For Review Only 20 Al., 2009)

Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

The species distribution pattern, composition and diversity of Rubiaceae along the altitudinal gradient of the Himalayas in Western Bhutan

Journal:For Songklanakarin Review Journal of Science Only and Technology Manuscript ID SJST-2018-0116.R1

Manuscript Type: Original Article

Date Submitted by the Author: 22-May-2018

Complete List of Authors: ., Tobgay; Prince of Songkla University, Biology; Daga Higher Secondary School, Science Sridith, Kitichate; Prince of Songkla University, Biology; Prince of Songkla University, Biology

Keyword: Rubiaceae, Bhutan, Altitude, Himalaya, Diversity

For Proof Read only Page 1 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 (Original Article) 4 5 6 The species distribution pattern, composition and diversity of Rubiaceae along the 7 8 altitudinal gradient of the Himalayas in Western Bhutan 9 10 1,2* 1 11 Tobgay and Kitichate Sridith 12 13 1 14 Department of Biology, Faculty of Science, Prince of Songkla University, Hat Yai, 15 16 Songkla, 90110 Thailand 17 18 19 2 Daga Higher SecondaryFor School, Review Dagana, Ministry Only of Education, Thimphu, 00975 20 21 Bhutan 22 23 24 *Corresponding author, email: [email protected] , Telephone +97517714036 25 26 27 Abstract 28 29 30 The study conducted along the altitudinal gradient of 300 – 3900 m asl. recorded a total 31 32 of 36 species (21 genera) and 46 species (29 genera) in the study area I and II 33 34 respectively. The sighting of only one species each for the majority of genera, including 35 36 Pavetta L., Oldenlandia L. and Argostemma Wall., which are described as the largest 37 38 39 genera of Rubiaceae, is unusual. While the species with narrow distribution range are 40 41 found concentrated at the lower altitude exhibiting endemic nature, the herbaceous 42 43 species exhibiting maximum distribution range are dominant at the higher altitudinal 44 45 range. The low altitudinal hump-shaped distribution pattern was observed in both the 46 47 study areas. The higher diversity indicated by Shanon-weiner and Simpson diversity 48 49 analysis is attributed to moderate mean precipitation, temperature and relative humidity. 50 51 52 The altitude and litter thickness contributed the maximum to the species composition 53 54 and distribution pattern based on CCA analysis. 55 56 57 58 1 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 2 of 25

1 2 3 Key words: Rubiaceae, Bhutan, Altitude, Himalaya, Diversity 4 5 6 1. Introduction 7 8 9 Comprising of around 13000 species belonging to 620 genera (Bremer & Erikson, 10 11 2009), Rubiaceae stands as the fourth largest angiosperm family (Block, Taylor, & 12 13 Huysman, 2009). Rubiaceae is found distributed all across the regions of the world 14 15 except Antarctica (Barbhuiya, Dutta, Das, & Baishya, 2014), exhibiting high species 16 17 diversity and richness proliferation from low to mid-altitude in humid forest (Davis et 18 19 For Review Only 20 al., 2009). 21 22 In spite of its abundance, the diversity of Rubiaceae is checked by high endemism 23 24 25 further aggravated by ecological sensitivity (Barbhuiya et al., 2014), thus increasing the 26 27 vulnerability to extinction, particularly the monotypic genera. The susceptibility of 28 29 Rubiaceae is further augmented by the presence of 72% of genera with lesser than 10 30 31 species and 211 monotypic genera (Davis et al., 2009). So, the perceived restricted 32 33 distribution of Rubiaceae observed in secondary forest highlights the ecological 34 35 sensitivity of the family and the immeasurable risk of extinction. This behavior of the 36 37 38 family limits their diversification in the disturbed places. 39 40 41 Against the backdrop of such ecological traits of Rubiaceae, the Himalayan Kingdom of 42 43 Bhutan, which is described as one of the ten biodiversity hotspots of the world (Myers, 44 45 Mittermeier, Mittermeier, Fonseca, & Kent, 2000) hosting around 7000 vascular plant 46 47 species comprising of 82 endemic species (Wangchuk, 2005) have recorded only 55 48 49 genera and 141 species (Grierson & Long, 1999) of Rubiaceae in the Flora of Bhutan. 50 51 Apart from its record in the Flora, no research concerning distribution pattern and 52 53 54 ecology about the family had been conducted till date in Bhutan. Such documentation is 55 56 57 58 2 59 60 For Proof Read only Page 3 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 necessary to assess the changes in plant diversity and to the conserve endemic and 4 5 endangered species. 6 7 8 Hence, the focus of the current study was to assess the distribution pattern, species 9 10 richness and the influences of environmental factors to the family. Particularly, the 11 12 study is aimed at (i) Exploring the distribution pattern of the species in the family, (ii) 13 14 Evaluating the species richness and diversity of the family, and (iii) Assessing the 15 16 17 influence of environmental factors on the distribution pattern and species diversity. 18 19 For Review Only 20 2. Methods 21 22 Study areas 23 24 25 The survey was conducted in two study areas along the Himalayan range of western 26 27 28 Bhutan showing altitudinal variations from 300 to 3900 m asl. (Figure1). 29 30 31 1. The area falling within Phuntsholing and Thujaydrak (referred to as study area I 32 33 hereafter) 34 35 2. The area falling within Dagachu and Dagala (referred to as study area II 36 37 hereafter) 38 39 Figure 1. 40 41 42 These areas cover subtropical forests (200 – 1000 m), warm broad-leaved forests (1100 43 44 – 2000 m), cool broad-leaved forests (2100 – 2900 m), conifer forests (3000 -3500 m) 45 46 and subalpine forests (3500 – 3900 m) according vegetation zonation done by Norbu et 47 48 49 al. (2003).The survey had focused on covering maximum range of area as Rubiaceae are 50 51 found to occupy wide range of habitats (Bremer & Erikson, 2009). The two areas 52 53 showing high elevational gradient were chosen based on the differences in rock 54 55 56 57 58 3 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 4 of 25

1 2 3 composition (Long, McQuarrie, Tobgay, Grujic, & Hollister, 2011), annual 4 5 precipitation, temperature and relative humidity (Table 1). 6 7 8 The climate data for the past ten years, recorded at the weather stations located in the 9 10 study areas, were obtained from the National Center for Hydrology and Metrology, 11 12 Thimphu Bhutan. The mean annual precipitation, the mean maximum and minimum 13 14 temperature and the mean relative humidity of the study areas are shown in table 1. 15 16 17 Table 1. 18 19 For Review Only 20 2.2 The landscape, study sites and data collection 21 22 23 The study sites are characterized by deep river valleys, slopes, ridges, thick forest, 24 25 thickets and alpine vegetation. The degree of anthropogenic influences along the 26 27 28 gradient in both the study areas is almost similar, characterized by the presence of road, 29 30 hydro-power plant and settlements. Therefore, it provides wide range of micro-habitats 31 32 from disturbed to intact for the plant species to occupy. 33 34 35 The field visits were made twice a month from March to December 2017 to cover whole 36 37 flowering period. The species presence and absence data were obtained at every 200 m 38 39 elevation interval along the gradient in both the study areas. Consequently, every 200 m 40 41 elevation stands were marked as reference point for the data collection and analysis. 42 43 Along these lines, a total of 38 sites, 19 sites in each study area were marked. 44 45 46 Nonetheless, the species encountered in other areas were also included in the nearest 47 48 reference point (Vetaas & Grytnes, 2002) for the purpose of assessing the diversity and 49 50 richness. The distribution range of each species was calculated following the method of 51 52 Subedi, Bhattarai, and Chauudhary (2015). 53 54 55 56 57 58 4 59 60 For Proof Read only Page 5 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 The information on location, altitude, latitude and longitude were gathered using 4 5 Garmin GPS, the soil pH was recorded using the plant sap pH meter and the litter 6 7 thickness was measured by ruler (Zhang, Zu, & Li, 2013) for every plant specimen 8 9 collected. The soil samples were collected and analyzed for Carbon, Nitrogen, 10 11 Phosphorus and Potassium at the National Soil Center, Simtokha, Thimphu Bhutan. 12 13 14 The plant specimens were identified using the Flora of Bhutan (Grierson & Long, 15 16 17 1999), Flowers of the Himalaya (Polunin & Stainton, 1984), Flowers of the Himalaya: a 18 19 supplement (Stainton,For 1984) Review and other available Only taxonomic literatures. The voucher 20 21 specimens were prepared following the methods of Bridson and Forman (1992) and 22 23 deposited at the National Herbarium, Serbithang, Thimphu Bhutan. 24 25 26 2.3 Data analysis 27 28 29 The species diversity indices, evenness and richness were calculated using Simpson 30 31 diversity index (Simpson, 1949) and Shanon-weiner index (Shanon & Weaver, 1949). 32 33 The metrological data and the species-altitude relationship data were analyzed through 34 35 direct computation in the Microsoft Excel 2007. The canonical correspondence analysis 36 37 38 (CCA) was used to elucidate the effect of environmental variables – pH, Carbon, 39 40 Nitrogen, Phosphorus, Potassium, litter thickness and altitude – to the species 41 42 composition. The Pearson correlation analysis was executed to analyze the relationship 43 44 between environmental variables and the species richness. The analyses were performed 45 46 by using Statistical Package for the Social Sciences (SPSS) version 24. 47 48 49 3. Results 50 51 52 Species Richness and diversity 53 54 55 56 57 58 5 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 6 of 25

1 2 3 A total of 36 species of Rubiaceae belonging to 21 genera and 46 species belonging to 4 5 29 genera were recorded from study area I and II respectively (Table 3A & B). Among 6 7 them were 7 species which could not be identified due to the lack of complete floral 8 9 characters. The species richness was higher for area II at 56.1% (46 species) against 10 11 43.9% (36 species) for area I of the total Rubiaceae species recorded in the two study 12 13 14 areas. The Simpson and Shanon-weiner diversity and evenness analysis (Table 2) 15 16 revealed that the species diversity and evenness too was little higher for the area II. 17 18 19 Overall, the highestFor species recordedReview was for Wendlandia Only DC. and Galium L. taxa with 20 21 five species each followed by Ophiorrhiza L. and Mussaenda L. taxa with four species 22 23 each. Majority of the taxa consisted of one or two species only as detailed in table 3A & 24 25 B. The Galium L. taxa, specifically Galium aparine L. had shown maximum 26 27 diversification from sub-tropical forests through alpine forests based on the eco-floristic 28 29 30 zonation by Ohsawa (1987). In total, 18 taxa are represented by only one species. 31 32 33 Table 2. 34 35 Both the study sites showed similar variation in species life form, and in general the 36 37 38 shrub contributed 39.1% to the composition while tree, herb and climber constituted 39 40 13.4%, 36.6% and 10.9% respectively. 41 42 43 Table 3A & B. 44 45 46 Patterns of Rubiaceae species distribution along the altitudinal gradient 47 48 49 The Rubiaceae species shows varying degree of proliferation between 300 m to 3900 m 50 51 asl. in both the study areas along the altitudinal gradient. But the rate of proliferation 52 53 along the gradient isn’t uniform, exhibiting a hump-shaped pattern followed by a 54 55 gradual decrease (Figure 2). The highest species recorded in both the study areas is at 56 57 58 6 59 60 For Proof Read only Page 7 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 around 1400 m asl. The species distribution pattern in area I exhibits gradual decrease 4 5 while the study area II displays sharp decreasing pattern beyond 1500 m asl. 6 7 8 Figure 2. 9 10 11 The impact of environmental factors on distribution pattern 12 13 14 The cumulative degree of variance explained by the first two dimensions of Canonical 15 16 Correspondence Analysis (CCA) is 69.5% for area I and 79.7% for area II. The chi- 17 18 square value for area I is 325.977 (P <0.001) and area II is 312.634 (P<0.001). The 19 For Review Only 20 quantitative explanatory variables – pH, carbon, nitrogen, phosphorus, potassium, 21 22 altitude and litter thickness – for both the study sites had shown varying degree of 23 24 25 impact on the Rubiaceae species distribution pattern (Figure 3A & B). 26 27 28 Figure 3. 29 30 31 In general, altitude and litter thickness had shown greater influence on the species 32 33 distribution pattern along the gradient. The degree of variance caused by altitude stands 34 35 at 95.7% and 98.8% for site I and II respectively, causing significant influence on the 36 37 pattern of species distribution. Cumulating to just 0.33% of variance in site I by carbon 38 39 and 0.06% in site II by nitrogen, these two edaphic factors have contributed least to the 40 41 overall variation of species distribution pattern. 42 43 44 The Pearson correlation analysis supports the CCA, explaining a statistically significant 45 46 negative relationship between species distribution pattern and altitude in both the areas, 47 48 49 r(35)= -.37, p =.05 for area I and r(45) = -.36, p =.05 for area II. The litter thickness at 50 51 r(35) = .31, p =.05 for area I and r (45) = .32, p = .05 for area II on the other hand had 52 53 shown a significant positive correlation to the species distribution pattern. The analysis 54 55 also reveals varying degree of correlation between different environmental factors. It is 56 57 58 7 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 8 of 25

1 2 3 interesting to note the significant negative correlation between altitude and litter 4 5 thickness (Table 4A & B). 6 7 8 Table 4 A & B. 9 10 11 4. Discussion 12 13 14 Species diversity along the elevation gradient of the two study sites 15 16 17 The higher species diversity for study area II revealed by Simpson and Shanon-weiner 18 19 diversity and evennessFor analysis Review may be an indication Only of the prevalence of a favorable 20 21 conditions for greater species diversification in this area. While other factors would 22 23 have had bearings on the higher species diversity, the moderate variation in temperature, 24 25 rainfall and relative humidity in the study area II compared to the study area I, may have 26 27 28 generated overreaching impact on the overall species diversity. It is evident from the 29 0 0 30 maximum mean temperature of 26.6 C and the minimum mean temperature of 3.8 C in 31 32 area I against a maximum mean temperature of 27.40C and minimum mean temperature 33 34 of 7.30C in area II, to conclude that the temperature range was favorable in area II for 35 36 greater physiological processes, growth, development and diversification of plant 37 38 species. Furthermore, moderate rainfall and relative humidity may have enabled better 39 40 41 conditions for the diversification. Conversely, in the area I, the extreme maximum and 42 43 minimum temperatures coupled by high variation in precipitation and relative humidity 44 45 along the gradient and across the year (Table 1) may have hindered the growth rate and 46 47 diversification as plant growth is found to cease below 50C and above 300C even when 48 49 other conditions are reliable (Haferkamp, 1988). 50 51 52 53 54 55 56 57 58 8 59 60 For Proof Read only Page 9 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 Patterns of Rubiaceae distribution along the altitudinal gradient 4 5 6 The predominance of Rubiaceae at lower altitude exhibiting a peak at around 1400 m 7 8 asl. conforms with the proposition of Davis et al. (2009). Hosting all forms of life, the 9 10 species composition at lower altitudinal range of 300 – 1500 m asl. stands at 86% 11 12 against 50% at the higher altitude range of 1600 – 3900 m asl. The dominance of 13 14 herbaceous species at higher altitude and the absence of tree species beyond 1900 m asl. 15 16 17 and shrub species above 2700 m asl. hints limited tolerances of these life forms to 18 19 reduced temperature,For precipitation Review and relative Only humidity. On the contrary, the 20 21 preeminence of herbaceous species at the higher altitude range may be fostered by the 22 23 ability of herbaceous perennials to undergo dormancy during unfavorable periods and 24 25 complete their life cycle within the short growing period. 26 27 28 Generally, the Rubiaceae taxa represented by one or two species and the taxa showing 29 30 narrow distribution range, such as Wendlandia grandis (Hook.f.) Cowan., Psilanthus 31 32 benghalensis (B. Heyne ex Schult.) J.-F. Leroy, Ixora undulata Roxb., Uncaria 33 34 35 sessilifructus Roxb. etc., are concentrated at the lower altitude. Such nature of species 36 37 might suggest the endemic character of these species and their susceptibility to 38 39 extinction due to various disturbance regimes. 40 41 42 The detection of only one species for 18 taxa is unusual for the family which is 43 44 described as fourth largest angiosperm family. The sighting of only one species for 45 46 Pavetta L., Oldenlandia L. and just two species for Argostemma Wall., is surprising as 47 48 these three taxa are described as top 20 largest genera in Rubiaceae (Davis et al., 2009). 49 50 51 Such findings may well be associated with current research artifacts or unfavorable 52 53 conditions caused by topography, climate and edaphic factors for these groups of taxa. 54 55 The maximum distribution range demonstrated by Galium L., particularly the Galium 56 57 58 9 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 10 of 25

1 2 3 aparine L., ranging from 900 to 3900 m asl. confirms high resilience of this taxa to 4 5 varying environmental conditions and topography. In contrast to species showing 6 7 narrow distribution range, such taxa are expected to express high tolerances to the affect 8 9 of disturbance and global climate change. 10 11 12 The impact of environmental factors on distribution pattern 13 14 15 The assessment of the impact of environmental factors on the species distribution 16 17 pattern and composition revealed that each factor have caused varying degree of 18 19 For Review Only 20 influences on the total species composition (Figure 3A & B). The cumulative degree of 21 22 variance explained by the first two dimensions of CCA at 69.5% for area I and 79.7% 23 24 for area II substantiates the degree of variance caused by the environmental factors to 25 26 the actual species distribution pattern. The chi-square value at 325.977 (P<0.001) for 27 28 area I and 312.634 (P<0.001) for area II validates the statistical significance. 29 30 Consequently, the environmental factors have fostered heterogeneity of conditions that 31 32 shapes overall species distribution pattern and diversity. 33 34 35 Different Rubiaceae species have been influenced differently by each environmental 36 37 38 factor as revealed by CCA ordination diagram (Figure 3A & B). But altitude and litter 39 40 thickness, in most cases, had shown larger influence on the species distribution pattern 41 42 along the gradient. The degree of variance caused by altitude stands at 95.7% and 43 44 98.8% for area I and II respectively, causing significant influence on the pattern of 45 46 species distribution and diversification. The overreaching impact of altitude on species 47 48 composition could be related to its impact on productivity (Bruun, 2006) and biotic 49 50 51 interactions (Lovett, Marshall, & Carr, 2006), which is described to decrease coherently 52 53 with the increasing altitude. The Pearson correlation analysis strengthens the CCA 54 55 findings, explaining a significant negative relationship between species distribution 56 57 58 10 59 60 For Proof Read only Page 11 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 pattern and altitude, r(35)= -.37, p=.05 for area I and r(45)= -.36, p=.05 for area II. This 4 5 result is in accord with the inference of other similar researches (Rahbek, 1995; 6 7 Lomolino, 2001; Jamtsho& Sridith, 2017) where species diversity is described to 8 9 decline along the altitudinal gradient. 10 11 12 The total degree of variance caused by litter thickness accounts to 94.8% for area I and 13 14 99.7% for area II on CCA ordination, exhibiting high degree of influence on the species 15 16 17 diversity and distribution pattern. It is further validated by Pearson correlation analysis, 18 19 r(35) = .31, p =.05 Forfor area I Reviewand r (45) = .32, p =Only .05 for area II, showing statistically 20 21 positive significance of litter thickness on the species distribution pattern. The 22 23 correlation analysis also exhibited varying degree of associations between different 24 25 environmental factors (Table 4A & B), which have eventually brought combined effect 26 27 on the overall species distribution pattern and composition. So, the significant negative 28 29 30 correlation between altitude and litter, r (35) = -.33, p=.05 for area I and r(45) = -.31, 31 32 p=.05 for area II, supports the idea of one factor influencing the other and the combined 33 34 control of all the factors on the overall species distribution pattern. 35 36 37 The other environmental factors like pH, carbon, nitrogen, phosphorus and potassium 38 39 had not shown statistically significant influence on the species composition. 40 41 Nonetheless, their combined effect is important in determining species diversity as these 42 43 nutrient elements play key role in the growth and development of plant species in 44 45 general. 46 47 48 5. Conclusion 49 50 51 The higher species richness and diversity observed at low to mid-altitude followed by 52 53 54 gradual decline above 1500 m asl. is in concordance with the results of earlier 55 56 researches. The species richness and diversity are found to be influenced by favorable 57 58 11 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 12 of 25

1 2 3 range of annual precipitation, temperature and relative humidity. In general, altitude and 4 5 litter thickness had shown supremacy over other factors in shaping distribution pattern 6 7 of Rubiaceae along the elevational gradient. The representation of 18 taxa, including 8 9 some of the taxa described as largest, by one species in the current research requires 10 11 further validation. The investigation into this gap may help to uncover the obscurity 12 13 14 which may have been caused by the current research design or the species diversity 15 16 restriction of those taxa powered by disturbance regime or ecological factors. 17 18 19 AcknowledgmentsFor Review Only 20 21 22 The authors are grateful to the Department of Forests and Park Services, Ministry of 23 24 Agriculture and Forests (MoAF), Bhutan, for the research permit; the National Soil 25 26 Centre, Simtokha for their generous support in analyzing the soil samples; the National 27 28 Centre for Hydrology and Metrology, Thimphu, Bhutan for providing the climate data; 29 30 and the Graduate School, Prince of Songkla University, Hat Yai, Songkla, Thailand for 31 32 providing financial support to the first author as well as the research funding of the 33 34 35 project through Thailand’s Education Hub for Southern Region of ASEAN Countries 36 37 (TEH-AC) funding and the Thailand Annual Budget of the year 2016. 38 39 40 References 41 42 43 Barbhuiya, H. A., Dutta, B. K., Das, A. K., & Baishya, A. K. (2014). The family 44 45 Rubiaceae in southern assam with special reference to endemic and 46 47 rediscovered plant taxa. Journal of Threatened Taxa, 6(4), 5649-5659. 48 49 50 Block, P., Taylor, C. M., & Huysmans, S. (2009). Third International Rubiaceae 51 52 Conference: Introduction. Annals of the Missouri Botanical Garden, 96(1), 53 54 1-3. 55 56 57 58 12 59 60 For Proof Read only Page 13 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 Bremer, B., & Erikson, T. (2009). Time tree of Rubiaceae: Phylogeny and dating the 4 5 family, subfamilies, and trees. International Journal of Plant Sciences, 170 6 7 (6), 766-793. 8 9 Bridson, D., & Forman, L. (1992). The Herbarium Handbook. 3rd Edition. Royal 10 11 Botanic Gardens, Kew, UK. 12 13 14 Bruun, H. H., Moen, J. V., Virtanen, R., Grytnes, J. A., Oksanen, L., & Angerbjorn, A. 15 16 (2006). Effects of altitude and topography on species richness of vascular 17 18 plants, bryophytes and lichens in alpine communities. Journal of Vegetation 19 For Review Only 20 Science, 17, 37-46. 21 22 Davis, A. P., Govaerts, R., Bridson, D. M., Ruhsam, M., Moat, J., & Brummitt, N. A. 23 24 (2009). A Global Assessment of Distribution , Diversity, Endemism, and 25 26 27 Taxonomic Effort in the Rubiaceae. Annals of the Missouri Botanical 28 29 Garden, 96(1), 68-78. 30 31 Grierson, A. J. C., & Long, D. G. (1999). Flora of Bhutan. Including a record of plants 32 33 from Sikkim and Darjeeling, Vol. 2 part 2 (pp. 733-834). The Royal Botanic 34 35 Garden Edinburgh EH3 5LR UK and the Royal Government of Bhutan. 36 37 Haferkamp, M. R. (1988). Environmental factors affecting plant productivity. Montana 38 39 40 Agr. Exp. Sta., Bozeman, p. 132. 41 42 Jamtsho, T., & Sridith, K. (2017). Species composition of the vegetation along the 43 44 Sherichu River, lower montane area of Eastern Bhutan. Songklanakarin J. 45 46 Sci. Technol, 39(3), 303-316. 47 48 Long, S., McQuarrie, N., Tobgay, T., Grujic, D., & Hollister, L. (2011). Geological 49 50 Map of Bhutan. Journal of Maps, 7(1), 184-192. 51 52 53 Lomolino, M. V. (2001). Elevation gradients of species –density: historical and 54 55 prospective views. Global Ecology and Biogeography, 10, 3-13. 56 57 58 13 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 14 of 25

1 2 3 Lovett, J. C., Marshall, A. R., & Carr, J. (2006). Changes in tropical forest vegetation 4 5 along an altitudinal gradient in the Udzungwa Mountains National Park, 6 7 Tanzania. African Journal of Ecology , 44, 478-490. 8 9 Myers, N., Mittermeier, R. A., Mittermeier, C. G., Fonseca, G. A. B., & Kent, J. (2000). 10 11 Biodiversity Hotspots for Conservation Priorities. Nature, 403, 24. 12 13 14 doi:10.1038/35002501 15 16 Norbu, C., Dorji, T., Dorj, T., Tamang, H. B., Tshering, K., & Hutcheon, A. (2003). A 17 18 provisional physiographic zonation of Bhuta. Journal of Bhutan Studies, p. 19 For Review Only 20 70. 21 22 Ohsawa, M. (1987). Life Zone Ecology of the Bhutan Himalaya (p.17). Laboratory of 23 24 Ecology, Chiba University, Japan. 25 26 27 Polunin, O., & Stainton, A. (1984). Flowers of the Himalaya. Oxford University Press. 28 29 Rahbek, C. (1995). The Elevational Gradient of Species Richness: A uniform pattern? 30 31 Ecography, 18 (2), 200-205. DOI: 10.1111/j.1600-0587.1995.tb00341.x 32 33 Shannon, C. E., & Weaver, W. (1949). The mathematical theory of communication (p. 34 35 117). Urbana: University of Illinois Press. 36 37 38 Simpson, E. H. (1949). Measurement of diversity. Nature, p. 163-688. 39 40 doi:10.1038/163688a0 41 42 43 Stainton, A. (1984). Flowers of the Himalaya: a supplement. Oxford University Press. 44 45 Subedi, S. C., Bhattarai, K. R., & Chauudhary, R. P. (2015). Distribution pattern of 46 47 48 Vascular plant species of mountains in Nepal and their fate against global 49 50 warming. Journal of Mountain Science, 12(6), 1345-1354. 51 52 Vetaas, O. R., & Grytnes, J. A. (2002). Distribution of vascular plant species and 53 54 endemic richness along the Himalayan elevation gradient in Nepal. Global 55 56 Ecology & Biogeography, II, 291-301. 57 58 14 59 60 For Proof Read only Page 15 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 3 Wangchuk, S. (2005). Biodiversity at its Best (pp. 52-57). In Bhutan-Land of the 4 5 Thunder Dragon. Publication by the Department of Tourism, Royal 6 7 Government of Bhutan. 8 9 Zhang, J. T., Xu, B., & Li, M. (2013). Vegetation patterns and species diversity along 10 11 elevational and disturbance gradients in the Baihua Mountain Reserve, 12 13 14 Beijing, China. Mountain Research and Development (MRD), 33(2), 170- 15 16 178. 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 15 59 60 For Proof Read only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . Page 16 of 25

1 2 3 Tables 4 Table 1. The annual mean temperature, rainfall and relative humidity calculated using the record of 5 6 past ten years. (T. Max =Maximum temperature, T.Min = Minimum temperature, RH = Relative 7 humidity) 8 9 10 11 Months 12 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 13 T. Max 17.8 19.4 21.9 23.9 25.1 25.8 26.3 26.6 26.1 24.7 21.6 18.3 14 T. Min. 3.8 6.0 8.4 10.9 13.2 16.3 17.1 17.2 16.3 12.4 8.1 5.1 15

Area I Rainfall 10.1 25.5 72.2 113.3 146.3 273.2 385.3 313.8 208.3 75.3 7.0 6.8 16 RH 66.2 61.2 64.9 65.0 66.8 71.9 77.8 80.6 79.4 73.3 66.4 64.3 17 T. Max 17.3 19.2 22.2 24.4 25.9 27.4 26.2 26.4 25.9 25.0 21.6 18.5 18 T. Min. 7.3 9.3 12.0 15.7 18.2 20.1 20.0 20.3 19.3 15.6 11.5 8.9 19 Rainfall 10.4 13.6For 28.9 Review 53.1 119.3 22.4 Only 416.5 280.1 197.3 99.2 9.6 2.5 20 AreaII 21 RH 75.8 75.9 73.9 75.2 79.5 86.8 89.6 88.9 86.6 81.2 78.4 78.0 22 23 24 25 26 27 28 Table 2. Species richness, diversity and evenness of the two study sites, 29 30 analyzed using Shanon-weiner and Simpson diversity index. 31 32 33 34 Species Shanon’s Shanon’s Simpson’s Simpson’s 35 Genera Richness Diversity Evenness Diversity Reciprocal 36 Index Area I 21 36 (43.9%) 2.99 0.83 0.94 24.99 37 38 Area II 29 46 (56.1%) 3.47 0.91 0.96 27.04 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 For Proof Read only

Tgay54 Tgay53 Tgay51 Tgay81

Number Voucher

3900 3900

3700 3700

3500 3500

X 3300 3300

X 3100 3100

X 2900 2900

X 2700 2700

X 2500 2500

X X 2300 2300

X X 2100 2100

X X X 1900 1900 X X X X

Reference Points 1700 1700

X X X X 1500 1500

X X X X udy areaudy (Phuntsholing-Thujaydrak) I 1300

X X X X 1100 1100

X X X 2 2 900 900

X X 700 700

For Proof Read only X 500 500

X X X X X X X X X X X X X X X X X X X X X X X X X X X X Tgay55 Tgay61 Tgay65 Tgay72 Tgay57

300 X X Tgay76 X X X X X X X X X X X X X X X X X X Tgay66 X X X X X X X X X X X X X X X Tgay77 X X X X X X Tgay80 Tgay82 Tgay83

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X X X X X ”N ”N 0

57’19.43”N 53’26.06”N 51’42.30”N, 01’39.80”N 29’13.4 57’19.43”N 57’19.43”N 35’37.72”E 35’37.39”E 32’15.46”E 29’20.80”N 34’28.96”E 19’20.53”N 07’46.67”E 24’38.81”E 57’16.30”N, 35’39.43”E 34’31.64”E 51’22.80”N, 24’04.20”E 28’55.50”N 36’59.20”E 29’13.40”N 35’13.20”E 35’13.20”E 04’34.90”N 32’42.40”E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Coordinates Coordinates For Review Only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . 26 89°36'39.92'' E 89°35'34.40'' E 26 27 27 89 89 89 89 89 89 91 89 89 89 89 89 89

Table 3 A. 3 Table Lists of Rubiaceae recordedinspecies St S S H H 27°02'47.76'' N H H form Life

aA s. eC. T 26°58'40.04'' N hC. S 26 eK. S 27 eA. S 27 HiT. LeS. ArS. H 26 G Abb. HeS. L L C ArV. C GaC. H 27 HeV. HeV. H 26

LuG. S 27

R.R.

(Wall.) (Wall.) (L.) (L.)

Ghose

Roxb.

L. GaA. H 27 Roxb. IxU. S 26

tetrasperma

curviflora

Scientific Name

ill hwaites Springate Edgew. R. Parker R. Lam. Wall. (Roxb.) Tirveng. Wall. Ceriscoides campanulata Roxb.) (Wall. ex T. Yamaz. H.J.P. Winkl. T M Galium craticulatumGalium Chassalia Chassalia Galium aparine

Galium Galium asperuloides Argostemma Argostemma verticillatum Hedyotis scandens Himalrandia Leptodermis stapfiana Argostemma Argostemma sarmentosum verticillataHedyotis Ixora undulata Luculia grandifolia Leptodermis kumaonensis Leptodermis amoena Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 25

Tgay33 Tgay62 Tgay69 Tgay71 Tgay45 Tgay34 Tgay84 Tgay89

Number Voucher

3900 3900

3700 3700

3500 3500

X 3300 3300

X 3100 3100

X 2900 2900

X 2700 2700

X 2500 2500

X X 2300 2300

X X X 2100 2100

X X X 1900 1900

X X X X

Reference Points 1700 1700

X X X X 1500 1500

X X X X X X 1300 1300

X X X X X X 1100 1100

X X X X X 3 3 900 900

X X X X X 700 700

For Proof Read only X X X 500 500

X X X X X X X X X X X X X X Tgay46 Tgay48

300 X X X X X X X X X X X X X X X X X X X X X Tgay63 X X X X X Tgay73 X X X X X X Tgay44 X X Tgay60 Tgay39 Tgay93

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X X X X

”N ”N ”N ”N, 0 0 0

7.71'' N 0 02’11.28”N 51’29.1 58’41.42”N 03’32.2 57’15.97’’N 03’36.60’’N 51’26.3 58’45.88”N 24’15.90”E 51’22.33”N 23’49.24”E 35’39.31”E 56’28.31”N 35’50.59”E 35’34.70”E 53’46.60’’N 26’12.40’’E 35’40.75’’E 03’42.40’’N 35’34.30’’E 35’33.40’’E 57’58.94’’N 35’59.05’’E 23’50.60”E 56’14.27”N 30’51.29”E 35’34.48”E 35’24.36”E 03’58.60”N 35’27.50”E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Coordinates Coordinates For Review Only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . 26 26 27 26 27 26 26 26°58' 89 89 89 89 89 89 89 89°35'28.78''E 89 89 89 89 89 89 89 89

S S S S S S 26 S 26 C H H H H 27 form Life

. eI. H 26 uSp N Sp2. H 27 Sp1. PsB. Abb. OlC. NeP. MuR. MuR. RuM. MoA MuF. M OpR.

L. (B.

Leroy

Roxb. L. ex ex L. OpM.

Wall. Hook.f. PaS S 26 bosa Kurz RuS. C 27 m (Wall. ex (Wall. ex alensis auct. RuC. C 26 h tifolia Lewis. Lewis.

Roxb.

(Wall.) Bennet

sp. angus ingrata

cientific Name sikkimensis S ook.f.) ook.f.) W.H. Oldenlandia cory rugosaOphiorrhiza sp1.Rubiaceae Ophiorrhiza mungosOphiorrhiza Heyne Schult.)ex J.-F. Fleming sp2.Rubiaceae Hook.f. H

Morinda Mussaenda frondosa Neohymenopogon Psilanthus beng manjithRubia Musaenda Musaenda Mussaenda roxburghii parasiticus Neanotis Pavetta subcapitata cordifoliaRubia Rubia 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tgay54 Tgay65 Number

Voucher

Tgay79 Tgay47 Tgay86

3900 3900 Number Voucher

3900 3900

3700 3700

3500 3500

3300 3300

3100 3100

2900 2900

2700 2700

2500 2500

2300 2300

X 2300 2300

2100 2100

X 2100 2100

1900 1900

X = Presence species = of Presence

1900 1900 X Reference Points

1700 1700

X Reference Points

1700 1700

X X 1500

1500 1500

X X 1300

1300 1300

X X 1100

X X

1100 1100

900 X X

4 4

X X

900 900 X X X 700

X X

700 700

500 500 For Proof Read only X X

500 500

X X 300 X X X X X X X Tgay78

X X X X X X X X X X X X X X X X Tgay66

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

300 X X X X X X X X X Tgay55 Tgay52 X

X X Tgay87

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X ”N ”N ”N 0 0 ”N ”N

04’39.5 04’34.9 59’22.00”N 59’22.00”N 55’36.60”E 52’31.60”E 56’16.90”N 00’52.30”E 53’24.60”E 10’35.21”N 46’00.76”E 0 0 0 0 0 0 0 0 0 0 Coordinates Coordinates 27 27 89 89 89 90 89 59’23.1 58’04.83”N 56’08.90”E 57’23.84”N 35’11.98”E 35’36.42”E 0 0 0 0 0 0

Coordinates Coordinates For Review Only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 . 89°33'24.69'' E 26 26 26°53'35.41'' N 89 89 89°26' E42.07'' 89

S H

Life T T H Form Form form Life

Table 3 B. Lists of Rubiaceae species recorded in Study area II (Dagachu-Dagala) area (Dagachu-Dagala) Study II recorded in species B. Lists of Rubiaceae Table 3

eS. T 26°57'16.09'' N aL. S 26 W. W. We ArS. ArS. H 26 Abb. C ArV. Abb. ChC. SpL. WeG. W SpM. H 26 )

Wall. Wight Wight Aubl

(

a r = Abbreviation of species name, T= Tree, S= Shrub, H= Herb, C= Climber, Climber, C= H= Shrub, S= Herb, T= Tree, name, species of Abbreviation = o l 1 L. GaA. H 27

curvif

Note. Note. cientific Name

S Arn. Wall. Spermacoce latifoliaSpermacoce Wendlandia grandis Wendlandia wallichi Chassalia Spermacoce mauritianaSpermacoce Gideon (Hook.f.) Cowan. Wendlandia speciosa & Scientific Name Wall. longisipnaCatunaregam Thwaites Galium aparine Cowan (Link) Tirveng.

Argostemma Argostemma verticillatum Argostemma Argostemma sarmentosum Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

Tgay76 Tgay53 Tgay51 Tgay57 Tgay50 Tgay46 Tgay73

Tgay67 Number Voucher

3900 3900

3700 3700

3500 3500

3300 3300

3100 3100

2900 2900

2700 2700

2500 2500

2300 2300

X 2100 2100

X X 1900 1900

X X

Reference Points 1700 1700

X X X 1500 1500

X X X X 1300 1300

X X X X X 1100 1100

X X X X X X 900 900 5 5

X X X X X X 700 700

X X X X X 500 500 For Proof Read only

X X X X 300 300 X X X X X X X X X Tgay69 X X X X X X X X X X X X X X Tgay77 Tgay85

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Tgay70 X X X X X X X X X Tgay72 X X Tgay49 Tgay56 Tgay71 X X X X

”N ”N ”N ”N ”N ”N ”N ”N 0 0 0 0 0 0

4.36''N 0 04’28.20”N 04’28.20”N 04’42.8 05’09.2 00’02.3 03’48.4 04’80.0 14’27.70”N 14’27.70”N 44’11.53”E 53’31.26”E 04’08.00”N 54’29.00”E 52’00.30”E 59’27.30”N 55’35.50”E 51’30.40”E 24’04.20”E 04’02.90”N 52’04.40”E 55’36.50”E 55’58.30”N 59’36.90”E 53’01.00”E 59’26.30”N 55’32.70”E 54’29.00”E 59’27.30”N 55’35.50”E 51’22.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coordinates Coordinates 6 90°00'53.78'' E 27°05' 27 27 27 27 27 27 89°051'7.15''E 89 89 89 89 89 89 89 89 For89 Review89 Only89 89 89 89 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

S 26 S S S S T T 26° 56'48.30''N H H H H Life Form Form

...... NeI. IxU. HiT. Abb. HeS. GaC. H 27 LuG. S 27 HeV. H 26 GaSp MyL. S 26 GaAs HySp MuM MuR. MuG. . .

L. OlC. H 26 b

R.R.

L. HeA. H 27

(L.) Rox Vahl (Wall.) (Wall.)

(Wall. ex (Wall. ex

Roxb.

Lewis a oxburghii r glabra

ingrata

sp.

tis aenda o

s n ill. cientific Name S Kuntze Wall. Wall. Galium Galium asperuloides Galium Edgew. craticulatumGalium Roxb.) (Wall. ex T. Yamaz Hook.f. Hook.f.) W.H. Oldenlandia corymbosa Sweet Lam. M

Hedyotis Hedyotis scandens Himalrandia tetrasperma Ixora undulat Mus Mussaenda Nea Hedyotis Hedyotis auricularia verticillataHedyotis flaccidumHymenodictyon Luculia gratissima Mussaenda macrophylla longifolia Mycetia 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tgay42 Tgay59 Tgay48 Tgay34 Tgay60 Tgay39 Tgay90 Tgay92

Number Voucher

3900 3900

3700 3700

3500 3500

3300 3300

X 3100 3100

X 2900 2900

X 2700 2700

X 2500 2500

X 2300 2300

X 2100 2100

X 1900 1900

Reference Points 1700 1700

X X 1500 1500

X 1300 1300

X X X 1100 1100

X X X X X 900 900 6 6

X X X X X 700 700

X X X X 500 500 For Proof Read only

X X X X X 300 300 X X X X X X X X X X X X X X X X X Tgay43 X X X X X X X X X X X Tgay88 Tgay58 Tgay84 Tgay89 Tgay91 Tgay79

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X X X X X X Tgay75 X X X X

N N N ”N ”N ” ”N '' N '' 0 0 0” 0 0

1'47.82''N 0'43.34'' N 0 0 04’35.4 56’52.1 05’09.9 26’19.3 03’59.32”N 03’59.32”N 53’19.93”E 04’36.00”N 52’11.70”E 53’27.20”E 05’09.20”N 51’30.40”E 00’10.80”E 03’48.40”N 53’01.00”E 51’31.40”E 04’39.00”N 52’06.20”E 04’31.44”N 53’31.69”E 55’49.10”E 59’25.70”N 55’33.90”E 02’15.15”N 54’11.05”E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coordinates Coordinates 27°04'33.08'' N 27° 27 26 27 27° 26 26°56'48.3 89°53'25.94'' E 89°54'24.48''E 89 90 89 89°55'19.52'' E 89 90°00'53.78'' E 89 For89 Review89 89 Only89 89 89 89 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

S S S S S C C H Life Form Form

iB. H 27 uM. C 27 Sp1. S 27 Sp3. T 26 PaS. Sp2. Sp4. R PsB. Abb. RuS. OpF. OxF. RuC. R PeSp. PeSp. S 27

SpL. H 27 (B.

OpM. H 27

ex ex Aubl.

Hook.f. L.

Kurz

L. L. PaF. C 27 alensis auct.

Roxb.

fasciculat

ikkimensis Sp.

s cientific Name leming S Ophiorrhiza fasiculataOphiorrhiza Oxyceros sp2.Rubiaceae sp4.Rubiaceae D.Don. D.Don. mugosOphiorrhiza (Roxb.) T. Yamaz. Pentas Heyne Schult.)ex J.-F. Leroy sp1.Rubiaceae sp3.Rubiaceae latifoliaSpermacoce Gomes F

Pavetta subcapitata Psilanthus beng cordifoliaRubia Rubia Paederia foetidaPaederia Richardia brasillensis manjithRubia Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

Tgay78 Tgay68 Tgay40 Tgay38 Tgay35

Number Voucher

3900 3900

3700 3700

3500 3500

3300 3300

3100 3100

2900 2900

2700 2700

2500 2500

2300 2300

2100 2100

1900 1900

Reference Points 1700 1700

= Presence of species species = of Presence

X 1500 1500

X X 1300 1300

X X X X 1100 1100

X X X X 900 900 7 7

X X X X X 700 700

X X X X X 500 500 For Proof Read only

X X X X X 300 300 X X X X X Tgay36 X X X X X X X X X Tgay37 X X X X X Tgay74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X X X X X X X Tgay41 X X X X ”N ”N ”N ”N ”N 0 0 0 0

02’15.44”N 05’09.2 03’53.5 04’01.2 03’52.8 54’24.39”E 59’23.10”N 56’08.90”E 51’30.40”E 03’48.40”N 53’01.00”E 53’03.80”E 53’21.50”E 53’17.50”E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coordinates Coordinates 7 89°53'32.86'' E 89°53'06.09'' E 27 27 27 2 27 89 89 89 89 89 89 For89 Review Only Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

T T T C H Life Form Form

eC. T 27°04'04.13'' N nS. C 27 SpS. SpS. S 26 Abb. U UnS. SpM. TaW. WeT. W WeH.

Roxb.

DC. WeP. T 27°03'56.37'' N

i i (Schult.) i

(Sm.) = Abbreviation of species name, T= Tree, S= Shrub, H= Herb, C= Climber, Climber, C= H= Shrub, S= Herb, T= Tree, name, species of Abbreviation = wallich

1 sessilifructus DC.

Note. Note. cientific Name

S Spermacoce mauritianaSpermacoce Tarenoidea Uncaria Wendlandia heynei Wendlandia tinchoria Hutch. DC.(Wall.) Gideon Gideon Spermadictyon suaveolons (Hook.f.) Tirveng. Sastre& scandensUncaria Wendlandia coriacea &Santapau Merchant. Wendlandia puberula (Roxb.) Roxb.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 23 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2

3 Table 4. Pearson Correlation Analysis among environmental variables (A. Study area I 4

5 and B. Study area II) 6 7 8 9 10 A 1 2 3 4 5 6 7 8 11 1 Species 12 2 pH .11 13 Carbon -.01 -.25 14 3 4 Nitrogen -.06 -.29 .65** 15 5 Phosphorus -.11 .36* .02 -.11 16 6 Potassium -.23 .11 .52** .59** .32 17 7 Altitude -.37* .01 .26 .06 .01 .19 18 8 Litter .31* .01 .11 .18 .24 .24 -.33* 19 N = 36. * P For< 0.05; **P

1 2 3 Figures 4 5 6 Figure 1. A map showing the study sites. A. Map of Bhutan; B. Map of Dagana, 7 Chukha and Thimphu districts (study areas), C. Study area I ( Phuntsholing- 8 9 Thujaydrak), D. Study area II ( Dagachu – Dagala. 10 11 12 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Figure 2. Species richness and distribution pattern of Rubiaceae along the altitudinal 36 gradient. ( Study area I and Study area II) 37 38 39 40 41 42

43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 For Proof Read only Page 25 of 25 Songklanakarin Journal of Science and Technology SJST-2018-0116.R1 .

1 2 Figure 3. The figure displays CCA ordination results showing the relationships between 3 environmental variables ( ) and species (+). The distribution of species on the ordination 4 diagram is related to environmental factors. The abbreviated species names are displayed in 5 6 the figure and the full name list, author and distribution range is given in table 3A & B. (A. 7 Study area I and B. Study area II) 8 9 10 11 12 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2 59 60 For Proof Read only