four parts These parts are the upper cortex, algae layer, medulla and lower cortex (Constantine et al., 2018)
The upper cortex is the outer and protective layer covering of the thallus while the algae layer is the photosynthetic area in the thallus and whereas the algae associates with fungal hyphae For the medulla, it is the central foundation of the whole structure of thallus having firm walls and capable of acquiring gases in lichens The fungal hyphae in the medulla layer is uncompact but on the lower cortex, it has compacted fungal hyphae (Lepp, 2011b) And whereas some hyphae from the bottom termed as rhizines serve as roots to stick into subtrates is present and when the lower cortex is absent in the structure, the hypothallus is formed from a small sheet of hyphae to do the role
Alongside with that, there are other structures such as hapter, holdfast cephalodia, cilia, pruina, cyphellae and pseudocyphellae in lichens Hapter and holdfast help lichens attach on subtrates besides rhizines Cephalodia are small structures found only when lichen has both cyanobacteria and green algae (Henskens et al., 2012) These small structures usually in the upper or lower part
7 of the thallus and their important function is to execute nitrogen fixation in the environment
Cilia are small hair-like structures and pruina is an icy white coating which usually obtains calcium oxalate Both cilia and pruina are found on margin of the lichens The cyphellae are pores observed in the lower surface of thallus while the pseudocyphellae are the tiny pores formed by the expansion of medulla layer on the lower cortex and it is like a line along the margins on the upper cortex (Lepp, 2011a).
Growth forms and reproduction
Lichens have three common growth forms which are crustose, foliose and fruticose (Figure 1) Crustose lichen is the most common growth form and this type of lichen is a crust-like lichen and firmly attached to its substrates while foliose lichen is the type of lichens that has flattened shapes It is also known as leaf-like lichen that loosely binds on its substrate Fruticose lichen shown in Figure
1 (Sharnoff, 2018) is a shrub like or hair like lichens that grows in substrate by only attachment of its lower part since it obtains multidimensional forms (Lepp, 2011b) The growth of these crustose, foliose and fruticose lichens per year is usually around 0.5-5 mm
Reproduction of lichens is done by sexual and asexual (vegetative) reproduction In sexual reproduction, only the fungal component is being reproduced and there are fruiting bodies that have structures which are commonly known as ascus and basidium Ascus and basidium structures are particularly like container of spores that are being produced by two fungal partners such as Ascomycetes and Basiodiomycetes Ascomycetes produce spores called ascopores and Basiodiomycetes carried out spores named as basidiospores
In addition, these fruiting bodies are distinguished by their different forms namely apothecia and perithecia Apothecia are cup-like fruiting bodies found in the upper surface and can be differentiated into three kinds: lecanorine, lecideine, and lirellae For lecanorine apothecia, it has algae and margin having the same color with its thallus However, there is no margin, algae and have different color when it is a lecideine apothecia(Smith, 1921).Lirellae apothecia is long in shape and specifically attain black colored margins On the other hand, perithecia are spherical-like fruiting bodies whereas it only has small opening unlike apothecia
Figure 1 Three types of lichens (a) crustose (b) foliose (c) fruticose
Asexual or vegetative reproduction for lichens involves three structures These include isidia, soredia and lobules and unlike from the sexual reproduction both the algal and fungal components are reproduced Isidia are barrel-shaped or coral-like structure, soredia are small and powdery group of hyphae and lobules are flat lobes in the thallus These structures detached on the thallus and reproduce by the environmental conditions and physical interactions, for example by wind and disturbance of animals (Smith, 1921).
Factors affecting the lichens
Lichens are composite organisms found on variety of habitats where there are substrate and environmental conditions for their growth The substrate conditions, for instance are surface types and components Lichens are generally having high frequency on rough, flaky and persistence barks and low frequency on smooth barks Likewise, for the surface components including the pH value and water content on the tree barks, lichens can survive even though there is little amount of water available and has moderate amount of acidity in the substrates (Lam et al., 2013)
Environmental conditions affecting the lichens were temperature, relative humidity, light intensity and elevation Both environments that have high and low temperature alter the richness of lichens because the reproduction and the food of lichens are limited by these temperatures The factor of humidity in general, influences the lichen growth at which point is more evident in high humid locations
10 than the places that have low humidity (Čabrajić, 2009).The light intensity upon lichens is also one of environmental conditions considered as the sunlight plays an important role for lichens to exist And for that, lichens are present in places that have enough exposure on sunlight (Lam et al., 2013) The growth and diversity of lichens is also influenced by elevation, as higher elevation the more diverse and species of lichens were observed (Kumar et al., 2014)
2.5 Lichens as biomonitor of air pollution
Both sensitivity and adaptive capability of the lichens is considered to be essential in monitoring environmental problems particularly the air pollution The sensitivity of lichens is caused by their structure which has the absence of an outer layer like epidermis or cuticle and roots In addition to that, the adaptive capability pertains on the structural variations of lichens depending on the environment it grows With that, lichens are exposed and notably influenced by their atmospheric conditions ascertaining their relevancy as an indicator of air pollution (Lawrey,
Crustose, foliose and fruticose lichens have their own level of tolerance on the changes in the environment Within these lichens, crustose lichens are known to be the most tolerant lichen species since it is still present even in areas that have polluted air Whereas, foliose lichens can only grow in partly polluted areas and fruticose lichens cannot live totally on places that has really small amount of pollutants since it requires completely clean air for it to grow (BBC, 2014) Likewise, the determination of sensitive (absent) and tolerant (present) species
11 within the three common lichens specifically helps in distinguishing the possible indicator species as well as the changes in the environment
With that, some studies evaluated the global environmental problem which is climate change by using the responses of lichens in terms of the diversity and abundance within the areas of study As for example, a sixteen –year (1993- 2009) study in US Pacific Northwest determined that the high elevation lichens were the most affected since the number of species and the distribution decreased comparing on the low elevation concluding that the warming greatly affect the cooler environments (Glavich et al., 2018) A study from North-western Alaska observed low number of lichens in the Arctic tundra (Joly et al., 2009)
And, a study on cold temperature species of lichens decline in both highland specifically in the mountains of Western and Central Europe and lowland areas (Shukla et al., 2018) Nevertheless, from some studies lichens were increasing in cold climates rather than decreasing A study on forests in Sweden whereas specific species were investigated lichens such as Bryoria spp., Usnea spp and Alectoria sarmentosa (Ach.) Ach The Usnea spp and Alectoria sarmentosa
(Ach.) Ach appeared to increase in the areas that have cold temperature than in warm temperature places while the Bryoria spp was not present all in both temperatures (Esseen et al., 2016) Moreover, climate change increased of either the extreme heat or rainfall, the growth and the diversity of lichens will still be affected
Therefore, lichens in local climate scale were considered to be influenced by the varying levels of microclimatic factors from each environment it grows In
12 a study situated in Badrinath valley in Western Himalaya, the lichens were identified to be affected by the temperature, altitude and relative humidity Similarly, distribution study of lichens was altered in the White Mountains of California because of the light intensity (Gupta et al, 2014)
And in accordance of the sensitivity and tolerance capability of lichens, these were used to assess a common environmental problem which is air pollution Air pollution pertains on the harmful and excessive amount of pollutants released in the air caused by the intensified human activities For instance, these activities were vehicle emissions, power plants, agricultural and industrial activities and from that, the common pollutants released are sulphur dioxide and nitrogen dioxide
With that, the possible availability and excessive amount of atmospheric pollutants such as nitrogen dioxide and sulphur dioxide were monitored by using lichens (Richardson ,1988) As both high amount of nitrogen dioxide and sulphur dioxide negatively affect the chlorophyll and eventually kills the lichen Due to that, studies about both atmospheric pollutant levels used particularly the sensitive and tolerant species of lichens on each pollutant
Based on that, there was a study that discovered new species of lichens and occurrence again of some species in London conforming the air quality became free from sulphur dioxide (Hawksworth & McManus, 1989) In United Kingdom, the nitrogen dioxide pollution was observed as five terricolous alpine lichen species were affected which were sensitive lichen species to nitrogen dioxide (Nash & Gries, 2002)
A study in Colombo and suburbs that determined the amount of sulphur dioxide and nitrogen dioxide pollution evaluated lichens as well as the disturbances found in the area of the study The pollution within the area was confirmed to be in high amount since there was a declined in number of lichens as well as the total absence of tropical lichens Considering that, these tropical lichens were generally known as pollution sensitive species in the study in area of Colombo and suburbs (Attanayaka et al 2007)
Muhammad et al (2018) identified the D picta species of lichens as the species associated with the vehicular emissions in Malaysia particularly in Bahut Pahut area Since, D picta was present in all locations whether it has high traffic or low traffic level and considered to be the tolerant lichen species among all the present lichens Alongside with that, a study in Chiang Mai, Thailand determined that P cocoes was tolerant to air pollution And also determined the sensitivity of
D picta which may be considered as a bioindicator of air pollution within the area and the tolerance of P.cocoes (Pimwong, 2002)
The device used in this method is called passive sampler wherein it performs molecular diffusion to collect the pollutants In this procedure, there is a free flow of analytes (heavy metals, inorganic and organic compounds) from the atmosphere to collecting media illustrated in Figure 2 (sigmaaldrich, 2010) These compounds can be in any matrices such as soil, water and air (Wongniramaikul,
2012) Furthermore, the average amount from the collected pollutants were estimated by using the Fick’s law of diffusion
Passive Sampling
The device used in this method is called passive sampler wherein it performs molecular diffusion to collect the pollutants In this procedure, there is a free flow of analytes (heavy metals, inorganic and organic compounds) from the atmosphere to collecting media illustrated in Figure 2 (sigmaaldrich, 2010) These compounds can be in any matrices such as soil, water and air (Wongniramaikul,
2012) Furthermore, the average amount from the collected pollutants were estimated by using the Fick’s law of diffusion
Passive Sampling is a physico- chemical method commonly used in air pollution monitoring This method is also considered to be cost- effective and flexible tool in assessing the amount of pollutants in any study area It is a cost- effective device because it does not require any maintenance for it to work properly and electricity to operate as the same time as it is a flexible tool by being a lightweight and simple device
Due to that, some studies emerged using passive sampling methods in their respective countries with the purpose of monitoring air pollutants and their amount which affects the air quality in the country In Malaysia, the results from the passive sampling proved the impact of the human activities, specifically the construction site and vehicle emissions on the air quality in the area Considering, three areas were chosen and among these, the farthest from the roads and the construction site showed a 14.0 ppbv which was the lowest amount while the area that was near to these activities displayed a 23.4 ppbv of nitrogen dioxide amount and the highest nitrogen dioxide amount recorded was 36 8 ppbv which were measured in the areas that was nearest to the activities (Chong et al., 2017) A monitoring study in the major highways in Abeokuta, Nigeria determined air pollutants like nitrogen dioxide, sulphur dioxide, carbon dioxide and other pollutants in both wet and dry seasons And all the pollutants appeared to be in high concentration in the dry season than wet season For instance, the Iyana Mortuary have the highest concentration of both nitrogen dioxide and sulphur dioxide This can be associated with thedomestic cooking, expanding of roads and traffic congestion Likewise, the high concentration of carbon dioxide in Kuto was
15 because of the vehicular emissions and particularly the concentration of this pollutant was higher than the WHO (2005) threshold limit value (TLV) of 500 ppm indicating the air quality in the area was not in a good condition anymore and can severely affect the health of the people (Olaynika et al., 2015)
Another study in Chiang Mai Thailand wherein the NO2 concentrations within the urban, suburban and rural areas in Chiang Mai, Thailand were investigated and determined the high concentration in urban areas compared to the suburban and rural areas Particularly, the area that displayed the highest concentration was evidently located near where there was a common and serious traffic problem
Some studies that both used passive sampling and lichens in evaluating the air quality were found A study in South Korea evaluated the sulphur dioxide, nitrogen dioxide in different types of forest (Quercus, coniferous and deciduous) The deciduous forest was located in the Korea Peninsula which was more prone on the impact of human activities as coniferous forest and the Quescus forest was located in Jeju island that obtain high relative humidity level Due to that, coniferous forest had the highest amount of nitrogen dioxide while deciduous forest had the highest concentration of sulphur dioxide and the Quercus forest had the lowest amount for both pollutants The results of the study showed a negative correlation of SO2 and NO2 concentrations to the diversity of lichens observed in the sites (Udeni et al., 2016) A study of Pomphueak (2005) in Chiang Mai, Thailand showed a positive correlation of the lichen diversity with theSO2 and
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 is a province in Northern Thailand and considered as the second – largest province in the country The province covers about 20,170.1057 sq (Chiang Mai Province official site, 2009) and the annual temperature is 25.6°C and annual rainfall in the province is 1184 mm(Climate-Data.Org, 2017) The climatic condition within the province is affected by tropical monsoons whereas three seasons were observed such as cold season that begins during November and
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 following materials were used in collecting and analyzing the lichens and nitrogen dioxide (passive sampling method) within the study Materials used for collecting data for lichens were compass, pens, recording forms, pocketknife, grid frame, basket, camera, small paper envelopes, dropper, measuring tape and for nitrogen dioxide were cool box and plastic zip lock bag The laboratory materials for lichens study were the following: slide and cover glass, hand lens, 25-600 ml beaker, 10 ml graduated cylinder, analytical balance, pH meter, compound microscope, stereo microscope, ultraviolet lamp, GPS (Global Positioning System), Lux meter and wet and dry thermometer For the passive sampling method, the materials were oven, ultrasonic bath, shelter, 10 ml polypropylene tubes, parafilm, Whatman (GF/A), 10 ml syringe, 50 ml beaker, 10 ml micropipettes, 25 - 250 ml volumetric flask, glass stirring rods and pipet bulb
The chemicals used for lichens study were deionized water, sodium hypochlorite, potassium hydroxide; K :10%; Lugol’s iodine: 5% For the passive sampling method, the chemicals used were the following: deionized water, phosphoric acid, Triethanolamine (TEA), N-(1- Napthyl) ethylenediamine dihydrochloride (NEDA) and Sulfanilamide; C6H8N2O2S
Methods
The study investigated 10 mango trees (Mangifera indica L.) trees within an area size of 2 km x 2 km Grid frame was used to count the frequency of lichens on the selected tree The grid frame size was about 20 x 50 cm 2 and had 10 subsquares, size of 10 x 10 cm 2 It is attached on the part of the tree trunk where covered with lichen most has many lichens and placed 150 m above ground by using a rope (Figure 4) Species and frequency of lichens found inside the grid frame were collected and listed in the recording forms by using hand lens To avoid misidentification of lichens within the grid frame, lichens that had a smaller diameter than 3 mm was not collected Whereas, lichens outside the frame were also determined and its frequency was given as one
The lichens that were not recognized in the field was collected in paper envelopes for further identification by using ultraviolet lamp, compound microscope and stereo microscope Morphology, growth, reproduction and dispersal of these lichens were identified by using key of Sipman (2003), key of Wolseley and Aquire- Hudson (1997) and key of Awasthi (1991) The circumference of the tree, type of bark, bark pH, aspects whereas the grid frame were placed in the tree trunk, degree of exposure of the tree in the sun and the description of the location, possible activities and establishments near the area whereas the distance of the trees was also determined in each site
22 b) Measurement of some environmental factors
Some factors were collected by using equipment such as Lux meter for light intensity, Global Positioning System (GPS) for altitude, dry thermometer for temperature and wet - dry thermometer for determination of the relative humidity by being hung at 1.5 meters above the ground The elevation from each site was adapted by using Google Earth c) Analysis of bark pH
The collection of barks without lichens was conducted at 1 meter above the ground These barks are collected inside the paper envelopes and stored inside the freezer After that, the barks were weighted in the analytical balance having 0.5 grams and be kept in the plastic cups Then, they are soaked with 5 ml of distilled water for 8 hours Electrochemical analyser for pH meter was used to measure the bark pH from each 10 trees in six sites
Figure 4 The position of a grid frame on selected tree
The sites were chosen from one of the 10 investigated trees from each site The safety and required exposure of the passive sampler was also considered to avoid cases of lost passive samplers within the study The following procedures were the following: a) 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 reagent B was 0.038 g of N-( 1- Naphthyl) ethylenediamine dihydrochloride was adjusted to volume by using 25 ml beaker The Saltzman reagent was placed on the freezer until the collection day b) Preparation of diffusion tube
The GF/A filter paper having a 110 diameter was cut into small pieces to fit in the polypropylene tubes then were soaked in the deionized water for three times and put in the oven to prevent it to be stretched and for it to be dry
Forty-eight polypropylene tubes were prepared and placed on six passive samplers having eight tubes each These eight polypropylene tubes were divided into 5 diffusion tubes represented as replication of the samples and 3 blank tubes served as the control samples in same condition (Figure 5) 20% of
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 tubes were positioned vertically in the passive sampler and was placed at 150 meters above the ground in one of the trees in each site The exposure period was during May 25 to June 1, 2018
On the collection of the passive samplers, the tubes were sealed with paraffin film and placed in plastic zip lock bag The tubes were kept in freezer until the extraction of the samples
Figure 5 Configuration of passive sampler
The extraction of the samples was done by adding 2 ml deionized water in the tubes using a syringe The tubes were being shaken for about 10 minutes before analysis e) Analysis of the sample
The samples were transferred to small bottles and added 2 ml Saltzman reagent (Figure 6) The colors of the samples were compared to the NO2 standard color chart and determined the rates of NO2 levels (Figure 7)
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
Since the focus of the study is lichen diversity, the diversity index that is used for analyzing the data is Shannon Diversity Index The Shannon Diversity Index (H') is another commonly used index that is used to illustrate the species diversity in a community as it accounts for both the abundance and the evenness of the species present
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
The similarity of the lichen communities within all the sites was done by using cluster analysis andanalyzed by Multivariate Package Program (MVSP) The MVSP program used Bray -Curtis Similarity Index and illustrated in a dendrogram Detrended correspondence analysis (DCA) illustrated the distribution of lichens in the study by 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 that include 38 species were discovered Thirty-eight species were composed of 24 crustose lichens and 14 foliose lichens List of the total of families, genera and species of lichens was demonstrated in Table 2 and the list of species and their frequencies in all study areas was presented in the Table 3
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 highest diversity index was found in Mae Tang while the highest richness was found both in Mae Tang and Mae on The lowest diversity index recorded was in Chiang Mai City and lowest richness was found in Doi Saket The richness and diversity index within the study areas were showed 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
(2005) and Kheawsalab (2010) Crustose lichens were abundant compared to foliose lichens in study areas Saipunkaew et al (2007) indicated same results in which the crustose lichens increased in number at sites that has an altitude of 250- 400 m whereas, this study was also performed in a similar range of altitude The highest richness and the dominance of lichen species were found in Family Physiaceae Family Caliciaceae had the lowest richness while both Families Caliciaceae and Malmideaceae had the lowest dominance in the study Pyxine cocoes, Hyperphyscia adglutinata and Dirinaria picta were the species that had high frequeny Pyxine cocoes had the highest frequency while Dirinaria picta from the species discussed found to had the lowest frequency.Some species such as Hyperphyscia adglutinata, Phyllopeltula cf corticola and Rinodina roboris were only found in the urban area that had high anthropogenic impacts which corresponded to 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
Physcia poncisii, Parmotrema praesoredium, Hyperphyscia pruinosa, Trypatheliem eluteriae and Nigrovothelium tropicum were found in suburban areas Letroutia transgressa and Letroutia aureola were only found only in Mae On Lichens in this genus were indicators of moist condition such as moist forest Mae On was located close to the mountainous areas with less human activities occurred in the area This may explain why lichens in this genus were found only in Mae On site (Wolseley and Aguirre- Hudson ,1997)
4.1.2 Similarity of lichen communities and distribution of lichens
The method in determining the similarity of lichen communities in the study was done by using cluster analysis The cluster analysis was based on Bray-Curtis similarity index due to the objective of finding the similarities between the individual samples with a wide range of scales in a dendrogram in Figure 8 In the dendrogram, group average was calculated by unweighted pair-group method with arithmetic averaging (UPGMA) From this method, the results distinctly indicated two groups within the six study sites and 30 % similarity were found between the groups The first group were comprised of San Pa Tong, Mae Rim, Mae Tang, Doi Saket and Mae On and the second group was specified on Chiang Mai City The lichen communities were distinctly different from that of the first group as the species were commonly found in areas that had high human activities
Distribution of lichens within the sites was determined by Detrended Correspondence Analysis (DCA) which corresponded on the results from the Bray- Curtis presented in Figure 9 Some species common on the first group usually found in
41 suburban areas (San Pa Tong, Mae Rim, Mae Tang, Doi Saket and Mae On) were the following: Chrysothrix sp., Pyxine cocoes, Dirinaria picta and Hyperphyscia adglutinata Distinct species in each site were Trypathelium eluteriae, Bactrospora dryina, and Pyrenula sp The second group found species commonly observed in urban areas found (Chiang Mai City) were Rinodina roboris, Phyllopeltula cf corticola
,Dirinaria picta and Pyxine cocoes
Hyperphyscia adglutinata was found well distributed in all sites although had a high frequency in the urban area Phyllopeltula cf corticola and Rinodina roboris were both only found in urban areas Hyperphyscia adglutinata, and Phyllopeltula cf corticola were identified as tolerant species to air pollution which corresponded on
(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 commonly affected by environmental factors such as elevation, light intensity, temperature, relative humidity and bark pH These environmental factors collected in the study observed to have no distinct difference in their values in each site and have similar land use patterns and human activities The collected data for light intensity, relative humidity and temperature from all sites cannot be compared as the collected data of all sites were only collected at different point of time and day but it should be done at least daily
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
Passive sampling method consists of five procedures including preparation of the solutions (20% Triethanolamine (TEA) solution, N-( 1- Naphthyl) ethylenediamine dihydrochloride solution (NEDA), sulfanilamide solution, ortho-phosphoric acid and saltzman reagent), preparation of the diffusion tubes where TEA solution was absorbed in GF/A filter paper and was placed on the bottom of the diffusion tubes , exposure of diffusion tubes , analysis of NO2 concentrations by using standard NO2 color chart (Bootdee, 2009).The passive sampling method was done in six sites where the passive sampler in each study sites The NO2 concentrations from all site ranges were evaluated
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 ranges of concentration of NO2 collected in the study ranges from 9.5 -19.1 ppbv and the study of Sransupphasirigul (2012) with the ranges from 0.21- 13.09 ppbv corresponded on low concentration of NO2 found in the study Furthermore, the NO2 concentrations measured were both measured during the rainy season One of the factors that found to affect the NO2 concentration was rainfall in which it caused the low concentration (Thammapanya, 2012) The samples that were affected by ants and soil were omitted to have unbiased results and were shown in Table 5
4.1.5 Correlation between nitrogen dioxide, environmental factors, species richness and lichens diversity
The correlation between the eight parameters: relative humidity, temperature, light intensity, bark pH, and elevation, NO2, species richness and lichen diversity were done by using Pearson correlation test (p