GRASSES OF THE NEELUM VALLEY AZAD JAMMU AND KASHMIR: SYSTEMATICS, ANATOMY AND PHYSIOLOGY

BY

KHAWAJA SHAFIQUE AHMAD Regd. No. 2011-ag-17 M. Phil. (UAAR)

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN BOTANY

DEPARTMENT OF BOTANY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE FAISALABAD 2015 DECLARATION

I hereby declare that the contents of the thesis “Grasses of the Neelum Valley Azad Jammu and Kashmir: Systematics, anatomy and physiology” are the product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/ equations/ formulae/ protocols, etc). I further declare that this work has not been submitted for award of any other degree/ diploma. The University may take action if the information provided is found inaccurate at any stage. (In case of any fault the scholar will be proceeded against as per HEC plagiarism policy).

Khawaja Shafique Ahmad 2011-ag-17

i To

The Controller of Examinations, University of Agriculture, Faisalabad.

“We, the Supervisory Committee, certify that the contents and form of thesis submitted by Mr. Khawaja Shafique Ahmad, Regd. #. 2011-ag-17, have been found satisfactory and recommend that it be processed for evaluation by the external Examiner (s) for the award of degree”

Supervisory Committee

1. Chairman ------Dr. Mansoor Hameed

2. Member ------Dr. Farooq Ahmad

3. Member ------Dr. Bushra Sadia

ii ACKNOWLEDGEMENT

Firstly, I offer my adoration to ALLAH Almighty who gave me the strength and courage to complete my thesis work as well as the opportunity to thank all those people through whom his grace was delivered to me. Indeed the words at my command are not adequate to convey the depth of my feeling and gratitude to my supervisor Dr. Mansoor Hameed, Associate Professor, Department of Botany, University of Agriculture, Faisalabad, the most valuable and inspiring guidance, concrete suggestions, constant encouragement, expect evaluation, enormous help and constructive criticism throughout the course of this investigation and preparation of this thesis. With reverence and gratitude, I would like to thank Dr. Farooq Ahmad Department of Botany, University of Agriculture, Faisalabad, whose help, stimulating suggestions, and encouragement helped me throughout the course. I am very much thankful to Dr. Bushra Sadia, Centre of Agriculture Biochemistry and Biotechnology, University of Agriculture, Faisalabad for her help, guidance and motivation during all the research work. Sincere thanks to Dr. Abdul Wahid (Chairman), Dr. Muhammad Sajid Aqeel Ahmad, Dr. Mumtaz Hussain, Dr. Shehbaz, my lab members, Dr. Riffat Batool, Dr. Noreen Akhtar, Sana Fatima, Mehvish Naseer and my friends especially Muhammad Salman, Aqeel Butt, Muhammad Asif Shehzad, Waqas Ahmad, Aqil Sarwar, Sharif Abdullah, Abdul Hameed and Khawaja Baber for their kindness and moral support during my study. Thanks for the friendship and memories. I thank my parents, for striving hard to provide a good education for my siblings and I always fall short of words and felt impossible to describe their support. You have contributed irreversibly to the person I have become. I can’t thank you enough. My dear brothers Saeed Shad and Rafique Parwana, my dear sisters, Zubaida and Ayesha and my nephews, Saad and Abdullah who always are excited to hear of my success and that inspires me to perform better and be successful. I acknowledge my entire family for providing me a very educated atmosphere. For any errors or inadequacies that may remain in this work, of course, the responsibility is entirely my own. Khawaja Shafique Ahmad

iii DEDICATED TO

MY

SUPERVISOR

DR. MANSOOR HAMEED

iv CONTENTS CHAPTER TITLE PAGE 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 8 3. MATERIALS AND METHODS 21 4. RESULTS 31 5. DISCUSSION 175 6. SUMMARY 185 LITERATURE CITED 197

v Chapter List of contents Page 1. Introduction 1 2. Review of literature 13 2.1. Distributional range of grasses 13 2.1.1. Distribution in Pakistan 13 2.1.2. Worldwide distribution 14 2.2. Phytosociology 16 2.3. Systematics 17 2.4. Anatomy 19 2.5. Physiology 23 2.6. Modifications for environmental stresses 24 3. Materials and methods 27 3.1. Site selection 27 3.2. Collection and preservation of grasses 27 3.3. Edaphology 27 3.3.1. Soil collection 27 3.3.2. Soil texture 29 3.3.3. Saturation percentage 29 3.3.4. Soil ph and electrical conductivity (ece) 29 3.3.5. Organic matter (Walbley) 29 3.3.6. Total Nitrogen (Kjeldahl method) 31 3.3.7. Phosphorus (Olsen method) 31 3.3.8. Soil ionic content (Flame photometric method) 32 3.4. Phytosociology 32 3.4.1. Vegetation sampling 32 3.4.2. Species association 33 3.5. Morphological parameters 33 3.6. Anatomical parameters 34 3.6.1. Root anatomy 34 3.6.2. Stem anatomy 34 3.6.3. Leaf-sheath anatomy 35 3.6.4. Leaf blade anatomy 35 3.7. Physiological Parameters 35 3.7.1. Analysis of nutrients 35 3.7.2. Determination of shoot and root ionic content 36 3.7.3. Chlorophyll contents 36 3.8. Cladistic analysis of Andropogonae tribe 36 3.8.1. Morphological data and selection of outgroups 36 3.8.2. Gene bank data 37 3.8.3. Phylogenetic analysis 37 3.9. Statistical analysis 38 4. Results 39 4.1. Soil analysis 39 4.1.1. Soil physiochemical characteristics 39 4.1.2. Soil RDA analysis 41 4.2. Phytosociology 41 4.2.1. Taxonomic diversity 41 4.2.2. Relative density 45 4.2.3. CCA analysis 48 4.2.4. Relative frequency 48 4.2.5. CCA analysis 51

vi 4.2.6. Relative cover 53 4.2.7. CCA analysis 54 4.2.8. Importance value 56 4.2.9. CCA analysis 57 4.2.10. Species association analysis 59 4.3. Morphology 69 4.3.1. Plant height (cm) 69 4.3.2. Root length (cm) 69 4.3.3. Root dry weight (g) 72 4.3.4. Shoot dry weight (g) 72 4.3.5. Ligule length (cm) 72 4.3.6. Tiller Plant-1 72 4.3.7. Leaves Plant-1 74 4.3.8. Inflorescence length (cm) 74 4.3.9. Spike length (cm) 74 4.3.10. Spikelet numbers 74 4.3.11. Multivariate cluster analysis 74 4.4. Anatomy 75 4.4.1. Root anatomy 75 4.4.1.1. Root cross sectional area (µm2) 75 4.4.1.2. Root hair length (µm) 75 4.4.1.3 Epidermal thickness (µm) 77 4.4.1.4. Cortical thickness (µm) 77 4.4.1.5. Cortical cell area (µm2) 77 4.4.1.6. Endodermal cell area (µm2) 80 4.4.1.7. Pericyle thickness (µm) 80 4.4.1.8. Metaxylem vessel number 80 4.4.1.9. Metaxylem cell area (µm2) 80 4.4.1.10. Pith area (µm) 80 4.4.1.11 Pith cell area (µm)2 82 4.4.1.12. Multivariate cluster analysis 82 4.4.2. Stem anatomy 84 4.4.2.1. Stem cross sectional area (mm2) 84 4.4.2.2. Epidermal thickness (µm) 84 4.4.2.3. Sclerenchyma thickness (µm) 84 4.4.2.4. Cortical cell area (µm2) 86 4.4.2.5. Vascular bundle area (µm2) 86 4.4.2.6. Metaxylem area (µm2) 86 4.4.2.7. Phloem area 86 4.4.2.8. Multivariate cluster analysis 86 4.4.3. Leaf sheath anatomy 88 4.4.3.1. Sheath thickness (µm) 88 4.4.3.2. Sclerenchyma thickness (µm) 88 4.4.3.3. Epidermal cell area (µm2) 88 4.4.3.4. Vascular bundle area 91 4.4.3.5. Multivariate cluster analysis 91 4.4.4. Leaf blade anatomy 93 4.4.4.1. Leaf blade thickness (µm) 93 4.4.4.2. Lower epidermal thickness (µm) 93 4.4.4.3. Upper epidermal thickness (µm) 93

vii 4.4.4.4. Sclerenchyma thickness (µm) 93 4.4.4.5. Mesophyll thickness (µm) 95 4.4.4.6. Bundle sheath thickness (µm) 95 4.4.4.7. Vascular bundle area (µm2) 95 4.4.4.8. Metaxylem vessel area (µm2) 95 4.4.4.9. Phloem area (µm2) 97 4.4.4.10. Bulliform cell area (µm2) 97 4.4.4.11. Adaxial stomatal area (µm2) 97 4.4.4.12. Abaxial stomatal area (µm2) 97 4.4.4.13. Adaxial stomatal number 97 4.4.4.14. Abaxial stomatal number 99 4.4.4.15. Multivariate cluster analysis 99 4.4.4.16. Leaf structural modifications in grasses 101 4.5. Physiology 121 4.5.1. Root Sodium (mg g-1) 121 4.5.2. Shoot Sodium (mg g-1) 121 4.5.3. Root Potassium (mg g-1) 121 4.5.4. Shoot Potassium (mg g-1) 124 4.5.5. Root Calcium (mg g-1) 124 4.5.6. Shoot Calcium (mg g-1) 124 4.5.7. Root Magnesium (mg g-1) 124 4.5.8. Shoot Magnesium (mg g-1) 125 4.5.9. Root Phosphorous (mg g-1) 125 4.5.10. Shoot Phosphorous (mg g-1) 125 4.5.11. Total chlorophyll Contents 125 4.5.12. Multivariate Cluster analysis 128 4.5.13. CCA analysis of root nutrients 128 4.5.14. CCA analysis of shoot nutrients 131 4.6. Cladistic analysis of tribe 131 5. Discussion 138 6. Summary 145 7. Literature cited 149

viii List of Figures Figure Title Page 1. Panoromic views of various study sites: a. Janawai, b. Taobut, c. Jura, 10 d, Kundal Shahi, e Sardari, and f. Halmat 2. Panoromic views of various study sites: a. , b. Dawarian, 11 c. Sharda, d, Kail, e. Kairan rest house, and f. Halmat 3. Grasses at various localities; a. Cholorophy content measurement, b. 12 Eleusine indica, c. Rostraria , d. Apluda mutica, e. Saccharum spontaneum, and f. Agrostis pilosa 4. Map of Neelum Valley, Azad Jammu and Kashmir showing collection 28 sites 5. Textural triangle to determine the soil texture classes 30 6. RDA ordination analysis of the soil physio-chemical characteristics 42 and grasses at different sites of the Neelum Valley, Azad Jammu and Kashmir 7. CCA ordination analysis of (a.) relative density, (b.) relative 49 frequency, (c.), relative cover, and (d.) importance value based distribution of grasses over different sites in Neelum Valley, Azad Jammu and Kashmir 8. Morphological characteristics (plant height, root length, root and shoot 71 dry weight, ligule length) of grasses of Neelum Valley, Azad Jammu and Kashmir 9. Morphological characteristics (tillers, inflorescence length, spikelet 72 length, number of spikelet) of grasses of Neelum Valley, Azad Jammu and Kashmir 10. Multivariate cluster analysis of the morphological characteristics of 76 grasses of Neelum Valley, Azad Jammu and Kashmir 11. Root anatomical characteristics (RCSA, root hair length, epidermal 77 thickness) of grasses of Neelum Valley, Azad Jammu and Kashmir 12. Root anatomical characteristics (cortical area, thickness, endodermal 78 cell area, pericyle thickness) of grasses of Neelum Valley, Azad Jammu and Kashmir 13. Root anatomical characteristics (metaxylem number and area, pith 81 area, pith cell area) of grasses of Neelum Valley, Azad Jammu and Kashmir 14. Multivariate cluster analysis of the root anatomical characteristics of 83 grasses of Neelum Valley, Azad Jammu and Kashmir 15. Stem anatomical characteristics (SCSA, epidermal and sclerenchyma 85 thickness, cortical cell area) of grasses of Neelum Valley, Azad Jammu and Kashmir 16. Stem anatomical characteristics (vascular bundle and metaxylem area, 87 phloem area) of grasses of Neelum Valley, Azad Jammu and Kashmir 17. Multivariate cluster analysis of the stem anatomical characteristics of 89 grasses of Neelum Valley, Azad Jammu and Kashmir 18. Sheath anatomical characteristics (sheath, schlerenchyma thickness, 90 epidermal cell area and vascular bundle area) of grasses of Neelum Valley, Azad Jammu and Kashmir 19. Multivariate cluster analysis of the leaf sheath anatomical 921 characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir 20. Leaf anatomical characteristics (blade, lower and upper epidermal 94

ix thickness, sclerenchyma area, mesophyll thickness,) of grasses of Neelum Valley, Azad Jammu and Kashmir 21 Leaf anatomical characteristics (bundle sheath thickness, vascular 96 bundle cell area, metaxylem area, bulliform cell and phloem area) of grasses of Neelum Valley, Azad Jammu and Kashmir 22. Leaf anatomical characteristics (adaxial and abaxial stomatal area and 98 number) of grasses of Neelum Valley, Azad Jammu and Kashmir 23. Multivariate cluster analysis of the leaf blade anatomical 100 characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir 24. Anatomical characteristics of some grasses of tribe Andropogoneae 102 from the Neelum Valley, Azad Jammu and Kashmir 25. Anatomical characteristics of some grasses of tribe Andropogoneae 103 from the Neelum Valley, Azad Jammu and Kashmir 26. Anatomical characteristics of some grasses of tribe Andropogoneae 105 from the Neelum Valley, Azad Jammu and Kashmir 27. Anatomical characteristics of some grasses of tribe Andropogoneae 106 and from the Neelum Valley, Azad Jammu and Kashmir 28. Anatomical characteristics of some grasses of tribe from 107 the Neelum Valley, Azad Jammu and Kashmir 29. Anatomical characteristics of some grasses of tribe and 108 from the Neelum Valley, Azad Jammu and Kashmir 30. Anatomical characteristics of some grasses of tribe Aveneae from the 110 Neelum Valley, Azad Jammu and Kashmir 31. Anatomical characteristics of some grasses of tribe Aveneae from the 111 Neelum Valley, Azad Jammu and Kashmir 32. Anatomical characteristics of some grasses of tribe Aveneae from the 112 Neelum Valley, Azad Jammu and Kashmir 33. Anatomical characteristics of some grasses of tribe Brachypodieae 113 from the Neelum Valley, Azad Jammu and Kashmir 34. Anatomical characteristics of some grasses of tribe from the 114 Neelum Valley, Azad Jammu and Kashmir 35. Anatomical characteristics of some grasses of tribe Paniceae from the 115 Neelum Valley, Azad Jammu and Kashmir 36. Anatomical characteristics of some grasses of tribe Paniceae from the 116 Neelum Valley, Azad Jammu and Kashmir 37. Anatomical characteristics of some grasses of tribe from the 117 Neelum Valley, Azad Jammu and Kashmir 38. Anatomical characteristics of some grasses of tribe Poeae from the 118 Neelum Valley, Azad Jammu and Kashm 39. Anatomical characteristics of some grasses of tribe Poeae from the 119 Neelum Valley, Azad Jammu and Kashmir 40. Anatomical characteristics of some grasses of tribe Poeae from the 120 Neelum Valley, Azad Jammu and Kashmir 41. Anatomical characteristics of some grasses of tribe from the 122 Neelum Valley, Azad Jammu and Kashmir 42. Root and shoot physiological characteristics (sodium and potassium) 123 of grasses of Neelum Valley, Azad Jammu and Kashmir 43. Root and shoot physiological characteristics (calcium and magnesium) 125 of grasses of Neelum Valley, Azad Jammu and Kashmir 44. Root and shoot physiological characteristics (root & shoot 127

x phosphorous and chlorophyll) of grasses of Neelum Valley, Azad Jammu and Kashmir 45. Multivariate cluster analysis of the root and shoot physiological 129 characteristics of grasses of Neelum Valley, Azad Jammu & Kahmir 46. RDA ordination analysis of the (a.) root and (b.) shoot ionic content 130 and the grasses at different sites at various sites of the Neelum Valley, Azad Jammu and Kashmir 47. The UPGMA tree based on 26 morphological characteristics 132 48. Phylogenetic tree based on combined 6 loci dataset. Numbers under 137 the branch are PP/BS values

xi List of Tables Table Title Page 1 Temperature (°C ) conditions of the study area during 2006 to 2014 in Neelum 2 Valley, Azad Jammu and Kashmir 2. Rainfall (mm) of the study area during the years of 2012-14 in Neelum Valley, 3 Azad Jammu and Kashmir 3. Humidity percentage (%) of the study area during the year of 2014 in Neelum 4 Valley, Azad Jammu and Kashmir 4. Soil physico-chemical characteristics of different sites in the Neelum Valley, 40 Azad Jammu and Kashmir 5. Meteorological and topographical data of sampling sites in Neelum Valley 42 Azad Jammu and Kashmir 6. Tribe wise synopsis for the grasses of the Neelum Valley Azad Jammu and 43 Kashmir 7. Relative density of native grasses at different sites in Neelum Valley, Azad 47 Jammu and Kashmir 8. Relative frequency of native grasses at different sites in Neelum Valley, Azad 52 Jammu and Kashmir 9. Relative cover of native grasses at different sites in Neelum Valley, Azad 55 Jammu and Kashmir 10. Importance value of native grasses at different sites in Neelum Valley, Azad 58 Jammu and Kashmir 11 Association analysis of some grasses at Chiliahana, Jura and Kundal Shahi 60 sites, Neelum Valley, Azad Jammu and Kashmir 12 Association analysis of some grasses at , Kairan and Nagdar sites, 62 Neelum Valley, Azad Jammu and Kashmir 13 Association analysis of some grasses at, Lawat, Dawarian and Dodonial, 64 Neelum Valley, Azad Jammu and Kashmir 14 Association analysis of some grasses at Sharda, Kail and Janawai, Neelum 66 Valley, Azad Jammu and Kashmir 15 Association analysis of some grasses at Sardari, Halmat and Tao Butt, Neelum 68 Valley, Azad Jammu and Kashmir 16 Anova of morpho-anatomy and physiological characteristics with F-ratio 70 17 Character states used in the cladistic analysis of Andropogoneae 134 18 Accession number of the gene markers from GeneBank 136

xii Abstract

Grasses belong to one of the most fascinating families of flowering , family with a wide range of diversity. Poaceae is a species-rich family that includes many economic plants, globally with about 10,000 species and 700 genera. The members of this group are present in all the conceivable suitable for the growth of the plant communities. Recent phylogenetic studies confirmed that multiple factors are involved indirectly that determine the grass diversity at large scales. A total of 52 species of grasses belonging to 10 tribes and 28 genera were recorded from 15 sampling sites in Neelum Valley, Azad Jammu and Kashmir. Physiochemical characteristics of the soil showed that most of the soil component varied significantly over different sites. The soil moisture content seemed to be closely related to the physical properties of the soil as well as to vegetation type. Morphological markers are helpful in the identification, differentiation and classification of the grasses at species, genus and tribe level. Significant variations in different morphological characters are observed in different genera of the same tribe and among the species of the same genus. Poaceae shows great variety in anatomical characteristics especially leaf anatomical parameters more than any other plant family and provides extensive data for systematic utilization. At root, stem and leaf level, anatomical characteristics of grasses showed significant variation among the tribes and within the species. Grasses showed angular prickles at the margin of the leaf in costal and intercostal zone, long cells with slightly sinuous walls, sharply pointed micro hairs and saddle, X or rounded shape silica bodies. Certain shapes of silica bodies were characteristic of grass subfamilies, e.g. dumbbell-shaped in panicoid grasses, saddle-shaped in most pooid grasses. Adaxial and abaxial surfaces of the leaf showed high number of ribs and ridges, with increase number of hairiness in most of the grasses. Tribe Paniceae, showed highly pointed angular bicelled prickles and micro hairs at the leaf margins that is characteristic feature of this tribe. Anatomical alterations such as enlarged succulence, sclerification, highly developed bulliform cells, endodermis in stem or roots and metaxylem area the indumentum of leaves and length and frequency of epidermal basis play an important role in the tolerance of various altitudinal stresses. The diversity in anatomical markers could be used to clarify the status of problematic taxa in different tribes. Presence of sclerenchyma strands on the abaxial side only makes the genus distinct it from the remaining species within the tribe. Saddle shaped silica bodies, microhairs and bulliform cells deeply penetrating the mesophyll were found the prominent characters of these tribes. The cladistics analysis of Andropogoneae showed Schizachyrium as the first branch within Andropogoneae, clustered with [Apluda+ Arthraxon]; then was sister to [Bothriochloa+ Heteropogon], collectively sister to the remaining crown clade ([Saccharum+Sorghum] + Capillipedium). Heteropogon spp. showed a close relationship with two

xiii Bothriochloa spp. whereas, Capillepidium was found much closer to the species of Sorghum and Saccharum. Phylogenetic analysis of molecular data showed Apluda (Apluda mutica) at the first branch within tribe Andropogoneae, sister to the remaining genera with robust support (PP = 1.00, BS = 100; or 1.00/100). Arthraxon (Arthraxon hispidus) was sister to the left 6 genera (Saccharum, Sorghum, Capillipedium Schizachyrium, Heteropogon, and Bothriochloa) with high PP value (0.96), but no bootstrap values. The Saccharum+Sorghum clade was sister to the crown clade (1.00/64) without PP and BS. Within the crown clade, Schizachyrium clustered with the left genera ([Heteropogon + Capillipedium] + Bothriochloa) as sister (0.97/53); Heteropogon was sister to the genus Bothriochloa with strong support values (1.00/91). Almost all morpho-anatomical and physiological characteristics are species specific and also specific in their degree of tolerance to either cold stress or drought. However, some specific modifications like amount of sclerification, size and shape of bulliform cells, presence of storage parenchyma, nature of pubescence and stomatal size and area can be related to environmental conditions. It is, therefore, concluded that certain anatomical characteristics like presence of silica bodies, surface appendages, bulliform cells and pattern of sclerification can safely be used as important tools for the identification at species or lower rank and formal taxonomic and nomenclatural changes should surely only be encouraged, particularly at the species level, when the lineages within a phylogeny correlate with morphological characters.

xiv CHAPTER 1

INTRODUCTION

Neelum Valley a temperate Himalayan region lies between 73°-75° E longitude and 32°-35° N latitude, covering an area of 3737 km2. It is situated in north-east of at an altitude of 900-6325 m above sea level (Mehmood et al., 2011). The area comprises deep valleys, high mountains, dissected small terraces, gentle to steep slopes and inclined spurs. Climate varies with altitude from sub-tropical to temperate (Anonymous, 1996). High variation in temperature has been recorded in the various parts of the valley. The maximum daily temperature varies from 20 to 30 °C during summers and 4-0 °C in winters (Table 1). Summer is mild hot and winter is very cold with heavy snowfall. June and July are the hottest, while December and February are the coldest months of the year. The mean annual rainfall is about 282 mm (Table 2). The maximum relative humidity (Table 3) is 90% and the minimum is 35% that is less during the day time and higher during night (Ahmad et al., 2012).

The higher elevated mountains area of the valley is rich in vegetation with a number of beautiful looking trees, shrubs and herbs making it an ideal valley for both domestic and international tourists (Dar, 2003). The characteristic species of lower hills (below 2000 m) are Pinus roxburrghii, Zanthoxylum alatum, Quercus dilatata, Pyrus pashia, Ficus palmata, Berberis lycium, Rubus fruticosus, and Clematis grata. Moist temperate occur between 2000-3000 m in moist depression and on gentle slope are dominated by Cedrus deodara, Pinus wallichiana, Quercus incana, and Ficus palmata. Low level blue pine forests are characterized by Quercus dilatata, Quercus incana, Populus ciliata, Machilus odoratissima, Sarcococca saligna, Pteris cretica, and Nepeta erecta (Malik et al., 1990; Ahmad et al., 2012). In sub-alpine and alpine region, vegetation consists of shrub formation often forming quite dense cover which is composed of limited number of herbaceous species, mostly deciduous and with small leaves but including dwarf Salix grandiflorum (Malik et al., 2001).

1 Table 1. Temperature (°C ) conditions of the study area during 2006 to 2014

Year Maximum Minimum

2006 29.6 °C 9.8 °C

2007 33.6 °C 9.5 °C

2008 32.8 °C 7.4 °C

2009 32.1 °C 8.2 °C

2010 35.0 °C 6.5 °C

2011 32.7 °C 7.1 °C

2012 35.8 °C 5.11 °C

2013 36.8 °C 9.7 °C

2014 36.2 °C 2.0 °C

2

Table 2. Rainfall (mm) of the study area during the years of 2013-14

2013 2014

Month Total Mean Mean Total Mean Mean rainfall max. min. rainfall max. Min.

January 201.1 19.6 2.8 175.1 14.4 3.3

February 89.2 21.3 4.8 272.1 12.5 4.9

March 88 29.5 9.2 145.6 21.6 10.4

April 112.6 30.4 14.5 61.9 28.6 13.6

May 97.0 35.5 16.7 63.9 30.8 16.2

June 88.1 35.9 21.9 40.6 37.8 21.9

July 278.9 36.3 22.8 197.4 34.2 22.9

August 282.3 34.1 22.4 226.4 35.2 22.0

September 132.3 33.6 20.4 143.0 34.6 20.6

October 132.8 26.3 12.8 0.0 0 0

November 29.7 24.3 7.4 19.1 24.0 6.3

December 6.50 17.7 4.8 0.0 17.6 0.9

3

Table 3. Humidity percentage (%) of the study area during the year of 2014

Months 300 AM 12 00 PM

January 90 63

February 89 68

March 84 58

April 68 35

May 71 44

June 60 38

July 84 58

August 84 54

September 83 47

October 84 50

November 78 44

December 87 51

Source: Meteorological observatory, Muzaffarabad

4 Grasses are economically very important that occupy a variety of types. In flowering plants, Poaceae is the 4th largest family in the world and comprises over 1,800 genera and 1,000 species, mostly having monotypic or diatypic genera (Osborne et al., 2011). Approximately 20% of the land is covered by grasses and they are the main component of the (Kellogg, 2001). Poaceae is an essential component of forests, , shrubs and ecosystems. Its members can tolerate extremities of environments, therefore, commonly used for studying systematic, ecology and genetics (Liu et al., 2006). Many specific characteristics have resulted in the emergence of several radiations inside the family

Poaceae, most noticeable are the annual habit and C4 photosynthetic pathway (Osman et al., 2011).

Grasses are not only ecologically but also geographically important. Most of are a source of human nutrition and many of them were domesticated in the old world (Clayton and Renvoize, 1986). Due to their considerable importance, now focus is on the development of plants, which have best agronomic traits through breeding strategies (Paterson et al., 2005). Grasses are not only beneficial to humans but also for other organisms, e.g., wildlife depends upon grass and habitats for protection, food and completion of their life cycle. Grass species inhabiting swamps and marshes grow together with the some aquatic species like Typha (cattails), and Carex and Cyperus (sedges) and give food and shelter for a variety of waterfowl and small animals species (Gould, 1969).

Grassland are very important because they provide many services like natural carbon sinks and provide livelihood, food source for livestock, etc. (Boval and Dixon, 2012). Some developed countries, viz., Germany, , Denmark, France, United States and Switzerland have planned and maintained . In Pakistan, no natural or artificial grasslands exist and very little research work is done on grasses (Husain et al., 1983; Ayaz, 1992; Ahmed et al., 2010). Grass patches are only reported in Khyber Pakhtun Khwah (KPK), Punjab, Sindh and (Husain et al., 2009).

Poaceae family is well known taxonomically and is classified on the basis of various morphological characteristics, particularly related to the inflorescence characters, but also to the micro-morphological as well as molecular characters (Ahmed et al., 2011). Majority of grasses are annual or perennial herbs, while very few are shrubs. Poaceae family is accepted

5 as a natural group and prominent characteristics are paleas, glumes, caryopses, lemmas and structural organization of the genome of chloroplast (GPWG, 2001). Most grass species have

C3 photosynthetic pathway, but the existence of C4 as well as CAM pathways in some species show evolutionary advancements, which are the characteristics of plants growing under stressful environmental conditions (Osmond et al., 1982; Ehleringer and Monson, 1993; Maherali et al., 2002).

In monocot plants, grasses are the largest, diverse and successful group (Zeb et al., 2011). Phylogenies have accumulated over the past 20 year, but mainly focus of these studies is on the groups below the subfamily level. Phylogenetic studies (e.g. Clark et al., 1995; GPWG, 2001; Duvall et al., 2007; Shaheen et al., 2012; Ullah et al., 2011) have recognized 12 subfamilies of family Poaceae composed on three species-poor lineages that are successively sister to all other grass species (, and ) and grouped the grasses in two main clades, that are known by their acronyms as BEP (Bambusoideae, Ehrhartoideae and ) and PACMAD (, , , , Aristidoideae and ) (Gibson, 2009).

Evolutionary patterns are not obvious all the time in the Poaceae family due to inadequate information about the availability of appropriate out groups. Grasses are difficult to identify, in particular some genera like Dicanthium, Saccharum, Chrysopogon and cannot be distinguished by morphological markers (Ahmad et al., 2011). It is difficult to identify the difference in morphological characteristics of family Poaceae to its sister groups because grass structures are so extremely derived that it is hard to find the homology, e.g., the caryopsis of the grass is homologous to the dry and indehiscent fruit of Ecdeiocolea (Rudall et al., 2005). Evolutionary patterns of grasses are highly complex and are influenced by hybridization, polyploidy, and reticulation. Fossil record has suggested that the Poaceae family is likely to be originated during the Late Cretaceous period in tropical areas of the world, approximately 55-70 million years before (Kellogg, 2001). The earliest grasses have particular feature like rhizomatous, wind-pollinated, herbaceous growth habit, broadleaved, six anthers and three stigmas (Clark et al., 1995). The caryopsis and bracteates inflorescence without palea and lemma were characteristics of most primitive grass lineages,

6 however, the spikelet, lemma and palea evolved in grasses before their divergence from the Pharoideae (Kellogg, 2001; GPWG, 2001 & 2012).

Taxonomic issues between species or between populations of same species can be solved by the use of anatomical studies (Naz et al., 2009). For this purpose anatomical markers, i.e., characters particularly used for species recognition, are used as the variation within the species, genera or a family is present (Ahmad et al., 2010). Anatomical features are of prime importance to researchers for the identification of small pieces of plant material, e.g., those present in animal’s gut, fossils or damaged objects (Al-Edany and Al-Saadi, 2012). The importance of anatomical markers in the systematic of plant groups has been investigated by Adedeji and Dloh (2004) in Hibiscus species, Celka et al. (2006) in Malva alcea, Zou et al. (2008) in Cercis species and Ahmad et al. (2011) in tribe Eragrostideae. In Poaceae and Cyperacea families, anatomical studies have resulted in change in nomenclature, e.g., existence of C4, C3 and many intermediate types in these two families (Koteyeva et al., 2011; Sage et al., 2014).

Anatomical alterations such as enlarged succulence (Hameed et al., 2009), sclerification (Hameed et al., 2010), highly developed bulliform cells (Alvarez et al., 2008), endodermis in stem or roots (Balsamo et al., 2006) and metaxylem area (Vasellati et al., 2001) play an important role in the tolerance of various stresses.

Different kind of stresses in grasses stimulates modifications in growth, development, photosynthesis, and pigment composition, yield and water relations. However, different plant species and their different growth stages respond these modifications differently depending upon the severity of stress (Jaleel et al., 2009). Plants growing in cold environments are mostly exposed to below zero temperatures in the autumn, spring and winter seasons, so for successful survival in cold, plants do various physiological and molecular adjustments to reduce the frost damage, which may be lethal (Sandve and Fjellheim, 2010). Cold acclimation (CA) is a phenomenon in which plants growing in non-freezing temperatures do many biological adjustments to increase their potential of frost tolerance (Thomashow, 1999). The process of CA include biological alterations on numerous stages, e.g., modulation in expression of different genes levels (Gilmour et al., 1998), gathering and breakdown of

7 proteins (Kosmala et al., 2009) modification in sugar amount (Hisano et al., 2008) and alteration in the photosynthetic apparatus (Rapacz et al., 2008).

The evolutionary history of temperate grasses makes them an excellent model system for studying adaptations to cold and frost stress. During the last decade several research groups have focused their research on understanding the cold and freezing stress responses in forage grass species (Poeae), mainly Lolium and species, and recently also Phleum pratense. Species of the Poeae tribe are excellent models for plant adaptations to cold environment because of their adaptation to habitats in the northernmost part of the Northern hemisphere, i.e., the circumpolar arctic region (Sandve et al., 2011). The responses to cold stress include reduction in expansion of leaf, wilting, chlorosis and necrosis. Reproductive development of plants is also damaged due to chilling, e.g., flowers sterility (Jiang et al., 2011). The most harmful effect of freezing is membrane injury, which is due to the dehydration (Steponkus, 1984).

Neelum Valley has rich diversity in grasses and various habitats are available for their growth. No detailed study of grasses from taxonomic point of view at the species, genus and tribe level is carried out so far. Grasses look parallel in external morphology and many problematic genera are not easy to distinguish from each plant on the basis of external morphological characters only. There was a need to identify them and to distinguish them from each other. Anatomical characters are considered an important tool in for identification of different species and are helpful at the species and generic level. In the past, anatomical studies incorporation with morphological studies for the resolution of taxonomic problems of monocots has been used. A concerted effort is needed to produce a molecular phylogenetic study of the family that combined dense taxon sampling with a large and sufficiently complete molecular data set.

It is hypothesized that different genera and species of inhabiting grass species of the Valley must have very specific morpho-anatomical modifications to cope with harsh environmental conditions. The study was, therefore, conducted in the Neelum Valley (Fig. 1- 3) with a special focus on following objectives:

8  To explore the grass flora of Neelum Valley  To study the morpho-anatomical and physiological differences among grasses  To evaluate significance of anatomy in systematic botany  To investigate the adaptive features against different environmental stresses (aridity and low temperature, etc).

9

Fig. 1. Panoromic views of various study sites: a. Janawai, b. Taobut, c. Jura, d, Kundal Shahi, e Sardari, and f. Halmat

10

Fig. 2. Panoromic views of various study sites: a. Neelum River, b. Dawarian, c. Sharda, d, Kail, e Kairan rest house, and f. Halmat

11 Fig. 3. Grasses at various localities; a. Cholorophy content measurement, b. Eleusine indica, c. Rostraria , d, Apluda mutica, e. Saccharum spontaneum, and f. Agrostis pilosa

12 CHAPTER 2

REVIEW OF LITERATURE

2.1. Distributional range of grasses

2.1.1. Distribution in Pakistan

Grasses are the dominant vegetation, present over extensive area in very diverse ecological habitats due to their great adoptability for life. In Pakistan, grasses are distributed through the country. Cynodon dactylon is present throughout the Cholistan, Potohar, and Kashmir region is considered a first class fodder grass (Cope, 1982). Dactyloctenium aegyptium is present in near cultivated fields, shady places and moist soil and is more abundant and diverse species. Chrysopogon serrulatus is the most dominant species, in the low grazing and protected areas. In the saline soil species like Cenchrus ciliaris, arabicus, Dicanthium and Polypogon sp., are found. Saccharum spontaneum is very common along stream banks and margins of ponds. Grasses like Eulaliopsis bipinnata and Cymbopogan jwarancusa are abundant on mountains and rocky slopes and near sand stones (Ahmad et al., 2009).

The large forming grasses Saccharum spontaneum and Saccharum bengalense are recorded along water channels and their tussocks are useful for the nesting of animals and birds (Marwat et al., 2010). Other important grass species are Dactylocteniun scindicum, Dactyloctenium foveolatum, Sporobolus arabicus, that are highly palatable. Dactyloctinium scindicum and Aristida mutabilis are frequent in places where wind-blown soil has accumulated. Vetiveria zizanoides has been declared as endangered grass in the Salt rang of Pakistan is almost near to extinction requires conservation. Some grasses are endemic, that are restricted to a particular area due to over grazing and lack of proper conservation plan i.e. Paspalidium flavidum, , Lolium temulentum and Chloris dolicostachya are more vulnerable (Chaudhary et al., 2001).

Weedy grasses are potentially important for every cropping system in the world. Many grassy weeds are among the most damaging weeds and most of them are widely

13 distributed of all flowering plants and form prominent feature of the flora of every continent (Marwat et al., 2010).

In family Poaceae, Chlorideae is one of the important tribe that is represented by 45 genera in the tropical regions. In Pakistan, 7 genera and 14 species have been reported in Salt Range of Pakistan (Ahmad et al., 2011). The genus is one the largest genera of the grasses, including more than 400 species. Within this large genus, two species, i.e., Panicum antidotale and P. turgidum occur in Cholistan of Pakistan. Of them P. antidotale is most promising, tall, erect and much branched perennial grass locally called as “Murrot or Bansi Ghaa” which yield a huge amount of palatable herbage for the livestock within the Cholistan desert (Akhtar and Arshad, 2006).

2.1.2. Worldwide distribution

Poaceae is one of the largest families of angiosperm which is present in every phytogeographic region in the world. Grasses have a wide range of diversity and play significant role in the lives of human beings and animals (Mitra and Mukherjee, 2005). Grasses dwell the earth in greater abundance than any other analogous group of plants, inhabited in warm, humid and tropical regions, marsh and swamp vegetation, and desert areas (Jones, 1999).

The world maps have shown the distribution of the main tribes of grasses in the total grass flora throughout the world with respect to their relationship to the climatic, historical, and taxonomic factors. The average percentage of the six largest tribes was as follows: Agrosteae (8.2 %), Eragrosteae (8.1 %), Andropogoneae (11.9 %), Festuceae (16.5 %), Aveneae (6.3 %), Paniceae (24.7 %). The distribution of these tribes can be explained by few notable factors. Of them, climatic factors are of key importance in relation to grass distribution and winter temperature has special significance (Hartley, 1950). The tribe Paniceae is widely distributed in the tropical, subtropical, and temperate regions of the world with approximately 3,270 species in 206 genera (CWPG, 2001). Arundineae encompass nearly 12 genera and 175 species and most of the grasses of this tribe are distributed in the tropical region of the old world (Clayton and Renvoize, 1986).

14 The overall distribution of C4 grasses is largely limited to warmer climates, and a strong positive correlation between C4 grass abundance, growing season, and temperature has been found at continental scales (Vogel et al., 1986), and along altitudinal gradients on tropical mountains across the world (Rundel, 1980). Precipitation gradients appear to have much less influence on C3 and C4 grass distributions. The C4 photosynthetic pathway is most prominent in grasses such as maize, , sorghum and switchgrass (Still et al., 2003)

Significant improvement has been made among the major grass lineages to resolve phylogenetic relationships, and most species belong to either the BEP or PACCMAD clades (Sanchez-Ken et al., 2007). The BEP clade includes the , rice and its relatives, and the Pooideae, a large group of 3300 species that has radiated broadly in open habitats of cold and temperate regions of the world. The PACCMAD clade consists of 5500 species that are ecologically diverse but largely restricted to open habitats in warmer regions (Sage, 2004).

There are about 10,000 grass species, and most of them are restricted to a single continent. The giant reed grass, Phragmites australis has broad geographic range of any which extends north to south in a wide range between latitudes 70° N and 40° S and is most abundant in the Old World temperate regions (Kellogg and Campbell, 1987). Humans activities often have played a key role in expanding the range of many of the grasses, including weeds such as Digitaria sanguinalis (crabgrass), Echinochloa crus-galli (barnyard grass, or cockspur), and (bluegrass). Endemism or geographic distribution is a very common factor among grasses, especially at the southern tips of continents and on mountain ranges (Clayton and Renvoize, 1986).

The nearly 800 genera of grasses fall into three distributional patterns and nearly three-quarters are confined to one of seven basic centres of distribution: Africa, Australia, Eurasia north of the Himalayas, South and Southeast Asia, North America, Temperate South America, and tropical America. About one-fifth of the genera cover even broader distribution patterns throughout temperate or tropical regions of the world (Davis and Soreng, 1993). Somewhat less than one-tenth of the genera have established discontinuous distributions on adjacent continents; 12 genera, for example, have such disjunctive distributions between North and South America, 11 genera show these patterns between North America and

15 Europe, and 7 genera are discontinuous between North America and northern Asia (Kellogg and Campbell, 1987).

2.2. Phytosociology

Phytosociological attributes such as community structure, species association and distribution pattern of flora of any specific region mainly dependent on soil physic-chemical characteristics. For example, in a saline desert conditions like the Cholistan Desert (Naz et al., 2010), relatively more salt tolerant species viz., Sporobolus ioclados with Aeluropus lagopoides, Haloxylon recurvum and Suaeda fruticosa are the dominant components of highly saline sites, whereas, moderately saline habitats support less tolerant species Fagonia indica, Cymbopogon jwarancusa and Ochthochloa compressa. Relatively high salt tolerant species like A. lagopoides, S. ioclados, S. fruticosa and H. recurvum showed a broad range of association as compared to the moderately salt tolerant species. Topographical situation in respect of altitude, slope and aspect greatly affect the distribution of plants by modifying light, temperature, and soil moisture conditions (Hussain and Ilahi, 1997; Champion et al., 1965).

Kufri in the Soon valley (the Salt Range) is dominated by grasses like Cynodon dactyon and Saccharum grifithii at drier habitats and Saccharum spontaneum along mountain springs (Ahmad et al. 2008). Some salt tolerant forage grasses i.e. Cynodon dactylon, Imperata cylandrica and Sporobolus arabicus were collected from same saline habitats of Salt Range and Sporobolas arabicus was considered most salt tolerant grass followed by C. dactylon and Imperata cylendrica (Hameed et al., 2010). Sporobolus arabicus is found dominating the saline habitats in the Salt Range by forming thick, dense patches (Chaudhry et al., 2001; Hameed et al., 2010). Its roots are exposed to both soil salinity and brine springs that are active during rainy seasons.

In the Salt Range seven species of tribe Aveneae belonging to 5 genera have been reported (Ahmad et al., 2009). Genus Avena and Polypogon Agrostis, Koeleria and Phalaris generally grow in the early spring. Agrostis, Koeleria and Polypogon are found in the shady and moist places, while Polypogon is also present in marshy places. Phalaris and Avena sp. are the serious weeds of fields.

16 2.3. Systematics

In early classification systems, Poaceae was divided into two subfaimilies the Paniceae, and a large and heterogeneous Poaceae based on gross morphological features of the inflorescence (Kellogg, 2000). Most taxonomic treatments of the Poaceae recognize six or seven major subfamilies of grasses, Bambusoideae, , Pooideae, Panicoideae, Arundinoideae, Chloridoideae, and Centothecoideae, which are further subdivided into 40 tribes consisting of 600-750 genera (Renvoize and Clayton, 1992). The scientific effort to distinguish properly, and ultimately to classify into natural groups, the 10 000 species of the grass family began with systematic attempts to group species according to the morphology of characteristic grass structures such as the spikelet. Eventually, this classical morphological approach to grass systematics was supplemented and complemented by data consisting of novel characters derived from detailed studies of anatomy (Watson and Dallwitz, 1992).

The most current description of subfamilies within the Poaceae was proposed in 2001 based on morphological, anatomical, cytological, and biochemical data, plus nuclear and chloroplast DNA sequence data (Mathews et al., 2000). The basal lineages comprise three subfamilies: the Pueloideae, Pharoideae, and Anomochloideae. The Bambusoideae, Ehrhartoideae, and Pooideae comprise the BEP clade. This group is equivalent to the Bambusoideae, Oryzoideae, plus Pooideae (BOP clade), but the subfamilial name of the Ehrhartoideae is given priority over the Oryzoideae. The remaining subfamilies (Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Aristidoideae, and Danthonioideae) form the PACCAD clade (GPWG, 2001).

Grasses have adapted to a wide range of environmental conditions from obligate aquatics to desert species due to vast range of variability in stature with several distinctive morphological features (Cody, 2000). Morphological characters have evolved from multiple independent origins within the grass family, and this homoplasy causes difficulty in reconstructing phylogenetic relationships in the Poaceae (Stebbins and Crampton, 1961). However, the inputs of large morphological data sets have provided the basis for grass taxonomy (Hilu, 2004; Watson and Dallwitz, 1992). Grasses are the only plant group with paleas, lemmas, glumes, and a caryopsis as a fruit type (GPWG, 2001). There is a remarkable diversity in almost every characteristic of grasses. For example, grasses range in size from

17 woody bamboos, such as sinicus that can attain a height of 30 m to Arctic grasses such as Phippsia algida which is only 2 to 15 cm high at maturity (Ohrnberger, 2002).

Inflorescences in the grasses ranges from spikes to many-branched panicles that may vary in the number of branches, the number of orders of branching, and the degree of elongation of axes (Doust and Kellogg, 2002). Most of the grasses have reproductive structures arranged into florets, which are organized within a spikelet structure. Each floret typically possesses a lemma, palea, lodicules, androecium, and gynoecium (GPWG, 2001). Lemmas are the sheath-like bracts lowest on the floret axis (Soreng and Davis, 1998). Paleas are considered prophylls, and are inserted above lemmas (GPWG, 2001). The caryopsis is a unique fruit type, similar to an achene in that both are dry indehiscent fruits; however, the pericarp is adnate to the seed coat in the caryopsis. Other examples of variability in the pericarp (caryopsis surface) in grasses include differences in textural patterns, including reticulate, verrucate, striate, substriate, tuberculate, regulate, echinate, psilate, lophate, and foveolate (Brandenburg, 2003).

There is significant role of silica bodies in taxonomy of grasses. Certain shapes of silica bodies are characteristic of grass subfamilies, e.g. dumbbell-shaped in panicoid grasses, saddle-shaped in most pooid grasses, and 12 vertically oriented silica bodies in the Bambusoideae (Rovner, 1988). Distribution and shape of silica bodies is taxonomically informative at the tribal level in the (Barkworth, 1981). The presence or absence of these silica bodies is valuable for differentiating species (Mejia-Saules and Bisby, 2003).

Hairs and prickles have also been inspected for taxonomic service. Microhairs are informative at the subfamilial level. Broad-tipped microhairs of the epidermis are restricted to the Chloridoideae (Amarasinghe and Watson, 1990), and microhairs are absent in the Pooideae, with the exception of Loefl. ex L. and L. (GPWG, 2001). At the generic level, morphology of epidermal papillae is useful for delineating groups within Sorghastrum Nash (Davila and Clark, 1990). Although cork cells have not received much attention, they may also provide taxonomic information. Cork cells are short cells containing deposits of organic material and suberized cell walls. The shape of cork cells in paleas were useful in differentiating among subgenera of (Acedo and Llamas, 2001).

18 A systematic study of eleven tribes of Gramineae; Andropogoneae, Aristideae, Arundineae, Aveneae, Brachypodieae, Bromeae, Eragrostideae, Paniceae, Poeae, Stipeae and Triticeae was carried out (Osman et al., 2011). The results were conducted to some numerical analysis aspects. The study deals with 34 species belonging to 25 genera 34 characters, including fruit morphology, fruit anatomy and palynology.

Morphological characteristics of different Digitaria species from Pakistan were investigated by Gillani et al. (2003). The aim was to know the taxonomic relationship between these Digitaria species. He identified a new sub species of Digitaria sanguinalies from Pakistan on the basis of presence of spines on the upper half margins of nerves of lower lemma only, while D. sangunalis had spines on the whole nerves of lower lemma.

Many problematic genera in grasses such as Bothriochloa and Dicanthium are difficult to distinguish on the basis of morphological characteristics. Similarly, Saccharum ravennae and Saccharum bengalense are also very similar in external morphology and S. bengalense is often confused with Arundo donax by non-taxonomists. Other species such as Chrysopogon serrulatus, Sorghum halepense and Cymbopogon jwarancusa are also not properly identified merely on the basis of morphological (vegetative and floral) characters (Ahmad et al., 2010).

2.4. Anatomy

Anatomical markers played a very significant role in the taxonomy and the identification of grasses. The diversity in anatomical markers could be used to clarify the status of problematic taxa in different tribes of grasses. Anatomical markers of taxonomic importance are the nature and size of macro- and micro-hairs, shape of bulliform cells, arrangement of vascular bundles and amount of sclerification present in the leaves, which may be used for resolving taxonomic problem of problematic genera within tribe Paniceae. Leaf anatomical characteristics of 17 species belonging to 9 genera of tribe Paniceae, native the Salt Range, Pakistan, was evaluated (Ahmad et al., 2015). Two Urochloa species, U. deflexa and U. ramosa, were distinguished from the rest of the species by the absence of median vascular bundles. Presence of sclerenchyma strands on the abaxial side only makes the genus Cenchrus distinct from the remaining species within the tribe. The distribution

19 pattern of bulliform cells proved to be helpful in the differentiation among Cenchrus species. Large macrohairs with deep penetration on the adaxial surface are characteristic of Digitaria nodosa. Saddle shaped silica bodies, microhairs and bulliform cells deeply penetrating the mesophyll are the prominent characters of this tribe, which justify the placing all these species in the same tribe.

The leaf epidermal traits such as, epidermal cells, stomata and hairs have proved to be an important tool in delimitation of taxa in many plant families (Stenglein et al., 2003). Presence of sclerenchyma and bundle sheath (Kranz sheath), the width of sclerenchyma, the indumentum of leaves and length and frequency of epidermal basis are features of prime importance that can identify relationship among the genera of Poaceae (Ahmad et al., 2010).

In the desert of northwest China, four ecotypes of common reed (Phragmites communis) were investigated namely swamp reed (SR), low-salt reed (LSMR), high-salt meadow reed (HSMR), and reed (DR). for anatomical and anatomical and chemical characteristics of the foliar vascular bundles. Compared to SR, the three terrestrial ecotypes, LSMR, HSMR and DR, showed highly increased bundle sheath cell area, lower xylem and phloem areas, lower xylem/phloem ratios, and higher number of leaf veins. Highly significant variation was observed in the lignification and suberization of the xylem and sclerenchyma cell walls of the four ecotypes, but the phloem and bundle sheath cell walls were generally similar. These results suggest that the adaptation of common reed, a hydrophytic species, to saline or drought-prone prompts changes in the anatomical of the foliar vascular bundle tissues that could contribute to the high resistance of reeds to extreme habitats such as saline and drought-prone dunes (Chen et al., 2006).

Anatomical studies have been used successfully to clarify taxonomic status and help in the identification of different species. The differences in size and shape of prickles, short cells, silica bodies, and microhairs with basal and distal cells, hooks, stomata and long cells in genus Digitaria have proved helpful in identification of many species within this genus (Gillani et al., 2002). Ecotypes of six species of Saccharum officinarum were explored for the structural and functional modification of anatomical characteristics. It was noted that all ecotypes showed a remarkable variation in stem anatomical characteristics especially in

20 width and length of long cells, number and shape of cork cells, number of stomata and number of rows of long cells were different in different varieties (Elahi and Ashraf, 2002).

Many grass showed such as Cymbopogon citratus, Cynodon dactylon, Panicum summatrense and Vetiveria zizanoides taxa showed differences in short and long cells, silica bodies, macro and microhairs and shape of subsidiary cells and most of these characters are diagonostic and can be used for making keys (Chaudhary et al., 2001). In tribe Aveneae, Agrostis viridis seems quite similar to genus Polypogon and there is confusion in identifying Polypogon monospeliensis from Polypogon fugax and Avena fatua from Avena ludoviciana on the bases of morphological studies. Foliar epidermal studies may help in identification and to solve the taxonomic problems in tribe Aveneae (Ahmad et al., 2011).

Andropogoneae is a monophyletic tribe, which is morphologically diverse comprising a variety of high value C4 grasses (Kellogg, 2000). Taxonomic status of many grasses in this tribe can be solved by studying the variations in the internal anatomy of blade (Ahmad et al., 2010). Poaceae shows great variety in anatomical characteristics especially leaf anatomical parameters than in any other family and provides extensive data for systematic utilization (Ellis, 1979; Metcalfe, 1960). Nwokeocha (2008) investigated taxonomic potential of leaf epidermal characters in punctata and concluded that leaf anatomy should be used with non-anatomical characters before recognizing species.

The leaf blade of grasses is divided into the costal and intercostal zones which are composed of long cells and short cells, named for their degree of elongation. Generally, long cells comprise more surface area of the leaf blade. Both zones are more obvious in the grasses that have well-developed sclerenchyma strands associated with the vascular bundles just below the epidermis (Ellis, 1979). Cell walls of long cells vary in their degree of undulation and pitting. Anticlinal walls of long cells may be parallel, forming rectangular cells, angled outward to form hexagonal cells, or bowed outward to form inflated cells. Long cells show a high degree of phenotypic and developmental variation, thus taxonomic significance of these characters must be inferred with caution (Metcalfe, 1960).

Transverse sections of grass leaves are also helpful in the identification and taxonomic delimitation of grasses. Particularly that of lamina are often used the characterized

21 the bulliform cells position in relation to the vascular bundle for identification purposes. In the genus , distribution of sclerenchyma and bulliform cells have proved useful at a specific level (Khan, 1984).

In leaf anatomy, epidermal traits i.e. epidermal cells, stomata and hairs have been proven to be an important tool in delimitation of taxa in many plant families (Ditsch et al., 1995; Stenglein et al., 2003; Riaz et al., 2010). It is confirmed that leaf epidermal features can help to elucidate taxonomic relationships at different levels (Davila and Clark, 1990; Mejia and Bisbey, 2003) and these leaf epidermal characters are of great value in grass systematics and characterization of broad groups within the grasses, particularly subfamilies and tribes (Yousaf et al., 2008).

Presence of sparsly distributed microhairs and prickle hairs in Cenchrus citrates and papillae alongside their long cells in C. giganteus are their distinguishing characters of these grasses (Folorunso et al., 2007). Length of epidermal hairs, prickles, width of sclerenchyma strands and number of strands in Aegilops were found to be distinguishing characters among these species and were important from taxonomic point of view (Kharazian, 2006). Austrostipa is a stipoid grass that has normally xeromorphic characters, but studies showed that it has hydrophytous or amphibious characters and it was concluded that hydromorphic characters are adaptations that permit A. aristiglumis to maximize its growth, where a surplus amount of water is present (Arriaga and Jacobs, 2006).

There were significant variations in leaf lamina thickness of Brachypodium pinnatum growing in different microhabitat light regimes (full shade under oak canopy, half shade near shrubs, and in unshaded grassland) in situ. Mesophyll thickness was about 1.5 times greater in the grassland in situ than in oak subcanopy due to an additional layer of mesophyll cells and to 25-32% taller mesophyll cells. Mesophyll thickness and the proportion of veins plus sclerenchyma were lower for plants transplanted from either full or half shade to full sun than in situ plants in the grassland. Parenchymatous bundle sheath tended to be thicker in the grassland than in the two other microhabitats (Mojzes et al., 2005)

The stomatal complexes in the Poaceae are unique. The lumina of guard cells are enlarged at either end and constricted in the middle. Subsidiary cells lie in the same plane as

22 the guard cells, but the two cell types do not arise from the same mother cell (Metcalfe, 1960). Several types of subsidiary cells have been identified, using their shape. They may be triangular, parallel-sided, low dome-shape, tall dome shape, or variable. These differences are useful for taxonomic purposes at the subfamily level. For example, triangular subsidiary cells are common in the Panicoideae, and uncommon in other subfamilies (Ellis, 1979).

2.5. Physiology

In grasses the basic leaf-blade anatomy can be used to find out if a grass has C3 or C4 photosynthetic patterns of leaf anatomy. The two basic types of leaf-blade anatomy are the Kranz and non-Kranz patterns. This anatomical structure (Kranz anatomy) shows a special significance when viewed from the physiological and ecological points of view that the grasses with Kranz-type leaf-blade anatomy follow the C4 photosynthetic pathway and the grasses with the non-Kranz type of leaf-blade anatomy follow the C3 photosynthetic pathway (Chaudhary, 1989). Paniceae demonstrates unique variability of photosynthetic physiology and anatomy including both non-Kranz and Kranz species and all subtypes of the latter. Reasons for the success of grass are due to vegetative propagation, high seed production, perennating habit, protected vegetative shoot meristum, ability to produce new tillers and variations in their root system (Duvall et al., 2001).

Leaf anatomy is also taxonomically useful at the subfamily level in grasses. Anatomy is related to biochemistry, and C4-type grasses usually have Kranz anatomy. Radiate mesophyll is characteristic of C4 grasses, but C4 photosynthesis has multiple independent origins in the Poaceae (Kellogg, 2000; Giussani et al., 2001). Thus similarities in anatomy may be due to parallel evolution.

Dengler et al. (1994) measured quantitative anatomical characteristics, including cross-sectional areas (volumes) of all tissues and perimeters (surface areas) of chlorenchymatous tissues in transverse sections of leaf blades of 125 species of grasses (Poaceae). The species sample represents the major taxonomic groups and the range of photosynthetic pathway variation, including the 'classical' anatomical-biochemical types (the NADP-malic enzymic type, NADP-ME; the NAD-malic enzyme type, NAD-ME; and the PEP carboxykinase type, PCK) and species of Eragrostis, Panicum and Enneapogon that are

23 PCK-like structurally, but NAD-ME biochemically. They found new evidence that both mesophyll and bundle sheath tissues of C4 species have less surface area exposed to intercellular space and lower surface: volume ratios than in C3 species and, in C4 species, the ratio of PCR (bundle sheath) tissue surface adjacent to intercellular space:tissue volume ratio is strikingly lower than the comparable value for PCA tissue. Overall, the pattern of variation in quantitative leaf blade anatomy is complex, reflecting correlations with both taxonomic group and photosynthetic type, and no new diagnostic characters emerge that can be used to distinguish one biochemical type from another.

Taub (2000) compared the C4 grass flora and climatic records for 32 sites in the United States. It was found that the proportion of the grass flora that uses the NADP malic enzyme (NADP-ME) variant of C4 photosynthesis greatly increased with increasing annual precipitation, while the proportion using the NAD malic enzyme (NAD-ME) variant (and also the less common phosphoenolpyruvate carboxykinase [PCK] variant) decreased. However the association of grass subfamilies with annual precipitation was even stronger than for the C4 decarboxylation variants. Analysis of the patterns of distribution by partial correlation analysis showed that the correlations between the frequency of various C4 types and rainfall were solely due to the association of the C4 types with particular grass subfamilies.

2.6. Modifications for environmental stresses

The morphological, anatomical and biochemical traits of the leaves of yellow foxglove (Digitalis grandiflora Mill.) were studied and comparison of the available light with soil moisture revealed that both factors significantly influenced the morphological and anatomical variables of D. grandiflora. Leaf area, mass, leaf mass per area (LMA), surface area per unit dry mass (SLA), density and thickness varied greatly between leaves exposed to different light regimes. Leaves that developed in the shade were larger and thinner and had a greater SLA than those that developed in the half shade. In contrast, at higher light irradiances, at the edge, leaves tended to be thicker, with higher LMA and density. Stomatal density was higher in the half-shade leaves than in the full-shade ones. The considerable variations in leaf density and thickness recorded here confirm the very high variation in cell size and amounts of structural tissue within species (Kolodziejek, 2014).

24 A remarkable decrease in the rate of photosynthesis can be observed under moderate to severe salt stress could be due to stomatal closure (Ashraf, 2004). Stomatal closure is resulted due to higher levels of ABA under salt stress which ultimately decreases stomatal conductance (Niu et al., 2004; Merlot, 2001; Etehadnia et al., 2010). This decrease in stomatal conductance leads to a marked reduction in other gas exchange parameters such as internal CO2 concentration, transpiration rate and photosynthetic rate (Ashraf, 2004).

At high saline habitats, there was significant decrease in chlorophyll a and b, net CO2 assimilation, stomatal conductance, and transpiration rate with the increase in environmental stress. Salinity decreases the whole plant photosynthesis by restricting leaf area expansion (Netondo et al., 2004). Increasing the salt concentration reduces the growth as well as the chlorophyll, carotenoid and total carbohydrate contents (Mansour, 2004). Proline generally increases in leaves with increasing salinity, but the tolerant genotypes are more efficient in proline accumulation (Qian et al., 2001). The concentration of soluble sugars, glycine and betaine generally increase under salinity, but the increase is more pronounced in resistant species (Saneoka and Nagasaka, 2001).

+ - High intracellular concentrations of Na and Cl in perennial grasses may inhibit the activity of many enzymatic systems and some cellular processes, such as protein synthesis or mRNA processing in perennial grasses (Forment et al., 2002). Sodium interferes with the + 2+ uptake of essential cations, especially K and Ca and promotes oxidative stress through + + generation of reactive oxygen species (Zhu, 2001). Substantial differences in Na and K accumulation between salt-resistant species may be due to differences in the selective ion transport capacities at root level (Hester et al., 2001; Wang et al., 2002). Salt secreting + + species would be expected to have the weakest selective transport capacity for K over Na as most of the salt would have to be transported up to the stem and excluded from the leaf via + salt glands. Aeluropus lagopoides did show high selectivity for K by retaining greater - 2+ amounts of Cl and Mg in roots than in shoots. Urochondra setulosa shoots did not show + high K selectivity (Gulzar et al., 2003). Salinity induced inhibition of plant growth may + - occur due to the effects of high Na , Cl by decreasing the uptake of essential elements such

25 + - 2+ as P, K , NO3 , ion toxicity or osmotic stress (Zhu, 2001, 2002). Sporobolus spicatus was found to secrete 93% NaCl by weight of salts secreted by plants from 4 different sites while + 2+ 2+ 2- K , Ca , Mg and SO4 constituted only 5% of salts (Ramadan, 2001).

Ion ratios could be helpful in categorizing the physiological response of a plant (salt- excluding, salt-secreting or salt-diluting) in relation to ion selectivity under increasing substrate salt concentrations (Wang et al., 2002). However, the influence of various ion ratios on salt tolerance is quite complex and attempts to draw general conclusions have not been successful (Grieve et al., 2004). Sodium-potassium ion ratio is among the most important of + + these ion ratios and plants tend to maintain a low Na / K ratio in the cytoplasm and low + cytosolic Na content below some crucial value (Tyerman and Skerrett, 1999).

Plants in cold climates are frequently exposed to sub-zero temperatures in the autumn, winter, and spring seasons requires a series of molecular anatomical and physiological adaptations to minimize frost related damages that can be lethal. Cold acclimation (CA) is a process whereby plants in response to low but non-freezing temperatures undergo a range of biological changes in order to increase their frost tolerance (FT) and prepare for the winter season (Thomashow, 1999). The process of CA involves biological modifications on many levels, e.g. modulation of gene expression levels, accumulation and degradation of proteins, changes in sugar content, and changes in the photosynthetic machinery (Guy, 1990) The Poaeae grasses (temperate grasses) is a large and economically important tribe including cereals and forage grasses . As opposed to rice, which is adapted to warm and humid environments, Pooideae grasses radiated in cooler environments. Thus evolution of cold and frost stress responses, either through fine tuning of ancient abiotic stress responses or evolution of novel adaptations to cold environments must have been central for the Pooideae sub-family (Sandve et al., 2008, 2011).

26 CHAPTER 3

MATERIALS AND METHODS

3.1. Site selection

Fifteen ecological diverse sites were selected from the entire area of Neelum Valley, namely, Chiliahana (CH), Jura (JU), Kundal Shahi (KS), Athmuqam (AT), Kairan (KN), Nagdar (NG), Lawat (LW), Dawarian (DW), Dodonial (DD), Sharda (SH), Kail (KL), Janawai (JW), Sardari (SR), Halmat (HM), and Tao butt (TB). The site selection was based on the altitude, physiognomy, slope, aspect, habitat type soil texture and floristic composition of the area (Fig. 4).

3.2. Collection and preservation of grasses

Frequent field trips were carried out in different flowering seasons of the year for the collection of grasses. Each grass specimen was collected in triplicate with accession number, date of collection, altitude, locality and habitat. The specimens were then pressed, dried and preserved in 3% mercuric chloride in ethyl alcohol and mounted on herbarium sheets by using standard herbarium techniques and deposited in the herbarium of Botany Department, University of Agriculture, Faisalabad. For identification, all available floras and manuals were consulted, e.g., Stewart (1972), Cope (1982), Jacobs (1993), Chaudhary (1989) and Rechinger (1998) along with internet resources.

3.3. Edaphology

3.3.1. Soil collection

Two kg soil samples were collected from each site to depth of 15 cm and mixed to make a composite sample. It was stored in a polythene bag and labelled. These were analyzed for physic-chemical characteristics.

27

28 3.3.2. Soil texture

Soil texture was determined by hydrometer method. A 100 g soil sample was taken in a 400 ml glass container. 200 ml distilled water and 125 ml sodium hexa-metaphosphate solution (1%) were added into it. The sample was allowed to soak over-night. The sodium hexa-metaphosphate treated sample was transferred to a dispersion cup and the contents were mixed with electric mixer for 5 minutes. The contents were added to a one-litre sedimentation cylinder and the volume was made up to one-litre with distilled water. Silt and clay fractions were computed with the help of a Bouycous-hydrometer and sand was worked out by subtraction. The texture class was determined with the help of textural triangle (Brady, 1996, Fig. 5).

3.3.3. Saturation percentage

For saturation percentage, the soil was dried at 70 °C and 200 g of soil were taken for preparing saturation paste. The saturation percentage was determined by the formula:

SP (%) = Amount of water added (g) Mass of oven dried soil (g) x [100-Pw]

Where SP % is saturation percentage, PW is known water content

3.3.4. Soil pH and electrical conductivity (ECe)

The soil extract was used to determine pH and electrical conductivity (ECe) using a pH/EC meter (WTW series InoLab pH/Cond 720).

3.3.5. Organic matter (Walbley)

For organic matter litter was separated from each sample and expressed in percentage. It was determined by Walkley and Black’s titration method (Hussain, 1989)

Organic method %age= S-T x 6.7 S

29

Fig. 5. Textural triangle to determine the soil texture classes

30 Where,

S= blank reading

T= Volume used of FeSO4

3.3.6. Total Nitrogen (Kjeldahl method)

The nitrogen in the samples was converted to Ammonium (NH4) form by digestion with Sulfuric acid (H2SO4), The Ammonia (NH3) is distilled into boric acid and determine by titration with standard H2SO4. This procedure is employed in large, small and very small determinations. The contents of receiver were titrated with 0.1N of H2SO4 till the blue colour disappeared first and then drop by drop until it turned pink.

Percentage of nitrogen was calculated with the help of formula.

N% = (T-B) x N x 1.4/S

Where

T = sample titration, ml standard acid

B = blank titration

N = normality of acid

S = weight of sample in grams

3.3.7. Phosphorus (Olsen method)

50 grams of soil samples and 1 teaspoon of carbon black with 100 ml of 0.5 molar sodium bicarbonate was added in a 250 ml Erlenmeyer flask and shaken for 30 minutes. The contents of the flask were filtered and more carbon black was added to get a clear filtrate. An aliquot of the filtrate corresponding to a definite fraction of the original soil was placed in a 25 ml volumetric flask. 5 ml of ammonium molybdate was added to the flask and mixed. Washed down the neck of the flask to avoid direct contact of concentrated molybdate solution and Stanous Chloride (SnCI2) and 1.0 ml of dilute SnCI2 solution was added and mixed thoroughly. Colour intensity was read in a colorimeter within 10 minutes. After

31 addition of the SnCl2 solution using a 660 mg filters. A standard curve with 5 ml of the

NaHCO3 solution included with standard phosphate solution was prepared.

Parts per million of Phosphorus in soil=Parts per million of Phosphorus in solution (from curve) were calculated.

3.3.8. Soil ionic content (Flame photometric method)

In soil water extract, potassium usually occurs in low concentrations. The flame photometric method was used to estimate Na+, K+ and Ca2+ cations. For Cl- determination, plant samples (100 mg each) were ground and heated in 10 ml of H2O at 80 ºC till the volume was halved. Then it was brought to 10mL again by adding distilled water. Cl- content was determined with a chloride meter (Jenway, PCLM 3).

3.4. Phytosociology

3.4.1. Vegetation sampling

For phytosociology data, combination of systematic quadrats each of 1 m2 along with seventy-five transect lines were laid (each of 100 m long) at each study site, separated by 20 m from each other.

The following phytosociological attributes were measured in each case.

Density (%) = Total number of individuals of a species in all quadrates Total number of quadrates studied

Frequency (%) = No. of quadrats in which a species occur x 100 Total number of quadrats studied

Cover (%) = Area covered by a species in a quadrat x 100 Total area covered by all the species

Relative density (%) = Number of individuals of the species x 100 Number of individual of all species

Relative frequency (%) = Number of occurrence of the species x 100 Number of the occurance of all the species

32

Relative Cover (%) = Coverage/dominance of a particular species x 100 Total coverage/dominance for all the species in a stand

Importance value = Relative frequency + Relative density + Relative coverage

3.4.2. Species association

The ecological data was organized in contingency table with 2 grass species apearing binomially distributed (present or absent) (Hubalek, 1982) to calculate association between them. A chi-sequare test was applied (Dice, 1945; Ludwig and Reynolds, 1988) in order to test the significance of pair-wise associations. After sorting out the significant associations, the dice indice was used to express the strength of association between each pair of species.

3.5. Morphological parameters

Morphological characteristics such as, plant height (cm), root length (cm), fresh and dry weight of root and shoot (g), number of tiller plant-1, number of leaves plant-1, ligule length (cm), inflorescence length (cm), and spike length (cm) were studied and 5-7 grass specimens were observed for morphological studies. For dry weight, plants were oven-dried at 65 °C until constant weight was achieved.

For phylogenetic analysis, other morphological characteristics such as, root type (fiberous, stoloniferous, rhizomatous), culm shape (decumbent, erect or stout), culm surface (caespitose or solitary), and culm origin (glabrous, node bearing or scabrous), sheath type (smooth, hairy, papery or keeled), blade type, (attenuate, lanceolate or linear), blade shape, (convolute, flat, lanceolate or folded), blad surface (glabrous, scabrous), leaf apex (attenuate, acuminate, acute), leaf origin (cauline, basal or both), ligule type (memberanous fringed, memberanous truncate or ring of hairs, inflorescence type (panicle, raceme, spike), presence or absence of awn and branching pattern (solitary, paired, recemose) etc. were also recorded.

33 3.6. Anatomical parameters

For anatomical parameters double-stained permanent slides were prepared by free- hand sectioning technique. For root anatomy 2 cm piece from the root-shoot junction of the thickest root, and for leaves one cm piece from the leaf centre along the midrib were taken. The material was first preserved in FAA (formalin acetic alcohol) solution for 48 h, which contained v/v formalin 5%, acetic acid 10%, ethanol 50%, and distilled water 35%. The material was then transferred in acetic alcohol solution (v/v 25% acetic acid and 75% ethanol) for long-term preservation. The data were recorded using an ocular micrometer and photographs were taken by Carl-Ziess camera equipped microscope.

3.6.1. Root anatomy

Root cross sectional area (mm2) Epidermal thickness (µm) Cortical thickness (µm) Cortical cell area (µm2) Endodermal cell area (µm2) Pericyle thickness (µm) Metaxylem vessel number Metaxylem area (µm2) Pith area (µm) Pith cell area (µm2) 3.6.2. Stem anatomy Stem cross sectional area (mm2) Epidermis thickness (µm) Sclerenchyma thickness (µm) Cortical cell area (µm2) Metaxylem area (µm2) Phloem area (µm2) 3.6.3. Leaf-sheath anatomy Leaf-sheath thickness (µm) Sclerenchyma thickness (µm)

34 Epidermis cell area (µm2) Vascular tissue area (µm2) 3.6.4. Leaf blade anatomy Leaf blade thickness (µm) Lower epidermis thickness (µm) Upper epidermis thickness (µm) Bundle sheath cell thickness (µm) Bundle sheath area (µm2) Metaxylem vessel area (µm2) Phloem area (µm2) Bulliform cell area (µm2) Adaxial stomatal number Abaxial stomatal number Adaxial stomatal area (µm2) Abaxial stomatal area (µm2) 3.7. Physiological Parameters

3.7.1. Analysis of plant nutrients

Digestion

Dried ground material (0.5 g in each tube) was taken in digestion tubes and 5 mL of concentrated H2SO4 were added to each tube (Wolf, 1982). All the tubes were incubated overnight at room temperature. Then 0.5 mL of H2O2 (35%) was poured down the sides of the digestion tube, ported the tubes in a digestion block and heated at 350°C until fumes were produced. They were continued to heat for another 30 minutes. The digestion tubes were removed from the block and cooled. 0.5 mL of H2O2 was slowly added to each tube and placed the tubes back into the digestion block. The above step was repeated until the cooled digested material was colourless. The volume of the extract was made up to 50 mL. The extract was filtered and used for determining K+, P, Ca2+, Na+ and Cl- content.

35 3.7.2. Determination of shoot and root ionic content

Shoot ionic contents such as Na+, K+ and Ca2+ were determined with a flame photometer (Jenway, PFP-7) following Wolf (1982). A graded series of standards (ranging from 5 to 25 mg L-1) of Na+, K+ and Ca2+ were prepared and standard curves were drawn. The values of Na+, K+ and Ca2+ from flame photometer were compared with standard curves and total quantities were computed. For Mg2+ the prepared solution was titrated against Sodium Dihydrogen Ethylenediamine tetra acetate (EDTA) and the end point was wine red the color changed from red to blue or green. Meq/liter of Mg was calculated as: Meq/liter of Mg = ml of EDTA X normality of EDTA X 1000/ml. of sample.

3.7.3. Chlorophyll contents

Total chlorophyll contents (chlorophyll a & b, carotenoids) were determined directly by chlorophyll meter.

3.8. Cladistic analysis of Andropogonae tribe

3.8.1. Morphological data and selection of outgroups

For cladistics analysis, nine genera of Andropogoneae were included and two genera, Arundinella and Agrostis, were selected as outgroups. For outgroup selection Stefanovic et al. (1998) and Hart (1987) were followed. The morphological matrix was constructed following the data method described in (Morrone et al., 2012). Characters for morphological matrix were used mainly for a reasonable argument of similarity; character-state transformation was based on outgroups analysis and discrimination of genera. Twenty-six characters were considered and data matrix prepared for cladistic analysis. Each character was denoted by 0 (indicating plesiomorphic) and 1, 2, 3, 4 (indicating apomorphic) by the outgroup comparison method (Watrous and Wheeler, 1981). Cladistic analysis of the data was conducted using PAUP* 4.0b (Swofford, 2000). All characters were considered as equally weighted. Heuristic searches for most parsimonious trees used 100 random addition sequences.

36 3.8.2. Gene bank data

For molecular data, the existing GenBank sequence was used as a starting point for alignment, and all the available nucleotide sequences downloaded from GenBank (Table. 18). Each genus was covered by at least one available species. We collected 11 species which were available in deposited data of GenBank. Then, we included only one sequence per species, keeping the longest sequence or the most recently added if sequences were of the same length. In total, our GenBank data alignment consisted of 6 quality nuclear (ITS) and plastid loci (ndhF, rbcL, trnL-trnF, atpB-rbcL and matK). The selected taxon and gene loci are shown in Table 2. Some loci were unavailable in GenBank, the gene loss was 44% of plastid and Capillipedium lacked ITS. We combined the plastid and nuclear loci for phylogenetic analysis. Specially, the gene regions were difficult to align in many places or indels appearance in the alignments were excluded: trnL-trnF (24-25, 121-208, 235-238, 262-271, 322-325, 530-533, 541-544, 582-593, 598-602, 695-699, 721-766, 801-803, 861- 868,897-901), matK (71-94), atpB-rbcL (176-187, 609-624, 665-676), and ITS (36-40, 48- 55, 73-88, 100-105, 398-411, 510-513, 597-600).

3.8.3. Phylogenetic analysis

For phylogenetic analysis on the combined dataset using Maximum Likelihood (ML) as implemented in RAxML-VI-HPC (Stamatakis, 2006) were used. The ML analyses employed the GTRCAT nucleotide substitution model, with the default settings for the optimization of individual per-site substitution rates. Due to the difficulties of bootstrapping datasets with large amounts of non-randomly distributed missing data, we also used Bayesian Inference (BI) to assess nodal support values for our phylogenies. Bayesian analyses were performed in the program MrBayes v.3.2 (Huelsenbeck and Ronquist, 2001; Ronquist et al., 2012). The default settings (GTR + G + I model) analysis was used for the combined dataset. For the BI analysis, we ran two separate analyses for 2 million generations each with tempt 0.05, then removed burin of 25% generations.

37 3.9. Statistical analysis

The data for different ecological attributes were analyzed using Conoco Computer Package for Window [version 5]. The data was also subjected to Conical Corresponding Analysis (CCA) and Redundency analysis (RDA), keeping grasses variable and cites as co- variable and vice versa. The data was subjected to multivariate analysis (cluster analysis) and analysis of variance (ANOVA) using completely randomized design (CRD). The LSD values (5%) were used to test the significance of mean values.

38 CHAPTER 4

RESULTS

4.1. Soil analysis

4.1.1. Soil physiochemical characteristics

Most of the soil characteristics varied significantly at P < 0.005 except soil organic matter and Ca2+ showed non-significant difference among the all study sites. The soil moisture content seemed to be closely related to the physical properties of the soil as well as to vegetation type. The main factor affecting soil moisture was precipitation. A significant difference was also observed in Ec, saturation percentage and ionic contents of the soil in the analysis. Sharda site showed highest saturation percentage that was followed by Sardari and Halmat with. The main factor affecting soil moisture was precipitation. The pH of the soil varies from 4.6 to 8.2 mostly acidic and tends to be slightly basic with low content of exchangeable cations at Chilhana site. The maximum ECe value was recorded at Chilhana, followed by Jura. The lowest ECe value was recorded at Kail site. A significant difference in K+ content was recorded in soil collected from Halmat, following by Sharda, whereas, Dawarian showed lowest K+ content. Ca2+ content was found maximum at Dawarian while minimum content was recorded at Tao Butt. No significant differences in soil Ca2+ and organic matter was observed among the study sites. High content of phosphorus were found at Chilahana, whereas, minimum content was recorded at Dawarian. A significant different was also recorded in Cl- content as it was maximum at Sharda and low content was recorded at Sardari. Total nitrogen percentage was found non-significant among all sampling sites, as highest percentage was found at Kundal Shahi and lowest was found at Janawai. Soil texture depends on the relationship or percentage of sand, silt and clay present in the soil. The data analysis showed that there was no significant correlation between soil and plant communities (Table 4).

39

Table 4. Soil physico-chemical characteristics of different sites in the Neelum Valley, Azad Jammu & Kashmir

+ 2+ 3- - Soil S ECe K Ca PO4 Cl TN OM Soil Sites pH (%) (dS m-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1) (%) (%) texture CH 8.2a 46.64b 8.8a 8.47d 4.18ab 92.14a 0.6def 0.118b 2.38a SCL JR 6.9ab 26.93i 8.7a 8.97cd 3.7b 83.52f 0.8cdef 0.105b 2.09a SCL KS 5.8bcdef 38.76fg 6.6efg 9.93bc 4.58ab 92.01a 0.4 ef 5.55a 2.03a SL AT 4.6bcdef 46.32bc 5.8gh 9.43bcd 3.67a 88.66c 1.1bcde 0.111b 2.36a SL KR 5.7bcdef 30.64h 6.8def 9.04cd 4.34ab 80.93gh 1.3bcd 0.109b 2.22a L NG 6.2abcd 43.49cd 8.0abc 8.66d 3.78b 81.22g 1.8 ab 0.099b 2.06a CL def h 1bcd ab j cdef b a LW 4.9 30.85 6.8def 9. 4.52 77.45 0.9 0.107 2.04 SL DW 5.4cdef 40.38ef 7.6bcd 8.45d 4.86ab 71.08k 1cdef 0.121b 1.84a CL DD 5.7ef 30.52h 8.4ab 8.67d 4.22ab 80.06h 1.2bcd 0.10b 2.13a L SH 7.0abcde 52.34a 8.6a 10.02b 4.46ab 87.67d 2.0a 0.104b 2.42a SL KL 6.3cdef 40.06de 5.7h 8.58d 3.97ab 84.02f 1.5abc 0.116b 1.92a L JW 5.6f 37.15g 7.4cde 8.98cd 4.42b 86.04e 0.8cdef 0.096b 1.98a CL SR 7.2abcd 47.87b 7.7bc 11.22a 4.82ab 78.06j 0.3f 0.112b 1.88a L HL 7.9abcdef 47.06bc 6.4fgh 9.44bcd 3.67b 90.56b 0.4ef 0.103b 2.27a CL TB 7.4abc 45.05bcd 6.8def 8.89d 3.55ab 79.07i 0.7def 0.122b 1.98a CL F ratio 2.71** 58.9** 13** 4.6* 0.72ns 356** 4.08* 1007** 0.34ns LSD (5%) 1.3146 2.8850 0.8241 0.9704 1.8851 0.9254 0.7209 0.1293 0.9563

Legends: S (%) = saturation percentage, ECe = Electric conductivity, TN = Total nitrogen, OM = Organic matter, SCL = sandy clayey loam, SL = sandy loam, L = loam, CL = clayey loam Study sites: Chiliahana (CH), Jura (JR), Kundal Shahi (KS), Athmuqam (AT), Kairan (KR), Lawat (LW), Dawarian (DW), Dodonial (DD), Sharda (SH), Kail (KL), Janawai (JW), Sardari (SD), Halmat (HM), Tao butt (TB). Mean within rows sharing same letter are non-significant at P<0.05 level (n= 3) *,** = significant at 0.05 and 0.001 levels, respectively. Ns = non-significant

40 4.1.2. Soil RDA analysis

RDA ordination biplot (Fig. 6) showed a strong effect of soil ionic contents on distribution of grasses at different habitats. Rostraria pumila, Pennisetum orientale, Sorghum nitidum, and Arundinella sp. showed a strong association with the Kail site under strong influence of soil Ece. Distribution of Lolium temulentum, Poa nemoralis, and Saccharum spontaneum, at Chilhana site was seemed to be affected by the moisture contents. Distribution of Heteropogon contortus, Parapholis incurva, Setaria pumila, Agrostis viridis, Capillipedium parviflorum, Panicum humile; Koeleria cristata, Bothriochloa pertusa, Digitaria cruciata, Aristida funiculate, Hordeum glaucum, and Eleusine indica species, strongly associated to the Nagdar, Kundal Shahi, and Kairan sites, were affected by Ca2+ 3- content. Bothriochloa bladhii at Janawai site was controlled by PO4 content. Presence of Festuca kashmiriana, Poa argunensis, Poa falconeri, and Sorghum arundinaceum species at Sharda, Dodonial, and Lawat sites seemed to be influenced by K+ and N+ content. Aristida cyana, Aristida mutabilis, Cenchrus sp., Poa attenuata, were more associated with Sardari whereas, Panicum atrosanguineum with Jura, and Agrostis pilosula and Avena byzantina were associated with Athmuqam site. Distribution of Aristida funiculata, Koeleria macrantha species at Tao butt site was not influenced by any of the soil factor. 4.2. Phytosociology

4.2.1. Taxonomic diversity

A total of 52 species of grasses belonging to 10 tribes and 28 genera were recorded from 15 sampling sites in Neelum Valley, Azad Jammu and Kashmir (Table 5). Poaeae was the largest tribe (12 spp.); followed by Andropogoneae (11 spp.); Aveneae (9 spp.); Paniceae (8 spp.); Aristideae and Brachypodieae (3 spp. each), whereas, rest of the 5 tribes represents 2 or 1 species. Dodonial was species rich site with 15 species (28.85%), followed by Dawarian, Sharda, and Taobut (12 spp.), Kairan, Nagdar, and Halmat with 11 species (Table 5, 6).

41 1.0

Pom

Aru Arp Avb Agp Peo AT Son KL Rop Hom Paa Fes Arc EC JR Lot HM Pon Ces Pad Sap Arm Lop CH Pat Arn pH SR Kom S Arf OM Cl TB K Brs TN Sep Pah Ca P Soa Pai JW Cep NG KS SH DD Fek Roc Hec Agv Bob Cap Pof Poa Koa Apm Dic Avf LW KR Hog Fel Mie Poi DW Bra Bop Brd -0.6 Sci Saf Eli Cyd

-0.8 0.8

Fig. 6. RDA ordination analysis of the soil physio-chemical characteristics and grasses at different sites of the Neelum Valley, Azad Jammu and Kashmir Legends: Chiliahana (CH), Jura (JR), Kundal Shahi (KS), Athmuqam (AT), Kairan (KR), Lawat (LW), Dawarian (DW), Dodonial (DD), Sharda (SH), Kail (KL), Janawai (JW), Sardari (SD), Halmat (HM), Tao but (TB). Agp: Agrostis pilosula; Agv: Agrostis viridis; Apm: Apluda mutica; Arc: Aristida cyanantha; Arf: Aristida funiculate; Arm: Aristida mutabilis; Arp: Arthraxon prionodes; Arn: Arundinella nepalensis; Aru: Arundinella sp; Avb: Avena byzantine; Avf: Avena fatua; Bob: Bothriochloa bladhii; Bop: Bothriochloa pertusa; Brd: Brachypodium distachyon; Bra: Brachypodium sp; Brs: ; Cap: Capillipedium parviflorum; Cep Cenchrus pennisetiformis; Ces Cenchrus sp; Cyd: Cynodon dactylon; Dic: Digitaria cruciata; Eli: Eleusine indica; Fek: Festuca kashmiriana; Fel: Festuca levingei; Fes: Festuca simlensis; Hec: Heteropogon contortus; Hog: Hordeum glaucum; Hom: Hordeum marinum; Koa: Koeleria cristata; Kom: Koeleria macrantha; Lop: Lolium perenne; Lot: Lolium temulentum; Mie: Milium effusum; Paa: Panicum atrosanguineum; Pad: Panicum decompositum; Pah: Panicum humile; Pai: Parapholis incurve; Peo: Pennisetum orientale; Poi: Poa infirma; Poa: Poa argunensis; Pat: Poa attenuate; Pof: Poa falconeri; Pon: Poa nemoralis; Pom: Polypogon monspeliensis; Roc: Rostraria clarkeana; Rop: Rostraria pumila; Saf: Saccharum filifolium; Sap: Saccharum spontaneum; Sci: Schizachyrium impressum; Sep: Setaria pumila; Soa: Sorghum arundinaceum; Son: Sorghum nitidum.

42 Table 5. Meteorological and topographical data of sampling sites in Neelum Valley, Azad Jammu and Kashmir

Altitude Slope Observed Sampling sites Coordinates (m) (%) Aspect taxa %age 34º 23’ 56.1” N Chiliahana 73º 46’ 29.5” E 1100 60-70 Eastern 9 17.31 34º 29’ 26.9” N Jura 73º 50’ 04.8” E 1290 55-65 Western 11 21.15 34º 33’ 03.6” N Kundal Shahi 73º 50’ 52.2” E 1318 40-50 North-eastern 8 15.38 34º 35’ 33.9” N Athmuqam 73º 55’ 0.72” E 1403 20-35 Western 13 25.00 34º 38’ 54.3” N Kairan 73º 56’ 57.1” E 1499 20-25 Northern 11 21.15 34º 40’ 24.7” N Nagdar 73º 57’ 20.0” E 1555 50-60 North-western 11 21.15 34º 41’ 15.9” N Lawat 73º 58’ 11.3” E 1579 40-50 Southern 11 21.15 34º 43’ 25.1” N Dawarian 74º 59’ 58.1” E 1807 35-45 Northern 12 23.08 34º 41’ 58.78” N Dodonial 74º 06’ 03.09” E 2774 20-30 Southern 15 28.85 34º 47’ 03.9” N Sharda 74º 10’ 47.6” E 2014 25-35 East-western 12 23.08 34º 48’ 58.2” N Kail 74º 25’ 09.7” E 2047 15-25 North-western 9 17.31 34º 47’ 23.0” N Janawai 74º 33’ 56.0” E 2187 30-40 Western 8 15.38 34º 45’ 44.1” N Sardari 74º 38’ 17.3” E 2239 15-20 South-eastern 10 19.23 34º 46’ 42.6” N Halmat 74º 40’ 28.9” E 2267 5-15 Eastern 11 21.15 34º 43’ 42.7” N Tao butt 74º 43’ 26.3” E 2300 5-10 East-western 12 23.08

43 Table 6. Tribe wise synopsis of the grasses of Neelum Valley, Azad Jammu and Kashmir

Tribe Plant species Andropogoneae Apluda mutica L. Arthraxon prionodes (Steud.) Dandy Bothriochloa bladhii (Retz.) S. T. Blake Bothriochloa pertusa (L.) A. Camus. Capillipedium parviflorum (R. Br.) Stapf Heteropogon contortus (L.) P Beauv. Saccharum filifolium Nees ex Steud. Saccharum spontaneum L. Schizachyrium impressum (Hack.) A.Camus Sorghum arundinaceum (desv.) stapf Sorghum nitidum (Vahl) Pers. Aristideae Aristida cyanantha Nees ex Steud Aristida mutabilis Trin. Aristida funiculata Trin. & Rupr Arundineae Arundinella nepalensis Trin. Gram. Pan. Arundinella sp. Aveneae Agrostis pilosula Trin. Agrostis viridis Gouan Avena byzantina K. Koch Avena fatua L. Koeleria cristata (L.)Pers. Koeleria macrantha (Ledeb.) Schult. Polypogon monspeliensis (L.) Desf. Rostraria clarkeana (Domin) Holub Rostraria pumila (Desf.) Brachypodieae Brachypodium distachyon (L.) Beauv. Brachypodium sp. Brachypodium sylvaticum (Huds.) P. Beauv Cynodonteae Cynodon dactylon (L.) Pers. Eragrostideae Eleusine indica (L.) Gaertn. Paniceae Cenchrus pennisetiformis Hochst. & Steud. Cenchrus sp. Digitaria cruciata (Nees) A. Camus Panicum atrosanguineum Hochst. Panicum decompositum R.Br. Prodr. Panicum humile Nees ex Steude. Pennisetum orientale L.C. Rich. in Pers. Setaria pumila (Poiret) Roemer & Schultes Poeae Festuca levingei Stapf in Hook.f. Festuca simlensis (Stapf) E.B. Alexeev Festuca kashmiriana Stapf Lolium perenne L. Lolium temulentum L. Milium effusum L. Parapholis incurva (L.) C.E. Hubb Poa infirma Kunth Poa argunensis Roshev. Poa attenuata Trin. Poa falconeri Hook. f. Poa nemoralis L. Triticeae Hordeum glaucum (Steud.) Hordeum marinum L.

44 4.2.2. Relative density

Based on the relative density, Agrostis pilosa was rarely present at Jura and Athmuqam sites. It was absent from rest of the sites. Agrostis viridis showed the maximum density at Kundal Shahi. Apluda mutica was moderately present at Kairan and Halmat sites but was frequently present at Dawarian with high density. Aristida cynantha was very rare at Dodonial and Jura but showed moderate density at Athmuqam. Aristida funiculata was frequent at Kundal shahi also rarely present at Halmat. Aristida mutabilis showed very low density at Dodonial was also very at all other sites. Arthraxon prionodes showed high densities at Chilhana and Kail. It was also present at Athmuqam, Halmat and Taobutt with moderate densities except Nagdar site where it showed low density.

Arundinella nepalensis was moderately present at Nagdar and Halmat except Sharda and Sardari where it was rarely present. Arundinella sp., showed its maximum density at Kail. Avena byzantina showed its moderate density at only single site Athmuqam. Avena fatua was moderately present at Kairan, Janawai and Tao butt. Bothriochloa bladhii showed moderate and high densities at Nagdar and Janawai respectively. Bothriochloa pertusa was also among the grasses with maximum densities. Both Brachypodium distachyon and Brachypodium sp., showed their presence at four study sites. Brachypodium sylvaticum and Capillipedium parviflorum was frequently present together at Kundal Shahi and Chilhana sites respectively.

Cenchrus pennesitformis was frequent at Sardari and at Dodonial it was very rarely present. Cenchrus sp., was moderately present at Kundal Shahi and Sardari. Cynodon dactylon grass showed its maximum density at Dodonial, Sharda and Janawai. At Dodonial and Sharda it was rarely present. Digitaria cruciata was the only grass that showed its maximum density at more than one site. Eleusine indica was frequent at Kairan and Nagdar.

Festuca kashmiriana and F. levengei showed their moderate density at Lawat. At Tao butt, F. levengei was very rarely present. Festuca simlensis showed its maximum density at Sharda and at Kail and Sardari it was moderately present. Heteropogon contortus was abundant at Janawai and at Jura it was rarely present. Hordeum galcum was rarely present at

45 single site Kairan. Hordeum marinum was abundant at Chilhana and rare at Athmuqam site but was absent from rest of the sites.

Koeleria cristata was moderately present at Kairan only whereas, K. macrantha was rare at Chilhana and Lawat. Lolium perenne was rare at Dawarian and Kail. Lolium temulentum was frequent at Kail and Halmat. Milium effusum was moderately present at Nagdar and rare at kairan site. Panicum atrosanguineum was present with moderate density at Jura and Sardari sites. Athmuqam and Janawai it was rarely present. Panicum decompositum showed its moderate density at Halmat together with P. humile at Tao butt.

Parapholis incurva was moderately present at Jura and very rare at Kairan. Pennesitum orientale showed its maximum density at Sardari and Kail and Tao butt it was rarely present. Poa argunensis was moderately present at Sharda and least frequent at Dodonial. Poa attenuata showed it moderate density at only Sardari sites whereas, Poa falconeri was the grass with maximum density at Lawat. Both Poa infirma and P. nemoralis showed their moderate density at Kairan and Chilhana sites.

Polypogon monspeliensis was frequent at Athmuqam with moderate density at Jura and Kail. Rostraria clarkeana was present at Jura with moderate density but show very low density at Dodonial and Sharda. Rostraria pumila showed low density at Jura, Athmuqam and Dawarian, but at kail it was present with moderate density. Saccharum filifolium showed its maximum density at Kairan and was very rare at Dawarian.

Sacchrum spontaneum was only present at Chilhana with moderate density. Schizachyrium impressum showed moderate density at Sharda and at Athmuqam, Lawat and Dawarian it was present with low density. Setaria pumila was frequent at Kundal Shahi and moderately present at Janawai. Sorghum arundinaceum was frequent at Janawai but was very rare at Dodonial. Sorghum nitidum was abundantly present at only Kail site with maximum density (Table 7).

46 Table 7. Relative density of native grasses at different sites in Neelum Valley, Azad Jammu and Kashmir

0.0-4.0 4.1-8.0 8.1-12.0 12.1-16.0 >16 Very rare Rare Moderate Frequent Abundant

47 4.2.3. CCA analysis

The PCCA of density data with respect to sites as environmental data showed significant variation. A biplot of species density variable sites revealed that most of the species were centroid. Poa infirma, Koeleria cristata, and Hordeum glaucum showed a strong association with KR site. Eleusine indica, Saccharum filifolium, Milium effusum and Avena fatua were more associated with NG site. Bothriochola pertusa showed a close relationship with TB site. Aristida funiculata and Agostis viridis were closely related to KS site.

Arundinella nepalensis, Panicum humile, Digitaria cruciata, Apluda mutica, Heterpogon contortus, Panicum decompositum, Bothriochloa bladhii and Sorghum arundinaceum were strongly associated with JW, HM and DW sites. Schizachyrium impressum, Lolium temulentum and L. perenne was closely related with JU site. Pennesitum orientale, Brachypodium distachyon, Aristida mutabilis and Cenchrus pennesitiformis showed a close association with SR site. Festuca levengei, F. kashmiriana, Cynodon dactylon and Poa falconeri were more associated with LW site. Sorghum nitidum, Rostraria clarkeana and Poa attenuata showed a close relationship with DD site. Panicum atrosanuineum, Festuca simlensis, Arundinella sp., Agrostis pilosa, Cenchrus sp., were closely related with SH and AT sites. Brachypodium sp., Koeleria macrantha, Rostraria pumila, Capillipedium parviflorum, Hordeum marinum, Poa nemoralis and Saccharum spontaneum showed a close association with CH site (Fig. 7).

4.2.4. Relative frequency

The grass Agrostis pilosa showed its moderate presence at Athmuqam and Jura sites. Agrostis viridis was present at Kundal Shahi only where it showed its maximum frequency. Apluda mutica grass showed moderate frequency at Kairan, Nagdar, Dawarian and Halmat sites but was frequently present at Tao butt with rare frequency. Aristida cynantha showed low frequency at Dodonial and Jura but showed moderate frquecny at Athmuqam. Aristida funiculata showed high frequency at Kundal shahi but it was present at Halmat with low frequency.

48 a. b.

c. d.

Fig. 7. CCA ordination analysis of (a.) relative density, (b.) relative frequency, (c.), relative cover, and (d.) importance value based distribution of grasses over different sites in Neelum Valley, Azad Jammu and Kashmir

Key to sites: Chiliahana (CH), Jura (JR), Kundal Shahi (KS), Athmuqam (AT), Kairan (KR), Lawat (LW), Dawarian (DW), Dodonial (DD), Sharda (SH), Kail (KL), Janawai (JW), Sardari (SD), Halmat (HM), Tao but (TB).

Key to species: Agp: Agrostis pilosula; Agv: Agrostis viridis; Apm: Apluda mutica; Arc: Aristida cyanantha; Arf: Aristida funiculate; Arm: Aristida mutabilis; Arp: Arthraxon prionodes; Arn: Arundinella nepalensis; Aru: Arundinella sp; Avb: Avena byzantine; Avf: Avena fatua; Bob: Bothriochloa bladhii; Bop: Bothriochloa pertusa; Brd: Brachypodium distachyon; Bra: Brachypodium sp; Brs: Brachypodium sylvaticum; Cap: Capillipedium parviflorum; Cep Cenchrus pennisetiformis; Ces Cenchrus sp; Cyd: Cynodon dactylon; Dic: Digitaria cruciata; Eli: Eleusine indica; Fek: Festuca kashmiriana; Fel: Festuca levingei; Fes: Festuca simlensis; Hec: Heteropogon contortus; Hog: Hordeum glaucum; Hom: Hordeum marinum; Koa: Koeleria cristata; Kom: Koeleria macrantha; Lop: Lolium perenne; Lot: Lolium temulentum; Mie: Milium effusum; Paa: Panicum atrosanguineum; Pad: Panicum decompositum; Pah: Panicum humile; Pai: Parapholis incurve; Peo: Pennisetum orientale; Poi: Poa infirma; Poa: Poa argunensis; Pat: Poa attenuate; Pof: Poa falconeri; Pon: Poa nemoralis; Pom: Polypogon monspeliensis; Roc: Rostraria clarkeana; Rop: Rostraria pumila; Saf: Saccharum filifolium; Sap: Saccharum spontaneum; Sci: Schizachyrium impressum; Sep: Setaria pumila; Soa: Sorghum arundinaceum; Son: Sorghum nitidum.

49 Aristida mutabilis showed very low frequency at Dodonial but was present at Athmuqam and Sardari with moderate frequency. Arthraxon prionodes showed high frequency at Kail with moderate frequency at Athmuqam and Nagdar. At Halmat, Tao butt and Chilhana it showed relatively less frequency. Arundinella nepalensis showed moderate frequency present at Nagdar and Halmat and Sardari. Arundinella sp., showed its maximum frequency at Kail site only. Avena fatua was moderately present at Kairan and Tao butt with moderate frequency but at Janawai it showed relatively high frequency. Bothriochloa bladhii showed high frequency at Janawai with moderate frequency at Nagdar.

Bothriochloa pertusa was present at Kairan and Dawarian with moderate and low density respectively. Both Brachypodium distachyon and Brachypodium sp., showed their consistent presence at four study sites. Brachypodium sylvaticum and Capillipedium parviflorum was present together at Kundal Shahi and Chilhana sites. Cenchrus pennesitformis was present at Jura, Sharda and Sardari with moderate frequency but at Lawat and Dodonial this grass showed relatively less frequency. Cenchrus sp., at Athmuqam, Dodonial and Sardari was present with moderate frequency whereas at Sharda it showed less frequency. Cynodon dactylon grass showed its maximum frequency at Dodonial, with moderate frequency at Lawat, Dawarian and Sardari. Digitaria cruciata showed high frequency at Kundal Shahi, Nagdar and Sharda.

Eleusine indica was present at Kairan with high frequency and Nagdar and Dawarian with moderate frequency. Festuca Kashmiriana and F. levengei showed their moderate density at Lawat. Festuca simlensis showed its maximum density at Sharda, Kail and Halmat. Heteropogon contortus showed maximum frequency at Janawai and Hordeum galcum showed moderate frequency at Kairan only. Hordeum marinum was present with maximum frequency at Chilhana. Koeleria cristata and K. macrantha showed moderate frequency at Kairan and Chilhana respectively. Lolium perenne showed its maximum frequency at Kail whereas, temulentum was the only grass that showed its maximum frequency at more than one site. Milium effusum was present at Kairan and Nagdar with moderate frequency. Panicum atrosanguineum at four sites and interestingly it showed moderate frequency at all sites. Panicum decompositum showed its moderate frequency at Halmat and Tao butt. Parapholis incurva was moderately present at Jura and very rare at Kairan.

50 Pennesitum orientale was consistent at Sardari and Kail and Tao butt with moderate frequency. Poa argunensis, Poa attenuata and Poa falconeri showed theri maximum frequency at Sharda, Sardari and Lawat respectively. Poa infirma and P. nemoralis was present with moderate and high frequency at Kairan and Chilhana respectively. Polypogon monspeliensis was frequent at Athmuqam, Jura and Kail. Rostraria clarkeana and Rostraria pumila showed moderate frequency at Jura, Saccharum filifolium showed its maximum frequency at Kairan whereas Sacchrum spontaneum was with high frequency at Chilhana. Schizachyrium impressum showed moderate frequency at Athmuqam, Lawat and Dawarian. Setaria pumila showed low frequency at Kairan site. Sorghum arundinaceum showed high frequency at Janawai whereas; Sorghum nitidum was among the grasses that showed highest frequencies (Table 8).

4.2.5. CCA analysis

The PCCA of frequency data with respect to sites as environmental data showed significant variations along axis 1. A biplot of species frequency variable sites revealed that Poa nemoralis, Hordeum marinum, Saccharum spontaneum, Capillipedium parviflorum, Koeleria macrantha, and Brachypodium sylvaticum were strongly associated with CH site. Aristida funiculata and Agrostis viridis showed a close association with KS site. Saccharum filifolium, Digitaria cruciata, Setaria pumila, and Avena fatua were more associated with NG site. Poa infirma, Hordeum glacum, and Koeleria cristata were closely related with KR site. Eleusine indica, Bothriochloa pertusa, Apluda mutica were closely related with TB. Panicum humile, Heteropogon contortus, Panicum decompositum, Sorghum arundinaceum, Pennesitum orientale, Bothriochloa bladhii and Arundinella nepalensis all showed close association with JW, HM and DW sites. Cynodon dactylon, Brachypodium distachyon, Panicum atrosangiuneum, Lolium temulentum and L. perenne showed close association with JU and SD sites. Cenchrus pennesitiformis, Poa falconeri and Festuca kasmirian were in close relation with SH and LW sites. Rostraria clarkeana, Arundinella sp., Sorghum nitidum, Aristida mutabilis, Festuca simlensis and Aristida cynantha were closely associated with AT and KL sites (Fig. 7).

Table 8. Relative frequency of native grasses at different sites in Neelum Valley, Azad Jammu & Kashmir

51

0.0-4.0 4.1-8.0 8.1-12.0 12.1-16.0 >16 Very rare Rare Moderate Frequent Abundant

52 4.2.6. Relative cover

Agrostis pilosa and Agrostis viridis showed high percent cover at Athmuqam and Kundal Shahi respectively. Apluda mutica showed high relative cover at Dawarian and Halmat. Aristida cynantha occupied high cover area at Jura and Dodonial. Aristida funiculata showed high frequency at Kundal shahi but it was present at Halmat with low frequency. Aristida mutabilis showed high cover at Athmuqam. Arthraxon prionodes showed high cover area at Kail. Arundinella nepalensis and Arundinella sp., showed its maximum cover area at Nagdar and Kail sites respectively. Avena byzantina and Avena fatua showed their maximum cover Athmuqam and Janwai and Tao butt respectively.

Bothriochloa bladhii showed the maximum cover at Janawai and Bothriochloa pertusa was present at Nagdar and Tao butt. Both Brachypodium distachyon, and Brachypodium sp., showed their consistent presence at four study sites where Brachypodium sp., showed relatively low cover at Dodonial. Brachypodium sylvaticum and Capillipedium parviflorum showed their maximum cover at Kundal Shahi and Chilhana sites. Cenchrus pennesitformis showed low cover values at Dodonial. Cynodon dactylon grass showed its maximum cover at Dodonial, with moderate cover at Sharda. Digitaria cruciata was the only grass that showed high cover at Kundal Shahi, Nagdar and Sharda.

Eleusine indica was present at Kairan with high cover. Festuca Kashmiriana showed high cover at Sardari and F. levengei was present at Sharda with moderate cover. Festuca simlensis showed its maximum cover at Sharda. Heteropogon contortus showed maximum cover at Dodonial. Hordeum glacum showed moderat cover value at Kairan and Hordeum marinum was present at Chilahana with high cover. Koeleria cristata showed low cover area at Kairan and K. macrantha showed showed high cover at Nagdar. Lolium perenne showed its maximum cover at Tao butt whereas, temulentum was the only grass that showed its maximum cover at Kail and Halmat. Milium effusum was present at Nagdar with moderate cover. Panicum atrosanguineum was present at Jura and Sardari with moderate cover.

Panicum decompositum showed its moderate cover at Dodonial, Halmat and Tao butt. Parapholis incurva was moderately present at Jura. Pennesitum orientale was with high cover at Sardari. Poa argunensis, Poa attenuata and Poa falconeri showed their maximum

53 cover at Sharda, Sardari and Lawat respectively. Poa infirma and P. nemoralis was present with moderate and high cover at Kairan and Chilhana respectively. Polypogon monspeliensis was present with high cover at Athmuqam. Rostraria clarkeana showed high cover at Jura and low at Sharda. Rostraria pumila showed high cover at Kail and relatively low cover at Jura, Athmuqam and Dawaraian. Saccharum filifolium showed its maximum cover at Kairan whereas Sacchrum spontaneum was with high cover at Chilhana. Schizachyrium impressum showed moderate cover at Athmuqam with relative low cover at Athmuqam, Lawat and Dawarian. Setaria pumila showed low cover at Kundal Shahi. Sorghum arundinaceum showed high cover at Janawai and low cover at Dodonial. Sorghum nitidum was present at Kail with relatively very high cover area (Table 9).

4.2.7. CCA analysis

The PCCA cover data with respect to sites as environmental data showed significant variations. Hordeum glacum, Koeleria cristata, Poa infirma, Eleusine indica, Milium effusum, Apluda mutica, Bothriochloa pertusa, and Saccharum filifolium showed strong association with KR and NG sites. Seteria pumila, Digitaria criciata, Herterpogon controtus, Panicum humile, Sorghum arundinaceum, Bothriochloa bladhii, Lolium perenne, Arundinella nepalensis, and Panicum decompositum showed strong association with TB, JW, DW, SR and HM sites.

Aristida funiculata and Agrostis viridis were closely related with KS site. Brachypodium distachyon, Koeleria macrantha, Capillipedium parviflorum, Poa nemoralis, Hordeum marinum, and Saccharum spontaneum showed close association with CH site. Brachypodium sp., Pennesitum orientale, Festuca kashmiriana, Rostraria clarkeana, Cenchrus pennesitiformis, Poa falconeri, Festuca levengei, F. simlensis, Aristida cynantha, Cynodon dactylon, Panicum temulentum, Rostraria pumila, Sorghum nitidum, Arundinella sp., and Cenchrus sp., showed strong associations with LW, SH, DD, AT, JU and KR sites (Fig. 7).

54 Table 9. Cover of native grasses at different sites in Neelum Valley, Azad Jammu and Kashmir

0.0-4.0 4.1-8.0 8.1-12.0 12.1-16.0 >16 Very rare Rare Moderate Frequent Abundant

55 4.2.8. Importance value

Agrostis pilosa showed low importance value (IV) at Jura and Athmuqam. Agrostis viridis showed high IV at Kundal Shahi. Apluda mutica showed high IV at Dawarian. Aristida cynantha and Aristida mutabilis showed low IV at Dodonial. Aristida funiculata showed high IV at Kundal shahi. Arthraxon prionodes showed high IV at Kail and Chilhana. Arundinella nepalensis and Arundinella sp., showed its maximum IV at Halmat and Kail sites respectively. Avena byzantina showed their maximum IV at Athmuqam and Avena fatua at Janwai and Tao butt. Bothriochloa bladhii showed the maximum IV at Janawai and Bothriochloa pertusa was present at Nagdar and Tao butt with high IV. Both Brachypodium distachyon and Brachypodium sp., showed moderate IV at Lawat, Dawarian and Sharda. But at Dodonial both grasses showed relatively low IV. Brachypodium sp., showed relatively low cover at Dodonial. Brachypodium sylvaticum and Capillipedium parviflorum showed their maximum IV at Kundal Shahi and Chilhana sites.

Cenchrus pennesitformis showed high IV at Jura, Lawat and Sardari. Cynodon dactylon grass showed its maximum maximum IV at Dodonial, with moderate IV at Lawat, Dawarian, Sharda and Janawai. Digitaria cruciata was the only grass that showed high IV at Kundal Shahi, Nagdar and Dawarian. Eleusine indica was present at Kairan and Nagdar with high IV. Festuca Kashmiriana showed high IV at Lawat and Sardari. F. levengei was present at Sharda with moderate IV and low at Tao butt. Festuca simlensis showed its maximum IV at Sharda and Kail sites. Heteropogon contortus showed maximum IV Janawai and Nagdar. Hordeum glacum showed moderate IV at Kairan and Hordeum marinum was present at Chilahana. Koeleria cristata showed moderate IV at Kairan and K. macrantha at Nagdar. Lolium perenne showed its maximum IV at Kail whereas, temulentum was the only grass that showed its maximum IV at Kail and Halmat.

Milium effusum was present at Nagdar with moderate IV. Panicum atrosanguineum was present at Jura with moderate IV. Panicum decompositum showed its moderate cover at Dodonial, Halmat and Tao butt. Parapholis incurva was moderately IV present at Jura. Pennesitum orientale was with high IV at Sardari and Halmat. Poa argunensis, Poa attenuata and Poa falconeri showed their maximum IV at Sharda, Sardari and Lawat respectively. Poa infirma and P. nemoralis was present with moderate and high IV at Kairan

56 and Chilhana respectively. Polypogon monspeliensis was present with high IV at Athmuqam. Rostraria clarkeana showed high IV at Jura and low at Sharda. Rostraria pumila showed high IV at Kail and Jura but at Athmuqam and Dawaraian sites it showed relatively low IV. Saccharum filifolium showed its maximum IV at Kairan whereas Sacchrum spontaneum was with high IV at Chilhana. Schizachyrium impressum showed moderate IV at Athmuqam, Lawat and Sharda with relative moderate IV. Setaria pumila showed high IV at Kundal Shahi. Sorghum arundinaceum showed high IV at Janawai and low at Dodonial. Sorghum nitidum was present at Kail with relatively very high importance value (Table 10).

4.2.9. CCA analysis

The PCCA importance value data with respect to sites as environmental data showed significant variations. Brachypodium distachyon, Koeleria macrantha, Capillipedium parviflorum, Poa nemoralis, Hordeum marinum and Saccharum spontaneum showed close association with CH site. Aristida funiculata and Agrostis viridis were closely related with KS site. Hordeum glacum, Koeleria cristata and Poa infirma showed close association with KR site.

Eleusine indica, Milium effusum, Apluda mutica, Bothriochloa pertusa, and Saccharum filifolium, Seteria pumila, Digitaria criciata, Herterpogon controtus, Panicum humile, Sorghum arundinaceum, Bothriochloa bladhii, Lolium perenne, Arundinella nepalensis and Panicum decompositum showed strong association with TB, JW, DW, SR HM and NG sites. Brachypodium sp., Pennesitum orientale, Festuca kashmiriana, Rostraria clarkeana, Cenchrus pennesitiformis, Poa falconeri, Festuca levengei, F. simlensis, Aristida cynantha, Cynodon dactylon, Panicum temulentum, Rostraria pumila, Sorghum nitidum, Arundinella sp., and Cenchrus sp., showed strong associations with LW, SH, DD, AT, JU and KL sites (Fig. 7).

57 Table 10. Importance value of native grasses at different sites in Neelum Valley, Azad Jammu and Kashmir

0.0-10.0 10.1-20.0 20.1-30.0 30.1-40.0 >40.1 Very rare Rare Moderate Frequent Abundant

58 4.2.10. Species association analysis

Chilhana site At Chilhana site, Poa nemoralis showed highly significant association with Hordeum marinum, while significant with Capillipedium parviflorum and Arthraxon prionodes. Significant association of C. parviflorum was also observed with Brachypodium sylvaticum, Saccharum spontaneum. The grass A. prionodes showed significant association with B. sylvaticum. H. marinum showed highly strong association with S. spontaneum, B. sylvaticum and A. prionodes. Similarly, A. prionodes showed significant association with S. spontaneum at this study site. A non-significant association was recorded for Koeleria macrantha with all of the species in comparison (Table 11). Jura site At this site, Panicum humile showed significant association with Panicum atrosanguineum, Cenchrus pennisetiformis, Heterpogon controtus and Rostraria pumila. Polypogon monspeliensis showed highly significant association with C. pennisetiformis, while significant with P. atrosanguineum, H. contortus and R. clarkeana. Aristida cynantha was in significant association with Poa infirm. A highly significant association was found between C. pennisetiformis and P. atrosanguineum where this grass was significant with P. infirma, H. contortus, R. clarkeana and R. pumila. Poa infirma showed highly significant association with H. contortus while significant with P. atrosanganium, R. clarkeana and Arthraxon prionodes. A highly significant association P. atrosanguineum was found with that of H. controtus and R. clarkeana and also between H. controtus and R. clarkeana. Similar association was also noted in R. clarkeana and R. pumila. A highly significant association was found between R. pumila and Sorghum arundinaceum (Table 11). Kundal Shahi site At this site, Saccharum spontaneum showed highly significant association with Digitaria cruciata, Agrostis vidridis and Aristida funiculata, while significant with Brachypodium sylvaticum. A strong association was also observed between B. sylvaticum with that of D. cruciata and Agrostis viridis. At this site, D. cruciata showed highly significant association with Capillipedium parviflorum and A. viridis. Setaria pumila showed highly significant association with C. parviflorum while significant with A. viridis.

59 Table 11. Association analysis of some grasses at Chiliahana, Jura and Kundal Shahi sites, Neelum Valley, Azad Jammu and Kashmir

Chiliahana Pne Kma 3.89 Kma Cpa 4.55 3.09 Cpa Apr 4.03 1.97 3.56 Apr Hma 6.62 3.84 4.10 3.07 Hma Api 4.10 2.87 2.24 1.50 5.08 Api Bsy 2.68 2.50 4.25 4.25 4.03 2.57 Bsy Ssp 3.63 2.87 4.25 3.0 6.75 4.55 2.57 Jura Phu Pmo 4.03 Pmo Acy 2.93 3.22 Acy Cpe 5.24 5.08 2.94 Cpe Pin 4.03 4.10 4.25 4.25 Pin Pat 5.07 4.48 3.36 5.90 4.64 Pat Hco 4.25 4.10 2.87 4.25 5.30 4.96 Hco Rcl 4.55 4.25 3.89 4.55 4.25 4.91 5.30 Rcl Rpu 4.58 2.43 1.97 4.10 3.78 4.48 3.63 4.91 Rpu Api 4.03 4.01 4.25 3.56 4.25 4.12 4.01 4.03 4.01 Api Sar 2.24 1.19 1.19 2.24 3.07 3.36 2.43 4.02 6.22 2.69 Kundal Shahi Ssp Bsy 4.10 Bsy Dcr 5.39 5.39 Dcr Spu 3.72 3.56 4.44 Spu 15.9 Cpa Cpa 3.72 4.58 6.19 3 Sfi 3.09 3.89 3.36 2.69 2.43 Sfi Afu 6.75 4.55 4.91 4.03 4.25 5.08 Afu Avi 5.18 5.18 6.51 4.75 5.76 4.10 6.48

Legends: Pne: Poa nemoralis, Kma: Koeleria macrantha, Cpa: Capillipedium parviflorum, Apr: Arthraxon prionodes, Hma: Hordeum marinum, Api: Agrostis pilosa, Bsy: Brachypodium sylvaticum , Phu: Panicum humile, Pmo: Polypogon monspeliensis, Acy: Aristida cynantha, Cpe: Cenchrus pennesitiformis, Pin: Poa infirma, Pat: Panicum atrosanguineum, Hco: Heterpogon controtus, Rcl: Rostraria clarkeana, Rpu: Rostraria pumila, Api: Agrostis pilosa, Sar: Sorghum arundinaceum. Ssp: Saccharum spontaneum, Dcr: Digitaria criciata, Spu: Setaria pumila, Cpa: Capillipedium parviflorum, Sfi: Saccharum filifolium, Afu: Aristida funiculata, Avi: Avena byzantiana

P>0.05 P>0.01 P>0.001

60 A highly strong association was observed between C. parviflorum and A. viridis and less significant with A. funiculata. Saccharum filifolium showed highly strong association with A. funiculata and a highly significant association was also observed between A. funiculata and A. viridis (Table 11). Athmuqam site At this study site, Rostraria pumila showed a highly significant association with Hordium marinum and significant association with Agrostis pilosa. Polypogon monspeliensis showed highly strong association with Agrostis pilosa and Panicum atrosanguineum. Aristida cynantha showed significant association with Aristida mutabilis where it showed a non-significant association with rest of the grasses in comparison. Cenchus sp., was found in strong association with A. mutabilis and Avena byzantina. Festuca simlensis only showed a highly strong association with Schizycharium impressum which on the other hand showed non-significant association with rest of the grasses. There was no significant association of A. pilosa and Arthraxon prionodes with other grasses in comparison. Aristida mutabilis only showed significant association with A. byzantina. H. marinum, P. atrosanguineum and S. impressum showed non-significant association with the rest of the grasses in comparison (Table 12). Kairan site At Kairan site, Poa infirma showed a highly significant association with Setaria pumila and significant with Aristida mutabilis and Hordeum glaucum. Elusine indica showed non-significant association with most of the grasses except that of A. funiculata and Melium effusum where this grass showed highly significant association. Hordeum glaucum showed highly significant association with Koeleria cristata and less significant with M. effusum grass. Saccharum filifolium only showed association with A. mutabilis and no significant variation of this grass was recorded with rest of the grasses in comparison. Bothriochloa pertusa, Avena fatua, K. cristata, A. mutabilis, Poa infirma, and Setaria pumila showed non- significant association with the rest of the grasses in comparison, except the relatively low association between K. cristata and M. effusum (Table 12).

61 Table 12. Association analysis of some grasses at Athmuqam, Kairan and Nagdar sites, Neelum Valley, Azad Jammu and Kashmir

Athmuqam Rpu Pmo 1.19 Pmo Acy 1.19 1.56 Acy Csp 1.85 2.69 3.09 Csp Fsi 0.91 1.19 2.50 2.57 Fsi Api 1.19 4.62 2.56 2.43 1.19 Api Apr 4.61 1.56 2.56 2.87 2.50 1.56 Apr Amu 2.51 1.97 4.25 5.65 2.50 1.97 1.19 Amu Hma 10.36 2.56 2.56 1.85 1.19 1.56 5.25 1.97 Hma Pat 2.50 4.62 1.56 4.02 1.19 4.62 2.56 1.97 2.56 Pat Sim 1.19 2.56 2.56 2.43 6.25 1.56 2.56 3.22 2.56 1.56 Sim Aby 2.51 2.69 2.56 5.65 1.50 2.56 2.69 4.07 2.69 1.97 1.97 Kairan Pin Ein 2.94 Ein Hgl 4.25 3.72 Hgl Sfi 2.56 2.94 1.97 Sfi Bpe 2.50 3.86 1.97 1.56 Bpe Afa 1.56 8.50 1.97 1.56 1.56 Afa Kcr 2.69 3.72 9.97 2.69 1.97 1.97 Kcr Amu 7.13 3.56 2.93 4.25 2.69 2.69 2.49 Amu Pin 0.85 2.34 2.42 2.56 0.85 2.69 2.42 1.08 Pin Spu 5.25 2.24 1.50 2.50 2.50 1.19 2.51 1.50 0.65 Spu Mef 4.25 5.24 4.07 1.97 2.69 2.69 4.25 2.69 1.08 1.50 Nagdar Ein Bpe 2.69 Bpe Hco 2.49 4.25 Hco Sfi 2.42 1.50 1.08 Sfi Mef 1.08 2.82 2.69 0.85 Mef Dcr 2.69 0.91 3.56 2.34 3.09 Dcr Bbl 4.03 3.86 1.08 0.46 0.85 1.60 Bbl Kma 1.08 0.91 2.51 3.31 2.56 2.24 2.82 Kma Amu 4.61 5.25 1.97 0.85 1.56 2.68 2.82 1.19 Amu Ane 2.69 1.56 4.25 0.85 2.56 3.89 2.56 1.19 1.19 Ane Apr 2.51 2.51 4.25 0.85 1.56 2.68 0.85 1.50 1.56 1.56

Legends: Rpu: Rostraria pumila, Pmo: Polypogon monspeliensis, Acy: Aristida cynantha, Csp: Cenchrus sp., Fsi: Festuca simlensis, Api: Aristida pilosa, Apr: Arthraxon prionodes, Amu: Apluda mutica, Pat: Panicum atrosanguineum, Sim: Schizachyrium impressum, Aby: Avena byzantina, Ein: Eleusine indica, Hgl: Hordeum glaucum, Sfi: Saccharum filifolium, Bpe: Bothriochloa pertusa, Afa: Aristida funiculata, Kcr: Koeleria macrantha, Pin: Poa infirma, Spu: Setaria pumila, Mef: Milium effusum, Hco: Heteropogon contortus, Dcr: Digitaria cruciata, Bbl: Bothriochloa bladhii, Kma: Koeleria macrantha, Ane: Arundinella nepalensis, Apr: Arthraxon prionodes.

P>0.05 P>0.01 P>0.001

62 Nagdar site At Nagdar site, Elusine indica showed non-significant association with most of the grasses except that with Aristida mutabilis. Bothriochloa pertusa showed highly significant association with A. mutabilis and significant with Heteropogon controtus. There was a significant association between A. mutabilis, Arthraxon prionodes with that of H. controtus. All rest of the grasses in comparison such as, Saccharum filifolium, Melium effusum, Digitaria cristata, Bothriochloa bladhii, Koeleria macrantha, Apluda mutica and Arundinella nepalensis showed non-significant association (Table 12). Lawat site At Lawat site, Poa falconeri showed significant association with Cynodon dactylon and non-significant association with rest of the grasses. Cenchrus pennesitiformis, Festuca kashmiriana, Rostraria clarkeana, Brachypodium distachyon, Brachypodium sp., F. levengei and C. dactylon showed non-significant association with rest of the grasses in comparison. However, a highly significant association was recorded between L. temulentum and Schichyrium impresum (Table 13). Dawarian site At Dawarian site, Digitaria criciata, showed non-significant association with all other grasses in comparison except Schizachyrium impressum where a significant association was found between both grasses. Eleusine indica only showed a highly significant association with Bothriochloa pertusa. Aristida mutabilis, Bothriochloa pertusa, Cynodon dactylon, Heteropogon contortus, Rostraria pumila, Brachypodium distachtyon, Brachypidium sp., Schizachyrium impressum and Saccharum filifolium all showed non-significant association in comparison, but a highly significant association was observed between B. distachyon and Lolium perenne (Table 13).

63 Table 13. Association analysis of some grasses at, Lawat, Dawarian and Dodonial, Neelum Valley, Azad Jammu and Kashmir

Lawat Pfa Cpe 1.33 Cpe Fka 2.43 2.56 Fka Rcl 2.57 0.65 1.19 Rcl Lte 2.43 0.85 4.62 2.50 Lte Bdi 2.87 2.87 2.56 2.50 1.56 Bdi Bsp 4.02 2.43 2.56 4.62 2.56 1.19 Bsp Fle 4.02 2.87 1.56 1.56 2.56 2.56 1.56 Fle Cda 4.17 1.85 1.19 2.50 1.19 2.50 1.19 0.85 Cda Sim 2.57 1.85 1.19 1.19 5.25 2.56 1.19 2.56 0.91 Dawarian Dcr Ein 2.87 Ein Amu 3.63 3.22 Amu Bpe 2.57 2.50 2.69 Bpe Cda 2.93 2.56 2.93 1.19 Cda Hco 1.50 2.50 2.51 0.91 1.19 Hco Rpu 2.42 0.85 0.69 0.65 2.50 0.65 Rpu Bdi 2.42 1.19 1.97 1.19 1.56 2.50 2.56 Bdi Bsp 2.93 10.36 3.22 2.69 2.69 2.51 0.85 2.69 Bsp Sim 4.25 1.56 4.25 0.91 4.62 0.91 2.56 1.56 1.56 Sim Sfi 2.51 1.19 2.51 2.50 2.50 0.65 2.56 2.50 2.56 2.56 Sfi Lpe 2.42 0.85 2.42 0.65 0.65 0.65 2.82 6.37 0.85 0.85 2.82 Dodonial Cda Pde 3.68 Pde Csp 4.76 1.56 Csp Par 4.75 4.25 1.56 Par Fka 2.43 2.56 2.56 1.08 Fka Bdi 3.02 2.56 1.56 2.93 2.56 Bdi Bsp 1.45 0.54 0.54 0.69 0.30 0.46 Bsp Amu 1.92 0.54 2.88 0.69 0.30 0.54 0.85 Amu Sar 2.43 2.56 0.85 1.08 0.30 2.88 0.19 4.36 Sar Acy 1.45 0.54 0.54 0.69 0.46 0.85 0.30 0.30 0.30 Acy Cpe 2.43 0.85 0.85 5.32 0.30 0.54 4.36 0.19 0.30 10.61 Cpe Rcl 3.26 0.54 2.88 0.69 0.46 0.85 3.31 0.30 0.46 0.30 0.30 Rcl Fle 3.02 2.50 2.50 1.50 0.30 0.54 0.19 0.19 0.30 0.19 0.30 2.82 Fle Hco 3.68 2.56 2.56 2.69 0.65 1.19 0.41 0.41 0.65 0.41 0.65 0.41 2.50 Hco Fsi 2.27 2.56 0.85 1.08 0.85 1.56 0.54 2.56 0.54 2.88 2.50 0.54 0.46 0.85

Legends: Pfa: Poa falconeri, Cpe: Cenchrus pennesitiformis, Fka: Festuca kashmiriana, Rcl: Rostraria clarkeana, Lte: Lolium temulentum, Bdi: Brachypodium distachyon, Bsp: Brachypodium sp., Fle: Festuca levengei, Cda: Cynodon dactylon, Sim: Schizachyrium impressum, Dcr: Digitaria cruciata, Ein: Eleusine indica, Amu: Apluda mutica, Bpe: Bothriochloa pertusa, Cda: Cynodon dactylon, Hco: Heterpogon controtus, Rpu: Rostraria pumiola, Sfi: Saccharum filifolium, Lpe: Lolium perenne, Pde: Panicum decompositum, Csp: Cenchrus sp., Par: Panicum atrosanguineum, Sar: Sorghum arundinaceum Acy: Aristida cynantha, Fle: Festuca levengei, Hco: Fsi: Festuca simlensis.

P>0.05 P>0.01 P>0.001

64 Dodonial site At Dodonial site, Cynodon dactylon showed non-significant association with most of the grasses in comparison except that of Cenchrus sp. and Poa argunensis where association was found significant. Panicum decompositum, Cenchrus sp., Poa argunensis, Festuca kashmiriana, Brachypodium distachyon, Brachypodium sp., Sorghum arundinaceum, Cenchrus pennesitiformis, Rostraria clarkena and Festuca levengei showed no signification association between species comparison. However, a highly significant association was found between Aristida cynantha and C. pennesitiformis (Table 13). Sharda site At sharda, Poa argunensis showed highly significant association with Festuca levengei, Cynodon dactylon, Cenchrus sp. and Festuca simlensis. The grass F. levengi was found highly significant in comparison with Poa falconeri grass. Cynodon dactylon showed non-significant association with all of the grasses in comparison. Brachypodium distachyon showed highly significant association with F. simlensis which along with Rostraria clarkeana showed significant association with Poa falconeri. Aristida mutabilis showed highly significant association with F. simlensis. All other grasses such as Cenchrus pennesitiformis, Rostraria clarkeana, Cenchrus sp. and F. simlensis showed non-significant association in comparison (Table 14). Kail site At Kail, Sorghum nitidum showed highly significant association with Arthraxon prionodes, Rostraria pumila, Lolium temulentum, Polypogon monspeliensis and also significant association with Arundinella nepalensis and Festuca simlensis. A very highly significant association was showed by A. prionodes with R. pumila and L. perenne and significant with L. temulentum and Polypogon monspeliensis. A non-significant association was found in L. perenne with other grass. P. monspeliensis showed highly significant association with F. simlensis and significant with A. nepalensis grass which in turn showed significant association with F. simlensis (Table 14).

65 Table 14. Association analysis of some grasses at Sharda, Kail and Janawai, Neelum Valley, Azad Jammu and Kashmir

Sharda Par Fle 6.36 Fle Cda 4.91 3.09 Cda Bdi 4.06 3.56 4.25 Bdi Pfa 4.96 5.24 2.69 4.03 Pfa Sim 4.39 2.94 2.56 2.69 2.69 Sim Bsp 3.90 3.09 2.56 1.97 2.69 2.56 Bsp Amu 2.68 1.60 0.85 1.08 2.42 0.85 2.56 Amu Cpe 4.47 3.56 1.97 4.03 4.25 2.69 2.69 1.08 Cpe Rcl 3.74 2.34 1.19 2.51 5.32 2.50 1.19 2.82 2.51 Rcl Csp 5.65 2.68 0.85 1.50 1.50 0.85 2.50 0.41 2.51 2.58 Csp Fsi 6.09 5.34 3.68 5.26 4.26 4.76 4.17 5.11 4.26 3.02 3.27 Kail Sni Apr 6.00 Apr Rpu 4.75 7.46 Rpu Lte 5.67 4.71 3.56 Lte Lpe 3.50 5.56 1.97 3.89 Lpe Por 3.27 2.34 2.42 2.34 2.82 Por Pmo 5.25 4.25 2.93 4.10 4.02 1.33 Pmo Ane 4.42 3.56 2.49 3.56 1.97 1.08 4.01 Ane Fsi 4.42 3.72 2.49 3.72 1.97 1.08 5.65 4.25 Janawai Phu Sar 9.06 Sar Hco 8.17 5.51 Hco Spu 4.26 4.92 4.96 Spu Bbl 6.00 4.91 5.41 4.48 Bbl Afa 6.48 4.55 4.64 4.10 5.07 Afa Pat 4.17 2.94 3.52 2.43 3.84 5.03 Pat Cda 3.68 2.94 3.36 2.87 5.06 2.94 4.62

Legends: Par: Panicum atrosanguineum, Fle: Festuca levengei, Cda: Cynodon dactylon, Bdi: Brachypodium distachyon, Pfa: Poa falconeri, Sim: Schizachyrium impressum, Bsp: Brachypodium sp., Amu: Apluda mutica, Cpe: Cenchrus pennesitiformis, Rcl: Rostraria clarkeana, Csp: Cenchrus sp., Fsi: Festuca simlensis. Sni: Sorghum nitidum, Apr: Arthraxon prionodes, Rpu: Rostraria pumila, Lte: Lolim temulentum, Lpe: Lolium perenne, Por: Pennesitum orientale, Pmo: Polypogon monspeliensis, Ane: Arundinella nepalensis, Phu: Panicum humile, Sar: Sorghum arundinaceum, Hco: Heteropogon contortus, Spu: Setaria pumila, Bbl: Bothriochloa bladhii, Afa: Aristida funiculata, Pat: Panicum atrosanguineum, Cda: Cynodion dactylon.

P>0.05 P>0.01 P>0.001

66 Janawai site At Janawai, grasses showed significant association in comparison with most of the other grasses. Panicum humile showed highly significant association with most of the grasses in comparison specifically with that of Sorghum arundinaceum, Heteropogon contortus, Bothriochloa bladhii, Avena fatua and Panicum atrosanguineum. Sorghum arundinaceum showed highly significant association with H. contortus, Setaria pumila and B. bladhii. A strong association was also observed between H. contortus with that of S. pumila and B. bladhii. Setaria pumila on the other hand showed significant association with B. bladhii and A. fatua. Bothiochloa bladhii showed highly significant association with A. funiculata and C. dactylon. Aristida funiculata and P. atrosanguineum were found in significant association with P. atrosanguineum and C. dactylon respectively (Table 14). Sardari site At sardari, majority of the grasses showed significant association in comparison with the rest of grasses. Poa attenuata showed highly significant association with Aristida mutabilis and Arundinella nepalensis. Festuca kashmiriana showed highly strong association with Cenchrus sp. and A. nepalensis. Aristida mutabilis was found significant with A. nepalensis, Cenchrus pennesitiformis and Cenchrus sp. Panicum atrosanguineum showed highly significant association with Pennesitum orientale and significant with Brachypodium distachyon. Bothriochloa bladhii showed highly significant association with B. distachyon and P. orientalis. Arundinella nepalensis were with significant association with P.orientale, B. distachyon and Cenchus sp. Brachypodium distachyon show significant association with Cenchrus sp. A highly significant association was observed in P. orientale with that of Cenchrus sp. and C. pennesitiformis (Table 15). Halmat site At Halmat site, Panicum decompositum showed significant association with Festuca simlensis, Arthraxon prionodes, Pennesitum orientale and Lolium temulentum. Festuca simlensis showed significant association with Aristida mutabilis and Lolium temulentum. Apluda mutica showed non-significant association with most of the grasses in comparison except Panicum humile.

67 Table 15. Association analysis of some grasses at Sardari, Halmat and Tao Butt, Neelum Valley, Azad Jammu and Kashmir

Sardari Pat Fka 6.51 Fka Amu 6.63 4.95 Amu Pat 4.64 4.44 2.49 Pat Bbl 5.47 4.64 3.07 3.07 Bbl Ane 8.38 6.19 4.01 4.07 3.07 Ane Bdi 4.48 4.12 3.72 5.24 6.75 4.01 Bdi Por 5.39 3.96 3.07 6.63 6.75 4.58 3.56 Por Cpe 5.39 4.64 5.24 3.96 4.58 3.78 3.56 5.08 Cpe Csp 5.07 10.34 5.65 3.78 4.58 4.10 5.24 4.71 4.10 Halmat Pde Fsi 5.08 Fsi Apr 5.56 2.87 Apr Phu 3.09 2.87 4.62 Phu Dcr 3.56 3.07 1.56 1.97 Dcr Por 5.08 3.63 2.43 2.43 3.22 Por Amu 4.25 5.30 2.43 4.02 3.22 3.78 Amu Ane 4.03 3.22 2.69 1.97 2.93 5.65 3.07 Ane Afu 2.94 2.43 2.56 4.62 2.93 2.87 4.02 2.69 Afu Lte 6.62 4.64 3.52 3.36 6.63 4.96 4.96 4.95 3.84 Lte Kma 2.24 1.85 1.19 2.50 2.51 2.57 2.57 1.50 5.25 3.65 Tao Butt Bpe Sfi 2.51 Sfi Pde 2.69 1.19 Pde Afa 2.93 1.50 2.69 Afa Apr 2.51 5.25 2.56 2.69 Apr Por 2.69 1.19 2.56 1.97 2.50 Por Lte 1.50 0.91 1.19 1.50 2.58 5.25 Lte Phu 4.07 4.61 4.25 2.49 4.61 2.50 1.19 Phu Amu 1.08 0.65 2.56 1.08 0.65 2.50 2.50 2.51 Amu Fle 2.42 0.65 0.85 2.42 2.82 0.85 0.85 1.97 2.82 Fle Hco 1.08 0.65 2.87 5.32 2.82 0.85 2.56 1.97 0.65 0.65 Hco Lpe 1.50 4.61 1.56 2.69 6.25 5.25 4.25 1.97 1.08 0.85 2.56 Legends: Pat: Poa attenuata, Fka: Festuca kashmiriana, Amu: Aristida mutabilis, Pat: Panicum atrosanganium Bbl: Bothriochloa bladhii, Ane: Arundinella nepalensis, Bdi: Brachypodium distachyon, Por: Pennesitum orientale, Cpe: Cenchrus pennesitiformis, Csp: Cenchrus sp., Pde: Panicum decompositum, Fsi: Festuca simlensis, Apr: Arthraxon prionodes, Phu: Panicum humile, Dcr: Digitaria cruciata, Por: Pennesitum orientale, Amu: Aristida mutabilis, Ane: Arundinella nepalensis, Afu: Aristida funiculata, Lte: Lolium temulentum, Kma: Koeleria macrantha, Bpe: Bothriochloa pertusa, Sfi: Saccharum filifolium, Pde: Panicum decompositum, Afa: Avena fatua, Apr: Arthraxon prionodes, Por: Poa orientale, Lte: Lolium temulentum, Phu: Panicum humile, Amu: Aristida mutabilis, Fle: Festuca levengei, Hco: Heteropogon contortus, Lpe: Lolium perenne.

P>0.05 P>0.01 P>0.001

68 A significant association was showed by A. mutabilis and Aristida funiculata with P. humile. Digitaria cruciata showed highly significant association with L. temulentum and non-significant association with rest of the grasses in comparison. Pennesitum orientale showed significant association with A. nepalensis and L. temulentum. Aristida mutabilis showed significant association with A. funiculata and L. temulentum. A significant association was also observed in A. nepalensis and Aristida funiculata with L. temulentum and Koeleria macrantha respectively (Table 15). Tao butt At Taobut, Bothriochloa pertusa, Panicum humile and Festuca levengei showed non- significant association with rest of the grasses in comparison. Saccharum filifolium showed significant association with Arthraxon prionodes and P. humile. Aristida funiculata was found significant with Heteropogon contortus only. Arthraxon prionodes showed significant association with P. humile and L. perenne whereas, Lolium temulentum showed non- significant association with most of the grasses in comparison except L. perenne where association was found quite significant (Table 15). 4.3. Morphology

4.3.1. Plant height (cm)

Ananlysis of variance table (F-ratio) for all characteristics is presented in Table 16. Capillipedium parviflorum, Parapholis incurve, Avena byzantina, Sorghum arundinaceum, Panicum humile, Saacharum filifolium, Koeleria macranctha, and Saccharum spontaneum showed the maximum plant height. Cynodon dactylon, Poa nimoralis, Rostraria clarkeana, Poa falconeri, Cenchrus sp., Cenchrus pennisetiformis, and Eleusine indica, showed the minimum plant height (Fig. 8).

4.3.2. Root length (cm)

Saccharum spontaneum, Rostraria pumila, Apluda mutica, Heteropogon contortus and Arthraxon prionodes, showed the maximum root length showing high surface area for water and binding capacity into the soil whereas, Bothriochloa bladhii, Capillipedium parviflorum, and Bothriochloa pertusa, showed the minimum root length showing decreased surface area (Fig. 8).

69 Table 16. Anova of morpho-anatomy and physiological characteristics with F-ratio Root Stem Leaf blade Leaf sheath Characteristics Morphology Physiology anatomy anatomy anatomy anatomy Plant height 402.86** Root length 22.36** Root dry weight 10.12* Shoot dry weight 21.96** Ligule length 34.95** Tiller number 46.47** Leaves Plant-1 45.52** Inf. length 146.80** Spikelet length 49.93** Spikelet number 165.62** Awn length 100.55** Root Sodium 16.75* Shoot Sodium 30.67** Root Potassium 44.81** Shoot Potassium 37.47** Root Calcium 39.59** Shoot Calcium 35.05** Root Magnesium 83.99*** Shoot Magnesium 37.60** Root Phosphorous 16.49* Shoot Phosphorous 24.38** Total chlorophyll 118.27*** Root anatomy Cross sectional area 48.75** 22.97** Hair length 111.72** Epidermal thickness 9.86* 38.31** Cortical thickness 5.85* Cortical cell area 0.01ns 52.37** Endodermal cell area 13.14* Pericyle thickness 25.80** Metaxylem vessel number 65.30** 81.71** Metaxylem area 52.86** 47.39** Pith area 18.86* Pith cell area 31.87** Cross sectional area Epidermis thickness Sclerenchyma thickness 2.74ns 11.73* 23.20** Vascular bundle area 15.88* 31.78** Phloem area 20.26** 19.77* Sheath thickness 10.25* Sheath epidermis cell area 19.20* Blade thickness 25.54** Lower epidermis thickness 16.49* Upper epidermis thickness 1.61ns Mesophyl thickness 7.63* Bundle sheath thickness 11.03* Bundle sheath area 22.91** * = P> 0.05, ** = P>0.01, *** = P>0.001, ns = non -significant

70

Fig. 8. Morphological characteristics (plant height, root length, root and shoot dry weight, ligule length) of grasses of Neelum Valley, Azad Jammu and Kashmir Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

71 4.3.3. Root dry weight (g)

Bothriochloa pertusa, Festuca kashmiriana, Poa argunensis, Panicum atrosanguineum, Digitaria cruciata and Heteropogon controtus showed increased dry weight whereas Milium effusum, Avena fatua, and Saccharum spontaneum showed decrease dry weight (Fig. 8).

4.3.4. Shoot dry weight (g)

A highly significant increase in shoot dry weight was observed in Arundinella nepalensis, Saccharum spontaneum, S. filifolium and Parapholis incurva. A highly significant decrease in shoot dry weight was observed in Brachypodium distachyon, Panicum decompositum, Poa infirma, P. attenuata, and P. nimoralis (Fig. 8).

4.3.5. Ligule length (cm)

Polypogon monspeliensis, Panicum atrosanguineum, Sacchrum filifolium, Saccharum spontaneum, Agrostis pilosa, Sorghum arundinaceum, Eleusine indica and Parapholis incurva showed the maximum ligule length. A signicant decrease in ligule length was observed in Arthraxon prionodes, Cynodon dactylon, Poa argunensis, Poa nemoralis, Festuca levengei and Festuca simlensis (Fig. 8).

4.3.6. Tiller Plant-1

Aritida mutabilis, Hordeum glaucum, Milium effusum, Avena byzantina, Koeleeria macrantha, Poa nemoralis and Eleusine indica showed the maximum number of tillers among all other grasses. In Brachypodium distachyon, Cynodon dactylon, Digitaria cruciata, Panicum atrosanguineum and Festuca kasmiriana a significant decrease in number of tillers was observed (Fig. 9).

72

Fig. 9. Morphological characteristics (tillers, inflorescence length, spikelet length, number of spikelet) of grasses of Neelum Valley, Azad Jammu and Kashmir Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

73 4.3.7. Leaves plant-1 The maximum number of leaves was observed in Parpholis incurva, Capillipedium parviflorum, Sorghum arundinaceum and Panicum humile. Cynodon dactylon, Poa nemoralis and Aristida mutabilis grass showed significant reduction in terms of number of leaves per plant (Fig. 9).

4.3.8. Inflorescence length (cm)

A highly significant increase in terms of inflorescence length was observed in Arundinella bengalensis, Arundinella nepalensis, Saccharum filifolium, Avena byzantine, Brachypodium sylvaticum, Capillipedium parviflorum and Milium effusum. Grasses such as Cynodon dactylon, Rostraria clarkeana, Poa falconeri, Heteropogon contortus and Bothriochloa pertusa showed the minimum length of inflorescence (Fig. 9).

4.3.9. Spike length (cm)

A highly significant increase in spike length was recorded in grasses like Agrostis viridis, Avena fatua, Aristida cynantha, Koeleria macrantha, Brachypodium distachyon, Brachypodium sylvaticum and Lolium perenne. A significant decrease in spike length was noted in rest of the grasses (Fig. 9).

4.3.10. Spikelet numbers

A significant variation in spikelet number was recorded in among tribes and also within the species. In Aveneae, Polypogon monspeliensis show the maximum spikelet numbers among all other tribes. In tribe Paniceae, the maximum numbers of spikelet were recorded in Setaria pumila including other grass such as, Eleusine indica, Aristida mutabilis and Arundinella bengalensis. A significant decreasing trend was observed in tribe Aristideae, Brachypodieae and Poeae showed a considerable increase in spikelet numbers.

Brachypodium distachyon was the grass with minimum spikelet numbers (Fig. 9).

4.3.11. Multivariate cluster analysis

Multivariate analysis for morphological data matrix clustered the species into two main clades and sub clades. The first clade is further divided into two sub-clades where

74 Saccharum filifolium and Saccharum spontaneum, Sorghum arundinaceum and Sorghum nitidum were sister species, which showed close relationship with Pennesitum orientale and Schizachyrium impressum. In second sub-groups of the first clade, Arundinella sp. and Arundinella nepalensis were sister species that showed a very close relationship to Hordeum galacum and Hordeum marinum shared some common morphological characteristics. In second clade, Heteropogon contortus stems into a single branch showing less similarity with the rest of the species in the group. Apluda mutica and Arthraxon prionodes showed close association with two Brachypodium grasses that were sister with each other. However on the morphological bases, Poa nemoralis was much similar in characteristics with Cynodon dactylon than rest of the Poa grasses. Rostraria clarkeana showed a close relationship with Aristida mutabilis in the last group (Fig. 10).

4.4. Anatomy 4.4.1. Root anatomy

4.4.1.1. Root cross sectional area (µm2)

All root anatomical characteristics were found statistically significant (Table 16) at (P> 0.05, 0.01, 0.001). Root cross sectional area significant increased in some particular species among the tribes as it was the maximum in Arundinella bengalensis, Festuca levengei, Heteropogon contortus, Festuca kasmiriana, Schizachyrium impressum, Cynodon dactylon, Koeleria cristata and Lolium temulentum. A highly decreased root cross sectional area was observed in Bothriochloa blahdii, Aristida mutabilis, Avena fatua, Brachypodium sp., Hordium glacum, Poa argunensis, Digitaria cruciata, and Hordeum glaucum grass (Fig. 11).

4.4.1.2. Root hair length (µm)

Arista mutabilis showed highly increased root hair length among the all rest of the grasses showing the high surface area for water absorption. Other grasses where root length significantly increased were Saccharum filifolium, Poa nemoralis, P. infirma, P. nemoralis, Festuca kashmiriana, Capillipedium parviflorum, and Bothriocholoa pertusa. A highly decrease root length was observed in Heterpogon contortus, Polypogon monspeliensis, and Aristida funiculata which showed the decreased surface area for water absorption (Fig. 11).

75

Fig. 10. Multivariate cluster analysis of the morphological characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir

76 4.4.1.3. Epidermal thickness (µm)

A highly increased epidermal thickness was observed in Cenchrus pennisetiformis, Avena fatua, Saccharum spontaneum, and Lolium perenne that may be an adaptation to protect internal plant tissues from harsh outer environment and to minimize the water loss. A less decrease epidermal thickness was observed in Arthraxon prionodes, Bothriochloa pertusa, Koeleria macrantha, Brachypodium sp., B. distachyon, Pennesitum orientale, Panicum decompositum, Poa nemoralis and P. infirma (Fig. 11).

4.4.1.4. Cortical thickness (µm)

A significant increase in cortical thickness was observed in Rostraria clarkeana, Saccharum filifolium, Arundinella nepalensis, Capillipedium parviflorum, Panicum humile, Digitaria cruciata and.Pennesitum orientale. This increase in the thickness of cortical region is an important strtedgy of many desserts and temperate grasses for the food storage and water conservation. Comparitively to the other grasses, to these grasses, Aristida mutabilis, Brachypodium distachyon, Cynodon dactylon, Cenchrus pennisetiformis and Panicum decompositum showed significant decrease in cortical thickness region (Fig. 12).

4.4.1.5. Cortical cell area (µm2)

Root cortical cell area was the only characteristic that was found statistically non- significant among all other parameters (Table 16). A highly increased cortical cell area was recorded in Pennisetum orientale, Poa attenuata, Lolium temulentum, Avena fatua, Cynodon dactylon, Sorghum arundinaceum, L. perenne, Poa infirma and Rostraria clarkeana grass. A significant decrease in cortical cell area was prominent in grasses such as, Arthraxon prionodes, Panicum decompositum, Agrostis viridis, Saccharum spontaneum, Eleusine indidca, Brachypodium sylvaticam, and Setaria pumila (Fig. 12).

77

Fig. 11. Root anatomical characteristics (RCSA, root hair length, epidermal thickness) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

78

Fig. 12. Root anatomical characteristics (cortical area, thickness, endodermal cell area, pericyle thickness) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

79 4.4.1.6. Endodermal cell area (µm2) Brachypodium sp., Poa nemoralis, Agrostis viridis, Sorghum nitidum and Poa attenuata and Lolium perenne showed the maximum root endodermal cell area enhancing the radial flow of water. Bothriochloa blahdii, Aristida funiculata, and Sorghum arundinaceum showed the minimum endodermal cell area (Fig. 12).

4.4.1.7. Pericyle thickness (µm)

A significant increase was observed in pericyle thickness in Panicum humile, Avena byzantina, Bothriochloa pertusa, Poa falconeri and Cenchrus pennisetiformis comparatively to the other grasses where root pericyle thickness significantly decreased (Fig. 12).

4.4.1.8. Metaxylem vessel number

A significant increase in metaxylem vessel number was observed in Brachypodium distachyon, Cenchrus sp., Poa arugensis, Panicum humile, Koeleria macrantha, Poa infirma, Cynodon dactylon, Agrostis viridis, Parapholis incurve, and Festuca simlensis that confer the maximum absorption of water. A highly decrease metaxylem vessel numbers was recorded in grasses mainly that of Lolium temulentum, Heteropogon contortus, and Apluda mutica (Fig. 13).

4.4.1.9. Metaxylem cell area (µm2)

A significant increase in metaxylem cell area was observed in Capillipedium parviflorum, Panicum decompositum, P. atrosanguineum, Arundinella nepalensis, Avena byzantina, and Saccharum filifolium. Metaxylem cell area specifically in Rostraria pumila, Polypogon monspeliensis, Festuca kashmirian, Saccharum spontaneum and Bothriochloa pertusa showed a significant decrease in this parameter (Fig. 13).

4.4.1.10. Pith area (µm)

A highly developed pith region was recoreded in Agrostis pilosa, Aristida funiculata and Poa nemoralis where most of the grasses in all tribes showed significant decrease in pith area mainly that of Festuca levengei, F. simlensis, Pennesitum orientale, Poa infirma, Sorghum arundinaceum, Aristida cynanatha and A. mutabilis (Fig 13).

80

Fig. 13. Root anatomical characteristics (metaxylem number and area, pith area, pith cell area) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

81 4.4.1.11. Pith cell area (µm2)

Avena byzantine, Aristida funiculata, Poa falconeri, and Sorghum arundinaceum showed the maximum pith cell area. Whereas, Aristida mutabilis, Apluda mutica, Eleusine indica, Poa argunensis, and Hordeum marinum showed highly decreased pith cell area (Fig. 13).

4.4.1.12. Multivariate cluster analysis

Based on root anatomical data matrix, multivariate cluster analysis showed distinct grouping among all of the grasses. Hordeum marinum and Cenchrus sp., stem into a single branch showing less similarities with other species. Arundinella nepalensis, Eleusine indica, and Panicum humile were grouped together showing close relationship.

In adjacent clade, Avena fatua showed a close relationship with Festuca kashmiriana. Arundinella sp., showed close relationship with Cenchrus pennisetiformis clustered together, was in close relation with grasses presented in other groups. Poa attenuata and P. nemoralis were sister species showed a close relationship with Schizachyrium impressum, Setaria pumila, and Apluda mutica, Sorghum nitidum, Saccharum spontaneum, and Aristida funiculata.

Poa falconeri was in close relationship with Lolium temulentum, Koeleria argentea, Panicum decompositum, and Digitaria cruciata. Poa infirma showed close relationship with Capillipedium parviflorum, and both were shared some common characteristics with rest of the clade. Arthraxon prionodes and Aristida mutabilis group together showed close relationship (Fig. 14).

82

Fig. 14. Multivariate cluster analysis of the root anatomical characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir

83 4.4.2. Stem anatomy

4.4.2.1. Stem cross sectional area (mm2)

All stem anatomical characteristics were found statistically significant (Table 16) at (P> 0.05, 0.01, 0.001). A highly significant increase in stem cross sectional area was recoreded in Arundinella bengalensis, A. nepalensis, Hordeum glacum, Apluda mutica, Arthraxon prionodes, Aristida funiculata, and Hordeum marinum. In contrast, a decreased trend was also observed in many of the grass in stem cross sectional area where this parameter significantly show reduction in grass mainly that in Poa infirma, Panicum decompositum, Bothriochloa pertusa and Aristida mutabilis (Fig. 15).

4.4.2.2. Epidermal thickness (µm)

Most of the grass in within tribes showed generally a decreasing trend in epidermal thickness. It showed highly significant increase in Pennisetum orientale, Poa nemoralis, Brachypodium sp., Sorghum arundinaceum, Heterpogon contortus, Bothriochloa pertus, B. bladhii, Avena byzantina and Koeleria macrantha less favourable environment a high evaluation. On the other hand, Arundinella bengalensis, Milium effusum and Setaria pumila a highly reduced epidermal thickness was recorded in these grasses conferring the more favourable environment a lower altitude (Fig. 15).

4.4.2.3. Sclerenchyma thickness (µm)

Apluda mutica, Parpholis incurva, Poa falconeri, and Arundinella bengalensis showed highly increased sclerenchyma thickness. Poa infirma, Sorghum nitidum and most of the grasses in Paniceae tribe showed highly reduced sclerenchyma thickness comparatively to the other grasses within tribes. Intensive sclerification in most of the grasses might be an adaptive mechanism to cope with low temperature and other environment stresses (Fig. 15).

84

Fig. 15. Stem anatomical characteristics (SCSA, epidermal and sclerenchyma thickness, cortical cell area) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

85 4.4.2.4. Cortical cell area (µm2)

A highly developed cortical cell area was recorded in most of the grass such as Saccharum spontaneum, Agrostis viridis, Lolium perenne, Brachypodium distachyon, Brachypodium sp., Schizichyrium impresum and Poa falconeri grass. Many other grasses within the tribes such as Bothriochloa pertusa, Aristida funiculata, Cynodon dactylon, Festuca simlensis and Milium effusum etc. show highly decreased stem cortical area (Fig. 15).

4.4.2.5. Vascular bundle area (µm2)

A significant increase in vascular bundle area was observed in Arundinella nepalensis, Saccharum filifolium, S. spontaneum, Sorghum nitidum and Aristida funiculata. Rest of the grasses showed a significant decrease in terms of vascular bundle area (Fig. 16).

4.4.2.6. Metaxylem area (µm2)

Metaxylem area significantly increased in Poa attenuata, Parapholis incurva, Rostraria clarkeana, and Arthraxon prionodes. A highly decreased metaxylem area was recorded in Setaria pumila, Saccharum spontaneum, Schizachyrium impressum, and Sorghum arundinaceum (Fig. 16).

4.4.2.7. Phloem area

Phloem area greatly increased in Brachypodium sp., B. distachyon, Rostraria clarkeana, and Bothriocloa bladhii. A highly reduced phloem area was observed in Festuca simlensis grass (Fig. 16).

4.4.2.8. Multivariate cluster analysis

In this analysis, Aristida cynantha stems into a single branch where, Rostraria pumila shared some common characteristics with Saccharum spontaneumand Hordeum marinum. Milium effusum and Cenchrus pennisetifromis were sister species that were closely related to Festuca levengei. Avena byzantina and Apluda mutica were closely related to each other; both were in close relationship with other grasses in rest of the clades.

86

Fig. 16. Stem anatomical characteristics (vascular bundle and metaxylem area, phloem area) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

87 Poa argunensis and P. infirma form were closely related with pennisetum orientale, Hordeum glacum, and Heteropogon contortus. Brachypodium sylvaticum and B. distachyon closely related grasses were closely related to Aristida mutabilis. Koeleria macrantha was closely related with Agrostis viridis. Poa attenuata was closely related with Lolium temulentum. Digitaria cruciata, Parapholis incuva, and Bothriochloa bladhii were closely related grasses. Polypogon monspeliensis showed a close relationship with Cenchrus sp. (Fig.17).

4.4.3. Leaf sheath anatomy

4.4.3.1. Sheath thickness (µm)

All leaf sheath characteristics were found statistically significant (Table 16) at (P> 0.05, 0.01, 0.001). A geneally increasing trend was observed in most of the grasses like Heteropogon contortus, Saccharum filifolium, Cynodon dactylon, and Hordeum glacum which showed the maximum sheath thickness. The grass Arthraxon prionodes, Apluda mutica, and Saccharum spontaneum showed a significant reduction in terms of leaf sheath thickness (Fig. 18).

4.4.3.2. Sclerenchyma thickness (µm)

A significant increase in sclerenchyma thickness was recorded in Eleusine indica, Aristida cynantha, Hordeum marinum, Poa infirma, P. attenuata, Lolium temulentum, and Panicum humile. Avena fatua, Brachypodium sp., Hordeum glacum, and Saccharum filifolium showed a significant decrease in sclerenchyma thickness (Fig. 18).

4.4.3.3. Epidermal cell area (µm2)

A significant increase in epidermal cell area was recorded in majority of the grasses mainly that of Festuca simlensis, F. levengei, and Poa nemoralis conferring high potential of these grasses towards water conservation strategies. Arthraxon prionodes, Apluda mutica, Arundinella nepalensis, and Poa falconeri showed a considerable decrease in epidermal cell area (Fig. 18).

88

Fig. 17. Multivariate cluster analysis of the stem anatomical characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir

89

Fig. 18. Sheath anatomical characteristics (sheath, schlerenchyma thickness, epidermal cell area and vascular bundle area) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

90 4.4.3.4. Vascular bundle area

A significant increase in vascular bundle area was observed in Saccharum filifolium, Lolium perenne, Panicum humile, Koeleria macrantha, Arundinella macrantha, and Poa argunensis. Vascular bundle area significantly decreases in most of the grasses especially that of Arthraxon prionodes, Festuca simlensis, F. levengei, Agrostis viridis, and Avena byzantina (Fig. 18).

4.4.3.5. Multivariate cluster analysis

Multivariate cluster analysis showed a very distinct clustering on the basis of leaf sheath data matrix Lolium temulentum formed the first branch, representing some distinctive sheath characteristics. Bothriocholoa pertusa showed a close relation with Panicum humile and Agrostis viridis. Brachypodium distachyon showed close relationship with Apluda mutica, Arundinella sp., and Saccharum spontaneum. Arthraxon prionodes showed sister relationship with Milium effusum, where both grass showed a closed relationship with other grasses in the clade. All the Poa species were clustered together in the same clade showing close relationship except Poa falconeri that was in close association with Koeleria macrantha, and Elusine indica, where E. indica formed a single branch.

Heteropogon contortus was closely related with Digitaria cruciatea, and Arundinella bengalensis. Cynodon dactylon, Rostraria clarkeana, and Cenchrus sp., shared some common characteristics were cluster together. Steria pumila, Polypogon monspeliensis, Rostraria pumila, Sorghum arundinaceum, Hordeum glacum, Festuca levengei, Brachypodium sp., Sorghum nitidium, Bothriochloa bladhii were group together in the same clade were closely associated with each other. Festuca kashmiriana, Capillipedium parviflorum, Saccharum filifolium, Parapholis incuva, Cenchrus pennisetiformis, Schizachyrium impressum, Panicum atrosanguineum, and Avena byzantina were clustered together showed closed relationship (Fig. 19).

91

Fig. 19. Multivariate cluster analysis of the leaf sheath anatomical characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir

92 4.4.4. Leaf blade anatomy

4.4.4.1. Leaf blade thickness (µm)

All leaf blade characteristics were found statistically significant (Table 16) at (P> 0.05, 0.01, 0.001). A highly significant increase in leaf blade thickness was observed among tribes and within the grasses which might be an important adaptation of temperate and alpine grasses to cope with low temperature stress. Sorghum arundinaceum, Cenchrus sp., Poa attenuata, P. falconeri, Poa nemoralis and Lolium temulentum showed highly increase thickness in leaf blade. Capillipedium parviflorum, Festuca simlensis, F. levengei, Brachypodium sp., and Panicum atrosanguineum however showed a considerable reduction in blade thickness (Fig. 20).

4.4.4.2. Lower epidermal thickness (µm)

A significant, highly developed lower epidermal thickness was recorded in Cenchrus pennisetiformis, Saccharum spontaneum, and Avena fatua. Rest of the grasses among different tribes showed comparatively less developed epidermal area (Fig. 20).

4.4.4.3. Upper epidermal thickness (µm)

A significant increasing trend in terms of upper epidermal thickness was observed in most of the grass species especially in Arundinella nepalensis and Sorghum arundinaceum. Grasses such as Bothriochloa bladhii, B. pertusa, Capillipedium parviflorum, and Hordeum glacum showed greatly reduce upper epidermal thickness (Fig. 20).

4.4.4.4. Sclerenchyma thickness (µm)

Extensive sclerification was observered in Festuca kashmiriana, Arundinella nepalensis, Poa argunensis and Sorghum arundinaceum which is the characteristic feature of most of the grasses under abiotic stresses such as aridity, low temperature and salt stress. Saccharum filifolium, Arundinella bengalensis, Panicum decompositum and Setaria pumila showed highly reduced sclerenchyma thickness (Fig. 20).

93

Fig. 20. Leaf anatomical characteristics (blade, lower and upper epidermal thickness, sclerenchyma area, mesophyll thickness,) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

94 4.4.4.5. Mesophyll thickness (µm)

A highly increased mesophyll thickness was observed in most of the grasses, specifically that of Koeleria cristata, Lolium temulentum, and Digitaria cruciata. On the other hand, Hordium marinum, Panicum atrosanguineum and Cenchrus pennisetiformis showed highly reduced mesophyll thickness (Fig. 20).

4.4.4.6. Bundle sheath thickness (µm)

A significant increase in bundle sheath cell was observed in Cenchrus sp., C. pennisetifomis, Lolium perenne, L. temulentum, Saccharum arundinenaceum and

Heteropogon contortus that is characteristic feature of C4 grasses. Parapholis incurva and Panicum atrosanguineum comparatively showed less developed bundle sheath thickness (Fig. 21).

4.4.4.7. Vascular bundle area (µm2)

Poa infirma, P. nemoralis, Hordeum glacum, and Agrostis viridis showed highly increased vascular bundle area conferring the successful adaptation of these grasses in water deficient soil. A highly decreasing trend was observed in rest of the grasses within tribes (Fig. 21).

4.4.4.8. Metaxylem vessel area (µm2)

A significant increase in metaxylem vessel area was observed in Heteropogon contortus, Arundinella nepalensis, Lolium temulentum, and Brachypodium distachyon that help them high rate of conduction of water. A remarkable decrease in metaxylem area was recoreded in many of the grasses mainly Poa infirma Festuca levengei, Cynodon dactylon, Brachypodium sylvaticum, Aristida mutabilis and Arundinella bengalensis which showed highly reduced metaxylem area (Fig. 21).

95

Fig. 21. Leaf anatomical characteristics (bundle sheath thickness, vascular bundle cell area, metaxylem area, bulliform cell and phloem area) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

96 4.4.4.9. Phloem area (µm2)

Sorghum arundinaceum, Lolium temulentum, Poa infirma, Parapholis incurva and Festuca kashmiriana showed highly increased phloem area. Rostraria pumila, Koeleria macrantha, Arundinella bengalensis are the grasses which showed a considerable reduction in phloem area (Fig. 21).

4.4.4.10. Bulliform cell area (µm2)

A highly significant increase in bulliform cell area was observed in Brachypodium sp., Milium effusum, Polypogon monspeliensis, Bothriochloa bladhii, Aristida funiculata, and Agrostis pilosa grasses that play a very important role in turgor changes and help plant in respiration process. On the other hand, most of the grasses especially Capillipedium parviflorum, Avena fatua and Brachypodium sylvaticum etc. grasses showed highly reduced bulliform cell area (Fig. 21).

4.4.4.11. Adaxial stomatal area (µm2)

A highly significant increase in adaxial stomatal area was recorded in Sorghum nitidum, Hordeum glacum, H. marinum, Cenchrus sp., Rostraria pumila and rest of the most of the grasses. Whereas, in Koeleria macrantha and Polypogon monspeliensis grass it was great reduced (Fig. 22).

4.4.4.12. Abaxial stomatal area (µm2)

Abaxial stomatal area greatly increased in Lolium perenne, Cenchrus sp., Eleusine indica, and Saccharum spontaneum. On the other hand, a remarkable reduction in abaxial stomatal area was observed in Arundinella nepalensis and Cenchrus pennisetiformis (Fig. 22).

4.4.4.13. Adaxial stomatal number

A remarkable, highly significant increase in terms of stomatal number was observed in Rostraria clarkeana and Aristida cynantha on the adaxial side of the leaf. On the other hand, most of the grasses showed a highly decreasing trend in stomatal number on adaxial side of the leaf blade (Fig. 22).

97

Fig. 22. Leaf anatomical characteristics (adaxial and abaxial stomatal area and number) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

98 4.4.4.14. Abaxial stomatal number

Schizachyrium impressum, Cenchrus sp., Agrostis viridis, Brachypodium distachyon and Poa argunensis grass showed the maximum stomatal number on the abaxial side of the leaf surface. Aristida funiculata, Hordeum galaucum and Poa nemoralis showed highly reduced stomatal numbers that might be an adaption to check the water loss and retain maximum turgidity inside the tissues (Fig. 22).

4.4.4.15. Multivariate cluster analysis

Multivariate cluster analysis showed a very distinct clustering on the basis of leaf blade data matrix Bothriochloa bladhii formed the first branch, indicating some distinctive leaf blade characteristics. Festuca cruciata, Digitaria cruciata, and Cynodon dactylon were clustered into same group showing close relationship. Poa nemoralis was sister grass to P. falconeri. Rostraria clarkeana and R. Pumila grouped together showed a close association with Parpholis incuva and rest of the grasses in the clade. Both Sorghum nitidum and S. arundinaceum was sister species showed a close relation with Schizachyrium impressum and Aristida funiculata.

Hordeum glacum showed a close relationship with H. marinum and Heteropogon contortus. Poa infirma and P. argunensis were sister grasses and showed a close relationship with Panicum decompsitum, Lolium temulentum and Brachypodium distachyon. Both Bothriochloa pertusa and Pennisetum orientale form a single separate branch showing less similarity of leaf blade characteristics with other grasses. Avena fatua and A. byzantina clustered into single clade showed closed relationship with Capillipedium parviflorum, and rest of the grasses within clade. Panicum atrosanguineum and Koeleria argentea showed fewer similarities with rest of the species in the same clade. Poa attenuata was clustered with Agrostis viridis (Fig. 23).

99

Fig 23. Multivariate cluster analysis of the leaf blade anatomical characteristics of grasses of Neelum Valley, Azad Jammu and Kashmir

100 4.4.4.16. Leaf structural modifications in grasses

In tribe Andropogoneae, Apluda mutica short slightly smaller angular prickles at the margin of the leaf in intercostals zone. Long cells with slightly sinuous walls with sharply pointed micro hairs were also observed in the coastal and intercostals regions. Saddle or rounded shape silica bodies were observed at both costal and intercostal zones. Leaf surface was not covered with dense hairs. No distinct ribs and ridges were distinct at abaxial surface. In Arthraxon prionods, saddle shaped silica bodies were present at intercostals zone at both abaxial and adaxial surface of the leaf. In coastal zone star shaped silica bodies were present with slightly sinuous walls. No distinct ribs and ridges were distinct at the abaxial surface. Leaf surface was found with densely packed hairs (Fig. 24).

Bothriochloa bladhii grass showed the increase number of hairiness at leaf surface area. Adxial surface was almost flat with no distinct ribs and ridges micro hairs and prickles were not prominent at both coastal and inter coastal zone and at the margin of the leaf. Both coastal and intercoastal zones showed long cells with slightly sinuous walls. Bothriochloa pertusa showed relatively short cells and saddle shape silica bodies in coastal and inter coastal zone.

Micro hairs were usually absent. Leaf area hairiness was greatly reduced in case of this grass. Capillipedium parviflorum showed highly increased and pointed micro hairs and prickles at the both sides of adxial and abxial surfaces of the leaf. Micro hairs were also prominent at both coastal and intercostal zones with slightly sinuous walls. Both X and saddle shape silica bodies were present. Adaxial and abxial surfaces of the leaf showed high number of ribs and ridges.

At leaf surface increased number of hairiness was observed. Heteropogon Contortus showed increased leaf hairiness, usually long hairs but less in numbers at coastal and intercostals region. Round shaped silica bodies were seen with highly sinuous walls. Angular prickles were more prominent at adxial surface of the leaf (Fig. 24-25).

101

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Apluda mutica Arthraxon prionodes Bothriochloa bladhii Fig. 24. Anatomical characteristics of some grasses of tribe Andropogoneae from the Neelum Valley, Azad Jammu and Kashmir

102

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Bothriochloa pertusa Capillipedium parfviflorum Heteropogon contortus Fig. 25. Anatomical characteristics of some grasses of tribe Andropogoneae from the Neelum Valley, Azad Jammu and Kashmir

103 Saccharum Simlensis showed highly pointed prickles at coastal zones which were usually absent in Saccharum Spontaneum. In S. Spontaneum saddle shaped silica bodies with some time highly sinuous wall were present but in S. Simlensis silica bodies were absent. No distinct ribs and ridges were present in both grasses. Schizachyrium impressum micro hairs and prickles were absent at both abaxial and adaxial surface. Both coastal and intercostal zones were distinct with usually short cells. Smaller sharply pointed hairs were present at the margins of the leaf. In Sorghum arundinaceum, densely packed hairiness was recorded with small prickles at the margins of leaf. Sorghum natidium showed the maximum number of saddle shaped silica bodies at coastal zone with densely packed hairiness (Fig. 25-27).

In tribe Aristideae, no distinct ribs and ridges were observed in Aristide Cynantha. Micro hairs and prickles were usually absent and leaf hairiness was greatly reduced. Whereas in Aristideae funiculate, prickles and microhairs were very prominent saddle shaped silica bodies were highly present at coastal zone. The walls of the cells were highly sinuous. Leaf hairiness was greatly reduced in this grass. On the other hand Artida mutabilis showed long sharply pointed prickles with prominent sinuous walls at both coastal and intercostal. No distinct leaf hairs were observed at the leaf surface (Fig. 28).

Grasses in Arundineae tribe, highly sinuous wall, large size of angular prickles and micro hairs were observed in most of the species. Comparatively large saddle shaped silica bodies were present, distinct ribs and ridges were observed at abaxial and adaxial surface of Arundinella bengalensis. Higly reduced prickles and micro hairs were observed in A. Nepalensis with sharp microhiars at leaf margins were observed in this grass (Fig. 29).

In Aveneae tribe, small angular prickles and distinct ribs and ridges were recorded in Agrostis veridis and Agrostsis pilosa with long, slightly sinuous walled cells at coastal and intercostal zones. In Avena byzantine, highly increased micro hairs and prickles at both coastal and intercostal zones and also at abxial and adxial surface of the leaf were observed. Leaf hairiness was greatly reduced in this grass as compared to Avena fatua, whereas, densely packed hairiness was recorded throughout the leaf surface.

104

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Saccharumm filifolium Saccharum spontaneum Schizachyrium impressum Fig. 26. Anatomical characteristics of some grasses of tribe Andropogoneae from the Neelum Valley, Azad Jammu and Kashmir

105

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Sorghum arundinaceum Sorghum nitidium Cynodon dactylon Fig. 27. Anatomical characteristics of some grasses of tribe Andropogoneae and Cynodonteae from the Neelum Valley, Azad Jammu and Kashmir

106

Root

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Aristida cyanantha Aristida funiculata Aristida mutabilis Fig. 28. Anatomical characteristics of some grasses of tribe Aristideae from the Neelum Valley, Azad Jammu and Kashmir

107

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Arundinella bengalensis Arundinella nepalensis Eleusine indica Fig. 29. Anatomical characteristics of some grasses of tribe Arundineae and Eragrostideae from the Neelum Valley, Azad Jammu and Kashmir

108 A highly pointed keel was also observed in A. Fatua with slight ribs and ridges. No prominent ribs and ridges were observed in Koeleria Cristata and K. Nacratha. Sharply pointed prickles and microhairs were also prominent in polypogon monspeliensis with highly packed hairiness. Both Rostraria clarkeana and Rostraria pumila showed reduced hairiness. A distinct ribs and ridges were present in R. clarkeana (Fig. 30, 31, 32).

Grasses of tribe Brachypodeae, showed highly increased hairiness i.e. Brachypodium distachyon throughout the leaf surface. A highly prominent ribs and furrows were observed in brachypodium Sylvaticum. No distinct ribs, ridges hairiness were observed in B.distachyon and Brachypodium sp. In tribe Cynodonteae and Eragrostideae, Cynodon dactylon and Eleusine indica showed highly reduced hairiness with no prominent ribs and ridges at the abaxial and adaxial surface of the leaf (Fig. 27, 29). In the tribe Paniceae, Cenchrus pennesitiformis, Cenchrus sp., Digitaria cruciata, Panicum atrosanguineum, P. decompoitum and P. humile showed highly pointed angular bicelled prickles and micro hairs at the leaf margins that is characteristic feature of this tribe. Pennesitum Orientale and Setaria pumila showed relatively reduced hairiness but with long hairs and highly sinuous walls (Fig. 33, 34, 35, 36).

In tribe Poeae, Festuca Kashmiriana showed highly sinuous walls at coastal and intercoastal zones with round shaped silica bodies. Festuca levengei and Festuca Simlensis showed highly pointed small micro hairs at coastal zones. Leaf hairiness greatly reduced in both grasses. In Lolium perenne, Lolium temulentum and Milium effusum leaf hairiness highly increased. Both coastal and intercoastal zones showed long cells with highly sinuous walls, distinct ribs and ridges with angular prickles and micro hairs. No prominent ribs and ridges with highly sinuous walls at coastal and intercostal zones were observed in Parpholis incurve. Poa argunensis showed small pointed mircohairs at costal and inter costal zones with reduced hairiness at leaf surface. Distinct ribs and ridges with small microhairs were prominent in poa nemoralis and Poa infirma. No prominent ribs and ridges were observed in P. falconeri, P. attenuata and P. argunensis (Fig. 37, 38, 39, 40).

109

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Abaxial Abaxial epidermis

Adaxial Adaxial epidermis

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Agrostis pilosula Agrostis viridis Avena byzantina Fig. 30. Anatomical characteristics of some grasses of tribe Aveneae from the Neelum Valley, Azad Jammu and Kashmir

110

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Leaf Leaf

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Abaxial Abaxial epidermis

Adaxial Adaxial epidermis

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Avena fatua Koeleria cristata Koeleria macrantha Fig. 31. Anatomical characteristics of some grasses of tribe Aveneae from the Neelum Valley, Azad Jammu and Kashmir

111

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Polypogon monspeliensis Rostraria clarkeana Rostraria pumila Fig. 32. Anatomical characteristics of some grasses of tribe Aveneae from the Neelum Valley, Azad Jammu and Kashmir

112

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Brachypodium distachyon Brachypodium sp. Brachypodium sylvaticum Fig. 33. Anatomical characteristics of some grasses of tribe Brachypodieae from the Neelum Valley, Azad Jammu and Kashmir

113

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Abaxial Abaxial epidermis

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Cenchrus pennisetiformis Cenchrus sp. Digitaria cruciata Fig. 34. Anatomical characteristics of some grasses of tribe Paniceae from the Neelum Valley, Azad Jammu and Kashmir

114

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Panicum atrosanguineum Panicum decompositum Panicum humile Fig. 35. Anatomical characteristics of some grasses of tribe Paniceae from the Neelum Valley, Azad Jammu and Kashmir

115

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Pennisetum orientale Setaria pumila Fig. 36. Anatomical characteristics of some grasses of tribe Paniceae from the Neelum Valley, Azad Jammu and Kashmir

116

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Festuca kashmiriana Festuca levingei Festuca simlensis Fig. 37. Anatomical characteristics of some grasses of tribe Poeae from the Neelum Valley, Azad Jammu and Kashmir

117

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Abaxial Abaxial epidermis

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Lolium perenne Lolium temulentum Milium effusum Fig. 38. Anatomical characteristics of some grasses of tribe Poeae from the Neelum Valley, Azad Jammu and Kashmir

118

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Parapholis incurva Poa argunensis Poa attenuata Fig. 39. Anatomical characteristics of some grasses of tribe Poeae from the Neelum Valley, Azad Jammu and Kashmir

119

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Poa falconeri Poa infirma Poa nemoralis Fig. 40. Anatomical characteristics of some grasses of tribe Poeae from the Neelum Valley, Azad Jammu and Kashmir

120 In Triticeae tribe, Hordeum glaucum showed highly pointed microhairs at costal zones with round shaped silica bodies. Leaf hairiness was highly increased in Horeum glaucum as compared to H. marinum. No prominent ribs and ridges were observed in both grasses (Fig. 41).

4.5. Physiology

4.5.1. Root Sodium (mg g-1)

All physiological characteristics were found statistically significant (Table 16) at (P> 0.05, 0.01, 0.001). Root sodium significantly varies among the tribes and within the species. In Andropongoneae tribe, Hetropogon controtus, Arundinella bengalensis, Arundinella nepalensis and Schizachyrium impresum, Capillipedium parviflorum and Bothriochloa pertusa accumulated the maximum amount of sodium in roots. In Arundineae both showed the maximum sodium in their roots. Grasses from the rest of the tribes mainly Cynodon dactylon, Festuca kashmirian, Festuca levingei, Koeleria macrantha, Lolium temulentum, and Poa infirma also showed high root sodium contents. Avena fatua, Poa argunensis, Hordeum glacum, Digitaria cruciata, Bothriochloa bladhii accumulate relative small amount of sodium contents (Fig. 42)

4.5.2. Shoot Sodium (mg g-1)

Variation in shoot sodium content was recoreded relatively high ranging from the maximum value in Cynodon dactylon, and the minimum value in Brachypodium distachyon. Other species mainly Brachpodium sp., Festuca simlensis and Aristida mutabilis accumulated high amount of sodium in shoots then rest of the species (Fig. 42).

4.5.3. Root Potassium (mg g-1)

Polypogon monspeliensis, Avena fatua, Saccharum filifolium, Koeleria cristata and Milium effusum were the grasses with the maximum root K+ content. Arundinella bengalenis, Lolium temulentum and Pennisetum orientale, were the grasses that accumulate relatively small amount of K+ in roots. Grasses within tribe Agrostideae and Arundineae comparatively showed less K+ content in contrast to the grasses in the tribes (Fig. 42).

121

Root

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Abaxial Abaxial epidermis

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Hordeum glaucum Hordeum marinum Fig. 41. Anatomical characteristics of some grasses of tribe Triticeae from the Neelum Valley, Azad Jammu and Kashmir

122

Fig. 42. Root and shoot physiological characteristics (sodium and potassium) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

123 4.5.4. Shoot Potassium (mg g-1)

Majority of the grasses accumulated high concentration of K+ in their shoots. Aristida mutabilis has the maximum shoot K+, whereas Heteropogon contortus retain the minimum concentration of K+ in shoots. No significant variation in shoot K+ can be observed among the grasses of tribe Paniceae and Poeae (Fig. 42).

4.5.5. Root Calcium (mg g-1)

Grasses within the tribes accumulated relatively less amount of Ca2+ as compared to the shoot Ca2+ except Heteropogon contortus, Poa infirma which showed higher amount of root Ca2+ content in their roots. Avena byzantina, Lolium temulentum, Koeleria macrantha, Poa nimuralis, and Capillipedium parviflorum showed the less amount of Ca2+ in roots (Fig. 43).

4.5.6. Shoot Calcium (mg g-1)

Festuca levingei, Poa attenuata, Bothriochloa pertusa, Hordeum marinum, Sorghum nitidum, Elusine indica, and Festuca simlensis showed the maximum Ca2+ content in shoots. Arundinella bengalensis, Koeleria macrantha, Lolium temulentum, and Capillipedium parviflorum on the other hand, retain the minimum amount of Ca2+ in shoots (Fig. 43).

4.5.7. Root Magnesium (mg g-1)

Root Mg2+ showed significant variation within species. The maximum root Mg2+ content was found in Bothriocholoa pertusa that was followed by Festuca simlensis, Aristida mutabilis, Polypogon monspeliensis, Festuca levengei, Lolium temulentum, Festuca kashmiriana, Poa infirma, Rostraria clarkeana, and Eleusine indica. Saccharum species i.e. S. spontaneum and S. bengalensis showed the minimum root Mg2+ root contents (Fig. 43).

124

Fig. 43. Root and shoot physiological characteristics (calcium and magnesium) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

125 4.5.8. Shoot Magnesium (mg g-1) A significant increase in shoot Mg2+ content was observed within tribes and among species. Bothriocholoa pertusa, Lolium temulentum, Aristida mutabilis, Poa infirm, Bothriocholoa bladhii, Eleusine indica, Paraphalis incurve, Poa nimuralis, Arundinella bengalensis, Hordeum galcum, Arthraxon prionodes, Pennesitum orientale, Festuca simlensis, Festuca levengei, Setaria pumila, and Arundinella nepalensis, showed the maximum Mg2+ content in root. A significant decrease in Mg2+ content was observed in Polypogon monspeliensis (Fig. 43).

4.5.9. Root Phosphorous (mg g-1)

Polypogon monspeliensis, Brachypodium distachyon, Bothriochloa bladhii, Heteropogon contortus, Aristida funiculata, Aristida mutabilis accumulate the maximum 3- content of root phosphorous (PO4 ) in their roots. Digitaria cruciata and Aristida cyanatha 3- were the grass with minimum (PO4 ) content in roots (Fig. 44).

4.5.10. Shoot Phosphorous (mg g-1)

3- A significant increase in shoot phosphorous (PO4 ) was observed in Bothriochloa bladhii, Bothriochloa pertus, Arundinella bengalensis, Brachypodium sp., Brachypodium sylvaticum, Cenchrus pennesitiformis, Digitaria cruciata, Panicum decompositum, Festuca levengei, Festuca Kashmiriana, Hordeum marinum which showed the maximum 3- accumulation of phosphorous (PO4 ) content in their shoots. On the other hand, Heteropogon contortus, Panicum humile, Poa argunensis Polypogon monspeliensis and Avena byzantina 3- showed the minimum shoot phosphorous (PO4 ) content in shoots (Fig. 44).

4.5.11. Total chlorophyll Contents

A highly significant increase was observed in Arundinella nepalensis, Koeleria cristata, Hordeum marinum, Koeleria macrantha, Cenchrus sp., Poa infirma, Hordeum glacum, Milium effusum, Lolium temulentum, Setaria pumila, Parapholis incurva, and with respect to total chlorophyll content. Festuca lenvengei, Festuca Kashmiriana, Pennesitum orientale, and Panicum atrosanguineum, Poa argunensis, Digitaria cruciata had the minimum amount of total chlorophyll contents (Fig. 44).

126

Fig. 44. Root and shoot physiological characteristics (root & shoot phosphorous and chlorophyll) of grasses of Neelum Valley, Azad Jammu and Kashmir

Legends: Andopogoneae Aristideae Arundineae Aveneae Brachypodieae Cynodonteae Eragrostideae Paniceae Poeae Triticeae

127 4.5.12. Multivariate Cluster analysis

Multivariate cluster analysis showed a very distinct clustering on the basis of root and shoot ionic contents and total chlorophyll. All grasses were grouped into two main clades that were further divided into sub-clades. In first clade, Festuca levengei and Rostraria pumila are sister species and form a separate sub-clade showing less similarities with the rest of the sub-clades. Digitaria cruciata, Hordeum glacum and Festuca simlensis were clustered into single clade that is sister clade to three other clades sharing some common characteristics. In second clade, Aristida mutabilis, Brachypodium sylvaticum, and Arthraxon prionodes were sister to each other sharing single sub-clade, showing close relationship with the species in other sub-clades. Cenchrus pennesitiformis and Capillepedium parviflorum both stem into two separate branches showing some contrasting characteristics with the rest of the species in other sub-tribes (Fig. 45).

4.5.13. CCA analysis of root nutrients

The PCCA root data with respect to nutrients as environmental variable showed a highly significant variation along axis 1. The distribution of Hordeum marinum, Lolium perenne, Panicum atrosanuineum, Cenchrus pennesitiformis, Heteropogon contortus, Schizachyrium impressum, Panicum temulentum, Sorghum nitidum, Poa falconeri, and Apluda mutica were mainly controlled by the Ca2+ content. Arundinella sp., Festuca levengei, Aristida cynantha distribution was strongly controlled by Na+ content. The distribution of Lolium temulentum, Bothriochloa pertusa, Rostraria pumila, Polypogon 2+ monspeliensis, and Arthraxon prionodes was highly influenced by the presence of PO4 contents. Poa infirma, Agrostis viridis, and Aristida funiculata showed stong association with Mg2+ content. Panicum decompositum, Poa attenuata, and Avena fatua distribution was strongly controlled by K+ content. The distribution of the rest of the grasses didn’t seem to be dependent over these factors (Fig. 46).

128

Fig. 45. Multivariate cluster analysis of the root and shoot physiological characteristics of grasses of Neelum Valley, Azad Jammu and Kahmir

129

Brd 1.5 Cyd Ca 1.0 Agp Pon Lop Apm Pof Apm K Mie Paa Fel Hom Pof Poa Aru Cep Son Pom Lop Na Pat Arc Sci Sap Saf Hec Fek Fek Pah Paa Son Pat Arn Ca Arn Soa Brs Ces Ces Cap Arf Avf Brd Dic Arm Kom Cep Agv Sci Fes Mie Pah Hom Bra Eli Cyd Peo Pon Fes Koa Bop Poi Pad Arc Hec Cap Pai Avb Soa Pad Peo Na Lot Eli Kom Roc Bop Pai Rop PO4 Fel PO4 Poa K Rop Pom Bra Avf Hog Arp Koa Sap Sep Bob Mg Agp Saf Aru Arm Lot Sep Arp Brs Roc Agv Hog Bob Poi Mg Avb Dic

-1.0 Arf

-1.5 -1.5 1.0 -1.5 1.5

Fig. 46. RDA ordination analysis of the (a.) root and (b.) shoot ionic content and the grasses at different sites at various sites of the Neelum Valley, Azad Jammu and Kashmir Key to the sites: Chiliahana (CH), Jura (JR), Kundal Shahi (KS), Athmuqam (AT), Kairan (KR), Lawat (LW), Dawarian (DW), Dodonial (DD), Sharda (SH), Kail (KL), Janawai (JW), Sardari (SD), Halmat (HM), Tao but (TB). Key to species: Agp: Agrostis pilosula; Agv: Agrostis viridis; Apm: Apluda mutica; Arc: Aristida cyanantha; Arf: Aristida funiculate; Arm: Aristida mutabilis; Arp: Arthraxon prionodes; Arn: Arundinella nepalensis; Aru: Arundinella sp; Avb: Avena byzantine; Avf: Avena fatua; Bob: Bothriochloa bladhii; Bop: Bothriochloa pertusa; Brd: Brachypodium distachyon; Bra: Brachypodium sp; Brs: Brachypodium sylvaticum; Cap: Capillipedium parviflorum; Cep Cenchrus pennisetiformis; Ces Cenchrus sp; Cyd: Cynodon dactylon; Dic: Digitaria cruciata; Eli: Eleusine indica; Fek: Festuca kashmiriana; Fel: Festuca levingei; Fes: Festuca simlensis; Hec: Heteropogon contortus; Hog: Hordeum glaucum; Hom: Hordeum marinum; Koa: Koeleria cristata; Kom: Koeleria macrantha; Lop: Lolium perenne; Lot: Lolium temulentum; Mie: Milium effusum; Paa: Panicum atrosanguineum; Pad: Panicum decompositum; Pah: Panicum humile; Pai: Parapholis incurve; Peo: Pennisetum orientale; Poi: Poa infirma; Poa: Poa argunensis; Pat: Poa attenuate; Pof: Poa falconeri; Pon: Poa nemoralis; Pom: Polypogon monspeliensis; Roc: Rostraria clarkeana; Rop: Rostraria pumila; Saf: Saccharum filifolium; Sap: Saccharum spontaneum; Sci: Schizachyrium impressum; Sep: Setaria pumila; Soa: Sorghum arundinaceum; Son: Sorghum nitidum.

130 4.5.14. CCA analysis of shoot nutrients The PCCA for shoot data with respect to nutrients as environmental variable showed a significant variation.The distribution of Festua kashmiriana, Panicum temulentum, Cenchrus sp., Agrostis viridis, was strongly infuluenced by Ca2+ content. Polypogon monspeliensis, Saccharum filifolium, Poa attenuata, Milium effusum, and Apluda mutica was strongly associated with K+ content. The distribution of Avena byzantina, Pennesitum orientale, Bothriochloa bladhii, Arundinella sp., Lolium temulentum, and Rostraria 2+ 2+ clarkeana was strongly controlled by PO4 and Mg contents. Aristida cynantha, Festuca simlensis, Heteropogon controtus and Sorghum arundinaceum was controlled by Na+ content. The distribution of the rest of the grasses didn’t seem to be dependent over these factors (Fig. 46).

4.6. Cladistic analysis of tribe Andropogoneae

For cladistics analysis of Andropogoneae, 26 morphological characters were employed to build a UPGMA tree implement in PAUP* 4.0b (Table 17). The Schizachyrium was the first branch within Andropogoneae, clustered with [Apluda+ Arthraxon]; then was sister to [Bothriochloa+ Heteropogon], collectively sister to the remaining crown clade ([Saccharum+Sorghum]+Capillipedium). Heteropogon spp. showed a close relationship with two Bothriochloa spp. whereas, Capillepidium was found much closer to the species of Sorghum and Saccharum (Fig. 47).The members of tribe Andropogoneae showed variation in root system that was either rhizomatous or stolniferous (e.g. Apluda mutica, Arthraxon hispidus), fiberous (e.g. Bothriochloa pertusa, Bothriochloa bladhii) or typically stolniferous (Sorghum arundinaceum). Root length showed variation up to 4 cm and the maximum length was recorded in Sorghum arundinaceum, Sorghum nitidum, Saccharum spontaneum, and Saccharum sinense). Culm may be caespitose or solitary in origin, decumbent (A. mutica), erect or geniculate (B. pertusa, and Capillipedium parviflorum) in shape with glabrous (Heteropogon contortus, A. prionodes, S. arundinaceum) or node beard surface (Saccharum spontaneum). The species of genus Saccharum and Sorghum showed the maximum length of leaf blade. Apluda mutica, B. pertusa, H. contortus, and Sorghum arundinaceum showed cauline type of leaf origin, while rest of the species showed basal type of origin.

131

Saccharum sinense Saccharum spontaneum Sorghum arundinaceum Sorghum nitidum Capillipedium parviflorum Bothriochloa pertusa Bothriochloa bladhii Heteropogon contortus Apluda mutica Arthraxon hispidus Arundinella nepalensis Agrostis gigantea

Fig. 47. The UPGMA tree based on 26 morphological characteristics

132 Arthraxon hispidus, C. parviflorum, S. sinense, S. spontaneum, and Schizachyrium scoparium showed dense type of hairness on the adaxial side of the leaf blade with rough abaxial surface. Some species such as, S. nitidum, S. arundinaceum, and A. nepalensis showed dense layers of hairiness on the surface of leaf sheath, whereas, rest of the species were reported with sparse or no hairs. Majority of the species showed lanceolate or linear type of leaf blade except Apluda mutica that bears attenuate type of leaf blade.

Glabrous surface of leaf blade was reported mainly in C. parviflorum, Bothriochloa spp. H. contortus, S. sinense and S. scoparium, whereas in rest of the species leaf blade was found with scabrous surface. Apluda mutica and A. hispidus were found flat or lanceolate blade shape, respectively, whereas rest of the species had blade shape convolute or folded shape. Sorghum nitidum was the only species with slightly scabrid leaf sheath. All other species were reported with smooth, smooth and keeled, hairy or papery sheath. Heteropogon contortus, B. bladhii, S. spontaneum and A. nepalensis showed the minimum length of ligule, i.e., < 2 mm. In most of the species, ligule was found with membranous fringed except A. mutica and S. scoparium (Table 17).

Species showed variation in inflorescence length as the maximum length was recorded in A. nepalensis. Saccharum sinense showed the minimum floret length. Apluda mutica and H. contortus were reported with minimum number of spikelets. The spikelets were in pairs and nearly always born on a fragile rachis; they were two flowered and lower floret male was completely covered by a glume.

This character opens the way to evolutionary development. Most of the species were found with raceme type of inflorescence along with panicle and spike in some species. Tiller number was found maximum in A. mutica, A. hispidus, and H. contortus, etc. The maximum awn length was recorded in S. arundinaceum and S. nitidum. The spikelets occurred in pairs and nearly always born on a fragile rhachis; they were two flowered and lower floret male was completely covered by a glume. This character opens the way to evolutionary development (Table 17).

133 Table 17. Character states used in the cladistic analysis of Andropogoneae Characters Character states Root type Rhizome (0)/fibrous (1)/stolniferous (2) Root length (1) <2cm, (2) 2-3 cm, (3) 3-4 cm, (4) >4cm Culm origin Caespitose (0)/solitary (1) Culm shape decumbent (0)/erect (1)/geniculate (2) Culm surface Glabrous (0)/node beared (1) Culm length (1) <2cm, (2) 2-3 cm, (3) 3-4 cm, (4) >4cm Leaf Length (1) <2cm, (2) 2-3 cm, (3) 3-4 cm, (4) >4cm Leaf apex Attanuate (0)/acute (1)/acuminate (2) Leaf origin Cauline (0)/basal (1) Leaf hairiness – adaxial (1) none, (2) sparse, (3) dense Leaf roughness – abaxial (1) smooth (2) rough (3) very rough Leaf sheath hairiness (1) none, (2) sparse, (3) dense Blade type Attanuate (0)/lanceolate (1)/linear Blade surface Glabrous (0)/scabrous (1) Blade shape Flat (0)/lanceolate (1)/folded (2)/ convolute (3) Sheath type Smooth (0)/smooth & keeled (1)/hairy(2)/ papery (3)/slightly scabrid (4) Auricle Absent (0)/present (1) Ligule type memberanous fringed (0)/ring of hairs (1) Ligule Length (1) <2mm, (2) 2-3 mm, (3) 3-4 mm, (4) >4mm Inflorescence length (1) <2mm, (2) 2-3 mm, (3) 3-4 mm, (4) >4mm Spikelet length (1) <2cm, (2) 2-3 cm, (3) 3-4 cm, (4) >4cm Spikelet number (1) <15, (2) 15-20, (3) >20 Inflorescence type Panicle (0)/raceme (1)/spike (2) Tiller number (1) <15, (2) 15-20, (3) >20 Awn length (1) <2mm, (2) 2-3 mm, (3) 3-4 mm, (4) >4mm Habitat Annual (0)/perennial (1)

134 For molecular studies, the existing GenBank sequence was used as a starting point for alignment, and downloaded all the available nucleotide sequences from GenBank (Table. 18). A total of 7446 characters and 14 taxa (11 ingroups and 3 outgroups) were included in phylogeny reconstruction. Because of loss of many data and close relationship appearing within the tribe, the support values were not good both in the ML and BI trees. But, we got same monophyletic topology of ML and BI trees of tribe Andropogoneae. Here, we only showed the BI tree with PP and BS values. The Apluda (Apluda mutica) was at the first branch within tribe Andropogoneae, sister to the remaining genera with robust support (PP = 1.00, BS = 100; or 1.00/100). Arthraxon (Arthraxon hispidus) was sister to the left 6 genera (Saccharum, Sorghum, Capillipedium Schizachyrium, Heteropogon, and Bothriochloa) with high PP value (0.96), but no bootstrap values. The Saccharum+Sorghum clade was sister to the crown clade (1.00/64) without PP and BS. Within the crown clade, Schizachyrium clustered with the left genera ([Heteropogon+Capillipedium]+Bothriochloa) as sister (0.97/53); Heteropogon was sister to the genus Bothriochloa with strong support values (1.00/91; Fig. 48).

135

136

Fig. 48. Phylogenetic tree based on combined 6 loci dataset. Numbers under the branch are PP/BS values

137 CHAPTER 5

DISCUSSION

Grasses a natural homogenous group of plants belong to the most fascinating families of flowering plants, Poaceae. They play a significant role in the lives of humans and animals with a wide range of diversity. The members of this group are present in all the conceivable habitats suitable for the growth of the plant communities. In Pakistan, the Gramineae is one of the dominant families, both on the basis of its number of genera and species (Mitra and Mukherjee, 2004). Poaceae is a species-rich family that includes many economic plants, globally with about 10,000 species and 700 genera (Crisp et al., 2009; Linder and Rudall, 2005). Recent phylogenetic studies confirmed that multiple factors are involved indirectly that determine the grass diversity at large scales (Edwards and Smith, 2010).

During the present research work, 15 ecologically different habitats in the Neelum Valley were selected to investigate soil plant relationship and comparative morpho-anatomy and physiology of all available grass species. Out of 52 species of grasses, Poaeae tribe was found the largest tribe and most of the grasses are radiated in cooler environments. The evolution of cold and frost stress responses, either through fine tuning of ancient abiotic stress responses or evolution of novel adaptations of these grasses to cold environments must have been central for the Pooideae (Sandve et al., 2008).

Plant distribution is controlled at large by climate and over small scale by environmental heterogeneity (Lavers and Field, 2006). Species diversity mainly depends on the occurrence, abundance and vegetation cover. There is a significant relationship between species diversity, distribution and moisture availability (Leathwick et al., 1998). A marked variation was recorded not only in the species composition, but also in the distributional pattern of grass species at different habitats. This represent clear picture of the influence of soil physico-chemical characteristics on community structure and composition of grasses. A broad range of association was observed between grasses and soil attributes in RDA analysis. The distributional pattern of these grass species seems to be strongly influenced by ECe, moisture contents and pH of the soil. Available moisture primarily depends upon soil physico-chemical characteristics and precipitation (Scholes et al., 1997), and this will

138 certainly change the species composition. Another factor affecting distributional patters of species is the altitude which again apparent in our studies. Elevation as an important factor has a multifaceted influence on the distribution pattern of the species (Zhao and Fang, 2006)

Morphological, anatomical and biochemical traits of grasses respond differently under various environmental factors depending upon intensity of light and available soil moisture (Kolodziejek, 2014). The response may also depend on grass species and degree of tolerance to different environmental stresses (Hameed et al., 2013). Therefore, occurrence of Lolium temulentum, Poa nemoralis, and Saccharum spontaneum, at CH site was likely to be affected by the moisture contents. Soil pH directly affects the availability of nutrient and species response to pH is mainly determined by moisture content (Gould and Walker, 2004). As was mentioned earlier, moisture contents specifically depend upon the composition of the soil, availability of nutrients and topography of the area (Michael et al., 2002), and the CH site supported grasses that require sufficient moisture for their survival. In particular, S. spontaneum, which is frequently found along the water channels (Marwat et al., 2012). Abiotic factors (e.g., light and shade, temperature, pH, water and nutrients availability) have a pronounced effect on the morphological traits of plants (Schlichting, 2002; Schlichting and Smith, 2002).

Morphological markers are helpful in the identification, differentiation and classification of the grasses at species, genus and tribe level. Morphological characters especially that of (inflorescence and spikelet) provides useful information for the identification of all levels of taxonomic ranks. Grasses particular, Arundinella bengalensis, A. nepalensis, Saccharum filifolium, Avena byzantine and Brachypodium sylvaticum showed the maximum inflorescence length. A highly significant variation was also observed in spikelet in Setaria pumila, Polypogon monspeliensis, Eleusine indica, Aristida mutabilis, and Arundinella bengalensis. In genus Avena, two species i.e., A. fatua and A. byzantina look morphologically very similar. Avena byzantina can be distinguished due to longer spikelet and awns while in Avena fatua spikelet and awns are shorter (Ahmad et al., 2010).

Transverse sections of grass leaves are also helpful in the identification and taxonomic delimitation of grasses (Ellis, 1986). A root, stem and leaf level, significant variations were observed in anatomical characteristics among the grasses within tribes. All

139 parameters varied significantly. Root cross sectional area and root hair length significantly increased in some particular grasses such as, Arundinella bengalensis and Arista mutabilis, which showed the maximum surface area for water conservation as this mainly depends on storage parenchyma. A highly increased epidermal thickness with extensive sclerefication was observed in most of the grasses particularly in Cenchrus pennisetiformis, Avena fatua, and Saccharum spontaneum, and this may be an important strategy to cope with low temperature stress and an adaptation to protect internal plant tissues from harsh outer environment and to minimize the water loss. Other important strategies are increased cortical area, intensive sclerification inside epidermis and around the vascular bundles modified vascular cylinder, thick leaf sheath and blade area (Hameed et al., 2013), well developed bulliform cells and low stomatal density (Alvarez et al., 2003; Jianjing et al., 2012).

The reduced vascular tissues, in particular metaxylem and protoxylem cross-sectional area can easily be correlated to efficient water uptake and moisture column maintenance. The reduced area of stomata can be regarded to as critical adaptation for semi-arid regions as smaller stomata can be maintained by lesser turgor, hence opening and closing according to environmental condition can be regulated efficiently. Reduction in leaf area is the principal strategy that makes it possible to attenuate the effects of the reduction in the availability of water under abiotic stress (Alem et al., 2002).

Knowledge of the leaf anatomy of grassland plants is crucial for understanding how these plants adapt to the environment. In the genus Brachypodium, distribution of sclerenchyma and bulliform cells proved useful at specific level (Khan, 1984). Occurrence of sclerenchyma and bundle sheath (Kranz Sheath), the width of sclerenchyma, the indumentum of leaves and length and frequency of epidermal basis are features of prime importance that can identify relationship among the genera of Poaceae (Dube and Morisset 1987; Jarves and Barkworth, 1992). Ellis (1986) pointed out that characters such as the thickness of the leaf, the number and arrangement of vascular bundles might be systematically useful, and characters such as the distribution of prickles may be relatively stable or environmentally variable. Ellis (1976) also observed that the position of vascular bundles in the blades appeared to be a useful diagnostic character above the generic level.

140 The leaf epidermal anatomy provides extensive taxonomic data related to grasses. Epidermal traits, i.e., epidermal cells, stomata and hairs have proved to be an important tool in delimitation of taxa in many plant families (Ditsch et al., 1995; Barthlott et al., 1998; Stenglein et al., 2003). It is confirmed that leaf epidermal features can help to elucidate taxonomic relationships at different levels (Metcalfe, 1960, Ellis, 1979, Palmer and Tucker, 1981; Davila and Clark, 1990, Cai and Wang, 1994; Mejia-Saules and Bisbey, 2003) and these leaf epidermal characters are of great value in grass systematics and characterization of broad groups within the grasses, particularly subfamilies and tribes. Modifications relating to leaf epidermis were relatively high in all grasses from Neelum Valley. Majority of the grasses studied were recorded with short and angular prickles at the margin of the leaf, long cells with slightly or highly sinuous walls, sharply pointed microhairs, saddle or rounded shape silica bodies, distinct ribs and ridges sometime present or absent at both abaxial and adaxial surface of the leaves. Most of the grasses were reported with densely packed hairiness at leaf surface. In tribe Paniceae, pointed angular bicelled prickles and micro hairs at the leaf margins is characteristic feature of this tribe. The presence of prickles with rounded base in Panicum maximum makes it, different from Panicum antidotale in which pointed projections are present on the upper leaf surface (Ahmad et al., 2015).

Microhairs and prickles have also been examined for taxonomic utility are very informative at the subfamilial level. Broad-tipped microhairs of the epidermis are restricted to the Chloridoideae (Amarasinghe and Watson, 1990), and microhairs are less visible in Pooideae, with the exception of Lygeum Loefl. ex L. and Nardus L. (GPWG, 2001). The significance and implications of silica bodies in taxonomy of grasses have been addressed. Certain shapes of silica bodies are characteristic of grass subfamilies, e.g. dumbbell-shaped in panicoid grasses, saddle-shaped in most pooid grasses, and 12 vertically oriented silica bodies in the Bambusoideae (Rovner, 1988). Distribution and shape of silica bodies is taxonomically informative at the tribal level in the Stipeae (Barkworth, 1981). The presence or absence of these Silica bodies is valuable for differentiating species (Mejia-Saules and Bisby, 2003).

141 The average length of microhairs is also an important character in identification, which is also in conformation (Shouliang et al., 1996). Different genera in the tribes look morphologically similar but anatomical studies are helpful in their differentiation and identification, when correlated with their morphological characters. Intercostal long cells in all the species of different genera in the tribe are with thin sinuous or moderately thick sinuous walls (Ahmad et al., 2011).

Grasses have been more rigorously subjected to cladistic analyses, using both morphological and molecular data, than many other groups. Classical early morphological studies for the whole Poaceae family was conducted by (Kellogg and Campbell, 1987) and its subfamilies by (Kellogg and Watson, 1993) using data collected by Watson and Dallwitz (1992), and Snow (1997). With the refinement of techniques in genomic analyses, many cladistic analyses based on molecular data were followed in the 1990s. Some of the best known and most frequently cited papers are Hamby and Zimmer (1988) using ribosomal RNA sequences, Doebley et al. (1990) using plastid rbcL sequence data, Davis and Soreng (1993) using plastid DNA restriction site variation, Nado et al. (1994) using plastid rps4 sequences, Clark et al. (1995) using plastid ndhF sequence data, Duvall and Morton (1996) using plastid rbcL sequences, Soreng and Davis (1998) using plastid DNA restriction site data, Barker et al. (1999) using plastid rpoC2 sequences, Hilu et al. (1999) using matK sequences, and Mathews et al. (1996) using the nuclear phytochrome gene family. A total of 26 morphological characters were selected, that were grouped as: root (2), culm (4), leaf (10), including leaf blade and leaf sheath, spikelet (5), auricle (1), ligule (2), tillers (1) and habitat (1). That is an agreement with the GPWG (2001). They assessed 46 structural characters that could be interpreted to be of use as measures of phylogenetic signal in the grasses. These characters can be grouped as follows: culm (2 characters), leaf (5), spikelet (10), floret (14), fruit and embryo (9), seedling (6). A major argument against a cladistic classification is that it is not always practical but the results suggest that some of them ‘‘may be useful for delimiting groups within tribes or subfamilies, but are too variable to be useful in delimiting subfamilies’’ (GPWG, 2001). The Cladistic analysis for moleclular data showed 7446 characters and 14 taxa (11 ingroups and 3 outgroups) were included in phylogeny reconstruction. Apluda mutica and Arthraxon hispidus formed were first and second branch within the tribe, respectively; that

142 was sister to the remaining genera. Within the crown clade, Schizachyrium clustered with the genera ([Heteropogon+Capillipedium]+Bothriochloa) as sister. Morphologically, Arthraxon is distinguished from all other genera within Andropogoneae by its lemmas with a sub-basal awn. Although several well supported groups have been identified, the present matrix with three non-coding markers (trnL–trnF, atpβ– rbcL and ITS) was insufficient to provide enough phylogenetic informative characters to resolve many evolutionary relationships at the intergeneric level in Andropogoneae. Adding more data and more taxa are the only ways of resolving these difficult groups (Hillis et al., 2003; Hodkinson et al., 2007; Pirie et al., 2008). Previous molecular studies showed two genera, Bothriochloa, and Capillipedium in a monophyletic group in the combined analysis (e.g., Spangler et al., 1999; Mathews et al., 2002). However, this relationship was not supported by our morphological data like most the other morphological studies (Clayton and Renvoize, 1986; Kellogg and Watson, 1993) and most studies preferred to keep these genera separate. A comparably comprehensive nuclear data set from the grasses is lacking. As concluding remarks, formal taxonomic and nomenclatural changes should surely only be encouraged, particularly at the species level, when the lineages within a phylogeny correlate with marker morphological characters. Much more thought and research though need to be directed toward establishing the exact nature of morphological characters and their interpretation in reflecting phylogeny. Furthermore, we can improve phylogenetic understanding within Andropogoneae and among subfamilies in the PACMAD clade by increasing the sampling of taxa and by using morphological to study inter-relationships of taxa within this group of plants. It is further revealed that foliar epidermal studies assist in identification and differentiation of different grass species. Different anatomical characters such as absence or presence of prickles, nature of the microhiars, shape of silica bodies and size of stomatal complex are of prime importance in delimiting different taxa at the species and generic level.

It is concluded that almost all morpho-anatomical and physiological characteristics are species specific and not to a genus or tribe. Same was the case with their tolerance degree to either cold stress or drought. However, some specific modifications like amount of sclerification, size and shape of bulliform cells, presence of storage parenchyma, nature of

143 pubescence and stomatal size and area can be related to environmental conditions. It is, therefore, concluded that certain anatomical characteristics like presence of silica bodies, surface appendages, bulliform cells and pattern of sclerification can safely be used as important tools for the identification at species or lower rank.

144 CHAPTER 6

SUMMARY

Poaceae is a species-rich family that includes many economic plants, globally with about 10,000 species and 700 genera. Grasses are the most fascinating group among plant families with a wide range of diversity. The members of this group are present in all the conceivable habitats suitable for the growth of the plant communities and play a significant role in the humans and animals nutrition. In Pakistan, the Poaceae is one of the dominant families, both on the basis of its number of genera and species. Recent phylogenetic studies confirmed that multiple factors are involved indirectly that determine the grass diversity at large scales.

In this study, a total of 52 species of grasses belonging to 10 tribes and 28 genera were recorded from 15 sampling sites in Neelum Valley, Azad Jammu and Kashmir. Poaeae was the largest tribe (12 spp.); followed by Andropogoneae (11 spp.); Aveneae (9 spp.); Paniceae (8 spp.); Aristideae and Brachypodieae (3 spp. each), whereas, rest of the 5 tribes represents 2 or 1 species. Dodonial was species rich site with 15 species (28.85%), which was followed by Dawarian, Sharda, and Taobut with 12 species, and Kairan, Nagdar, and Halmat with 11 species each.

Physiochemical characteristics of the soil analysis showed that most of the soil component varied significantly at different study sites. The soil moisture content seemed to be closely related to the physical properties of the soil as well as to vegetation type. The main factor affecting soil moisture was precipitation. A significant difference was also observed in ECe, saturation percentage and ionic content of the soil in the analysis. RDA ordination indicated a strong effect of soil characteristics on distribution of grasses at different habitats.

Grasses showed a broad range of association that varies significantly over ecological diverse sites. A highly significant association between various grasses was recorded over Jura, Kundal Shahi, Athmuqam, Kairan, Sharda, Kail, Janawai Sardari, Halmat and Tao Butt sites. A less significant or no remarkable association was recorded among grasses over Nagdar, Lawat, Dawarian and Dodonial sites.

145 Morphological markers are helpful in the identification, differentiation and classification of the grasses at species, genus and tribe level. All grasses showed a highly significant variation in terms of morphological parameters such as, plant height, root length, root and shoot dry weight, number of leaver and number of tillers per plant, inflorescence length, and spikelet length and numbers. Multivariate analysis of the morphological data matrix clustered the species into two main clades and many sub clades. In first clade Saccharum filifolium and Saccharum spontaneum, Sorghum arundinaceum and S. nitidum were sister species, which showed close relationship with Pennesitum orientale and Schizachyrium impressum. In second sub-groups of the first clade, Arundinella sp. and A. nepalensis were sister species that showed a very close relationship to Hordeum galacum and H. marinum representing some common morphological characteristics. In second clade, Heteropogon contortus stems into a single branch showing less similarity with the rest of the species in the group. All the Poa species except Poa nemoralis were grouped together as sister grasses.

A root, stem and leaf level, significant variations were observed in anatomical characteristics among the grasses within tribes. All parameters varied significantly. Root cross sectional area and root hair length significantly increased in some particular grasses such as, Arundinella bengalensis and Arista mutabilis which showed the maximum surface area for water absorption. A highly increased epidermal thickness was observed in Cenchrus pennisetiformis, Avena fatua, and Saccharum spontaneum, which may be an adaptation to protect internal plant tissues from harsh outer environment and to minimize the water loss. Cortical thickness showed remarkable increase particularly in Rostraria clarkeana and Saccharum filifolium, which may be an important strategy of many temperate grasses for the food storage and water conservation. Brachypodium sp. showed the maximum root endodermal cell areas that facilitate the radial flow of water. Increase epidermal thickness and intensive sclerification in grasses might be an important adaptive mechanism to cope with low temperature and other environment stresses. Other important strategies are increased epidermal thickness, highly developed cortical area, high sclerification in epidermis and around the vascular bundle area, increased metaxylem vessels, reduced and thick leaf sheath and blade, well developed bulliform cells, and low number of stomatal

146 density. The less tolerant Arthraxon prionode and Apluda mutica showed a significant reduction in this parameter.

All grasses showed very specific structural and functional modification in the leaf surface region. Majority of the grasses studied were recorded with short and angular prickles at the margin of the leaf in intercostal zone, long cells with slightly or highly sinuous walls, sharply pointed microhairs, saddle or rounded shape silica bodies, distinct ribs and ridges sometime present or absent at both abaxial and adaxial surface of the leaves. Most of the grasses were reported with densely packed hairiness at leaf surface. In tribe Paniceae, pointed angular bicelled prickles and micro hairs at the leaf margins is characteristic feature of this tribe.

Physiological parameters also showed highly significant variation in terms of root and shoot ions accumulation and total chlorophyll content. Grasses mainly, Hetropogon controtus, Cynodon dactylon, Polypogon monspeliensis, Bothriocholoa pertusa, Brachypodium distachyon, Aristida funiculata and Arundinella nepalensis retain the maximum accumulation of sodium, potassium, magnesium, phosphorus and chlorophyll content within the plant body.

In cladistics analysis, Apluda mutica was at the first branch within tribe Andropogoneae, sister to the remaining genera with robust support (PP = 1.00, BS = 100; or 1.00/100). Arthraxon (Arthraxon hispidus) was sister to the left 6 genera (Saccharum, Sorghum, Capillipedium Schizachyrium, Heteropogon, and Bothriochloa) with high PP value (0.96). The Saccharum+Sorghum clade was sister to the crown clade (1.00/64) without PP and BS. Within the crown clade, Schizachyrium clustered with the left genera ([Heteropogon + Capillipedium] + Bothriochloa) as sister (0.97/53); Heteropogon was sister to the genus Bothriochloa with strong support values (1.00/91).

As concluding remarks, formal taxonomic and nomenclatural changes should surely only be encouraged, particularly at the species level, when the lineages within a phylogeny correlate with marker morphological characters and much more thought and research though need to be directed toward establishing the exact nature of morphological characters and their interpretation in reflecting phylogeny. Furthermore, we can improve phylogenetic

147 understanding within among subfamilies in the PACMAD clade by increasing the sampling of taxa and by using morphology to study inter-relationships of taxa within this group of plants. It is also revealed that the application of anatomical markers, such as increased sclerification, absence or presence of prickles on the abaxial and adaxial side, shape of silica bodies and size of stomatal complex are of prime importance in delimiting different taxa at the species and generic level. Overall, it is concluded that anatomical characteristics are important for taxonomic identification at species level or at lower rank, but are also of great ecological significance. Many characteristics are species specific.

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163 Appendex I Key for grasses

1. Inflorescences a single raceme or spike………………………………… a. Spikelets not awned i. Spikelets in pairs, one sessile and other pedicellate…………….. ii. Spikelet not in pairs Lolium, Triticum b. Spikelets awned i. Spikelets in pairs, one sessile, the other pedicellate Heteropogon ii. Spikelets not in pairs Lolium, Hordeum 2. Inflorescence a more or less spike like panicle………………….. a. Spiklets not awned, not markedly hairy……………………….. Phalaris, Sporobolus b. Spiklets not awned, but hairy, or with bristles or spines……………..Setaria, Cenchrus, Pennesitum c. Spiketets awned………………………………… i. Spikelets awned but panicle loose……………………… Aristida ii. Spikelets awned but anicle dense, awn short to long…… …… Polypogon 3. Inflorescence open racemose, with a pairs of dissimilar spikelets Capillipedium, Bothriochloa, Sorghum 4. Inflorescence an open panicle, branches terminating in single spikelet…………. a. Spikelets not awned i. Spikelets obvious with more than one fully developed floret…… Poa, Festuca ii. Spikelets with one fully developed floret……………………… Agrostis, Panicum, Setaria b. Spiketets awned…………………………………………………………………… i. Spikelets with more than one fully developed floret…………… Arundo, Avena ii. Spikelets with one fully developed floret……………………… Arudinella, Aristida, 5. Inflorescence a digitate or subdigitate panicle…………………………………… a. Spikelets in dissimilar sessile and pedicellate pair………………… Arthraxon b. Spikelets similar, more than one fully developed floret per spikelets……….. Elusine c. One fully developed floret per spikelets but not awned………………Cynodon

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