Pollen Morphology of Selected Plants from Erkwit (Eastern Sudan)

ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ

A thesis submitted to the University of Khartoum in fulfillment of the requirements for the degree of Master of Science in Botany

Hind Abdel Wahab Farah Ahmed B.Sc. Honours Botany University of Khartoum, 2004

Supervisor: Dr. Dafaalla Ali Ibrahim

Department of Botany Faculty of Science University of Khartoum

May, 2008

ﺑِﺴْﻢِ اﻟﻠﱠﻪِ اﻟﺮﱠﺣْﻤَﻦِ اﻟﺮﱠﺣِﻴﻢِ

ﻗﺎل ﺗﻌﺎﻟﻰ:

﴿وَأَرْﺳَﻠْﻨَﺎ اﻟﺮﱢﻳَﺎحَ ﻟَﻮَاﻗِﺢَ ﻓَﺄَﻧﺰَﻟْﻨَﺎ ﻣِﻦَ اﻟﺴﱠﻤَﺎء ﻣَﺎء ﻓَﺄَﺳْﻘَﻴْﻨَﺎآُﻤُﻮﻩُ وَﻣَﺎ أَ ﻧ ﺘُ ﻢْ ﻟَ ﻪُ ﺑﺨَﺎزِﻧِﻴﻦَ﴾

ﺳﻮرة اﻟﺤﺠﺮ اﻻﻳﺔ (22)

DEDECATION

To the Soul of my father…

To my mother…

To my sister …

To my best friend…

With love,,,,

Acknowledgements

Praise is to Allah (the almighty who gave me the health, patience and power to carry out this work) and to the blessed profit Mohammed (piece and praise be upon him). This research is supported by a grant from the Association for The Promotion of Scientific Innovation to whom I am deeply indebted and thankful. I would like to thank The Sudanese National Heritage Society through whom I got the research grant. This study was conducted as M.Sc. research at Khartoum University, Faculty of Science, Botany Department under the supervision of Dr. Dafaalla Ali Ibrahim to whom I wish to express my grateful thanks for his encouragement, continuous support and valuable comments during this study. So I’m greatly indebted to him. I am also grateful to Dr. Saad Eldeen Elsammani for his kind support, to my uncle Dr. Abubakr Mohammed Ibrahim for his suggestions. My thanks are extended to the members of Biofertilization Department, National Center of Research and members of Botany Department, University of Khartoum, Faculty of Science whom supplied the space and time to conduct this research, deep special thank to Mohamed Abdalla, Ali and Randa and to my friends and colleagues for assisting and encouragement me on this work. Finally, thanks to all persons named or unnamed who helped me in one way or another during the period of this study.

CONTENTS

iii

2.3.5. Size …………….………..…….…….…………………..…... 17 Dedication 2.3.5.1. …………………………………………….Effect of different treatments in pollen grain...... size …………. i17

Acknowledgements …………………………………………………… ii

ContentsCHAPTER ………………………………………… THREE: MATERIALS AND METHODS………...... iii

List of Figures …………………………………………..…....……..... vi 3.1. The study area ……………………………………………………. 19 List of Tables …… ……………………………………..…....……...... vii 3.1.1 Topography and geology …….…..……..……………………. 19 Abbreviation ………………………………….………...... ……..... viii 3.1.2. Rainfall ……..……………..………………………………….. 20 Abstract ………………………………………..……………...... … Ix 3.1.3. Vegetation …………..……….……………………………….. 20 Abstract in Arabic …………………………………………………… iv 3.2. Chemical ………………………………………………………….. 23

3.2.1. 10 % Potassium Hydroxide ……………..….……………...… 23 CHAPTER ONE: INTRODUCTION 3.2.2. Acetolysis mixture ………………………….…………...... 23 Introduction …………….…………………..………………………… 1 3.2.3. Glacial acetic acid ……………………………….……..…...… 23 Objective of the study …………………...……..…………………….. 3 3.2.4. Basic fuchsin …………………………..……………..……… 23

CHAPTER 3.2.5. Glycerin TWO: ……………..…………………….….……...……… LITERATURE REVIEW 23

3.2.6. 10 % Hydrochloric Acid …….………..………..……….…… 23 2.1. Palynology and Pollen analysis …….…………….……….……… 4 3.2.7. Lycopodium sp. spores tablets………………….….……….... 23 2.2. Pollen taxonomy ……..………………………………………..… 6 3.3. Equipment ………………………………..………….…………... 23 2.3. Pollen morphology …………………………………………..…… 7 3.4. Sampling ……………………………………………….…….…... 24 3.5. 2.3.1. The Shape Pollen ………………………………………………..………. Preparation ……………………..………….……...... 824 3.6. Measurements ………………….……………………….…….…. 26 2.3.2. Apertures ………………………………………….………… 10 3.7. 2.3.3. Species Exine description stratification...……………………………………….. and terminology ………………….………….… 1327

2.3.4. Sculpturing types (Ornamentation) ……………..….………… 15

CHAPTER FOUR: RESULTS

Family: Anacardiaceae ……………………………………….……...... 28

Family: Apocynaceae ……………………………….…..……..…...... 29

Family: Arecaceae …………………………………………..………… 30

Family: Asclepiadaceae ……………………………………..………… 31

Family: Balanitacea (Simarubaceae)……………… …………………… 32

Family: Capparidaceae ……………………………………..………… 33

Family: Ebenaceae …………………………………………..………… 36

Family: Euphorbiaceae ……………….……………………………….. 37

Family: Fabaceae (Leguminosae)………...………………..…………… 42

Family: Moraceae ………………………………..…………………….. 46

Family: Olacaceae ……………………………………………………… 49

Family: Oleaceae …………………………………………………..….. 50

Family: Rhamnaceae …………………………………………...…...... 51

Family: Salvadoraceae ………………………………………………… 52

Family: Sapindaceae …………………………………………..………. 53

Family: Tamaricaceae ……………..…………………………..……… 54

Family: Tiliaceae ………………………………………………...…….. 55

CHAPTER FIVE: DISCUSSION

Discussion ………………………………………………………….… 59 CONCLUSIONS……………………………………………………... 64 REFERENCES……………………………………………………………65

List of Figures

Fig. (1) Polar (P) and equatorial (E) shape in pollen grains …………… 9

Fig. (2) Different kinds of pollen grains apertures ………………….… 12

Fig. (3) Exine Stratification of pollen grains …………………………... 14

Fig. (4) Pollen grains Sculpturing types ……………………….……… 16

Fig. (5) a. Size of grain before acetolysls …………………….….…… 62

Fig. (5) b. Size of grain after long time of acetolysls ………………… 62

Fig. (6) Effect of mounting in glycerin jelly ………………………..… 62

Fig. (7) a. Colour of grains after acetolysis …………………………… 63

Fig. (7) b. Colour of grains after staining …..…………………………. 63

List of Tables

Table: (1) Pollen shapes using the correction factor……….………….. 58

vii Abbreviations

-Abbreviations used in the text:

E = Size in equatorial view. P = Size in polar view. L = Correction factor. Engl. = English language. Ar. = Arabic language. D = Diameter. LM = Light microscope. H = Herbarium material. F = Fresh material. Syn. = Synonym/s. Vetn. Name/s = Vernacular name/s. SEM = Scanning electron microscope. K = Conversion factor.

-English equivalents of Arabic words: Jebel = Mountain. Khor = Seasonal stream.

viii

Abstract

Pollen morphological descriptions and photographic illustrations based on light microscope analysis are provided for 30 species belonging to 18 families from Erkwit area in the eastern of Sudan.

The pollen characteristics among species in the same family are similar to each other. The results indicate that pollen characters may be significant in taxonomical studies.

The effect of various chemicals on the size of pollen grains was studied. The size of acetolysed grains was affected by the treatment prior to acetolysis, and the measurements taken were corrected by a correction factor (L), The correlation between pollen size and chemical treatments, as suggested previously by (Christensen, 1946), is confirmed by our data.

ix Arabic abstract

ﻤﻠﺨﺹ ﺍﻻﻁﺭﻭﺤﺔ

ُﻓﺤﺼﺕ ﺍﻷ ﺸﻜﺎل ﺍﻟﻅﺎﻫﺭﻴﺔ ﻟﺤﺒﻭﺏ ﺍﻟﻠﻘﺎﺡ ﺒﻭﺍﺴﻁﺔ ﺍﻟﻤﺠﻬﺭ ﺍﻟﻀﻭﺌﻲ ﻟـ 30 ﻨﻭﻉ ﻴﻨﺘﻤـﻲ

ﻟـ18 ﻋﺎﺌﻠﺔ ﻤﻥ ﻤﻨﻁﻘﺔ ﺍﺭﻜﻭﻴﺕ ﻓﻲ ﺸﺭﻕ ﺍﻟﺴﻭﺩﺍﻥ ، ﻭﺘﻡ إﺭﻓﺎﻕ ﺍﻹﻴﻀﺎﺤﺎﺕ ﺍﻟﻔﻭﺘﻭﻏﺭﺍﻓﻴﺔ.

ﺘﺸﺎﺒﻬﺕ ﺨﺼﺎﺌﺹ ﺤﺒﻭﺏ ﺍﻟﻠﻘﺎﺡ ﻓﻲ ﺍﻷﻨﻭﺍﻉ ﺍﻟﺘﻲ ﺘﻨﺘﻤﻲ ﻟﻨﻔﺱ ﺍﻟﻌﺎﺌﻠﺔ ﻭﻫﺫﻩ ﺍﻟﻨﺘﺎﺌﺞ ﺫﺍﺕ

ﺃﻫﻤﻴﺔ ﻓﻲ ﺍﻟﺩﺭﺍﺴﺎﺕ ﺍﻟﺘﺼﻨﻴﻔﻴﺔ.

ُﺩﺭﺱ ﺘﺄﺜﻴﺭ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻜﻴﻤﻴﺎﺌﻴﺔ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻋﻠﻲ ﺤﺠﻡ ﺤﺒﻭﺏ ﺍﻟﻠﻘﺎﺡ، ﻭ ُﺠﺩ ﺃﻥ ﺤﺒﻭﺏ ﺍﻟﻠﻘـﺎﺡ

ﺍﻟﺘﻲ ﺃﺨﻀﻌﺕ ﻟﻠﻤﻌﺎﻤﻠﺔ ﺒﻭﺍﺴﻁﺔ ﺍﻟﺘﺤﻠﻴل ﺍﻟﺤﻤﻀﻲ ﻗﺩ ﺘﺄﺜﺭﺕ ﻓﻲ ﺍﻟﺤﺠـﻡ ﻟـﺫﻟﻙ ﺘـﻡ ﺘـﺼﺤﻴﺢ

ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺘﻲ ُﺍ ﺨﺫﺕ ﺒﻭﺍﺴﻁﺔ ﻤﻌﺎﻤل ﺍﻟﺘﺼﺤﻴﺢ (L) ، ﺍﻟﻌﻼﻗـﺔ ﺒـﻴﻥ ﺤﺠـﻡ ﺤﺒـﻭﺏ ﺍﻟﻠﻘـﺎﺡ

ﻭﺍﻟﻤﻌﺎﻤﻼﺕ ﺍﻟﻜﻴﻤﺎﺌﻴﺔ ﺍﹸ ﻗﺘﺭﺤﺕ ﻤﻥ ﻗﺒل ﻜﺭﻴﺴﺘﻴﻥ ﻓﻰ ﻋﺎﻡ 1946 ﻭﻗﺩ ﻭﺍﻓﻘﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻟﻤﺘﺤـﺼل

ﻋﻠﻴﻬﺎ ﻓﻲ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﻤﻘﺘﺭﺤﺎﺘﻬﺎ.

x ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ

CHAPTER ONE INTRODUCTION

Pollen grains are structures that house the male gametophyte generation of angiosperms and gymnosperms (seed plants). They are also the vehicles in which the male gamete genetic code is carried to the female gamete. Pollen grains develop in the anther in angiosperms. The pollen grains travels and deposits on the stigma of a receptive flower. In gymnosperms, pollen develops in the male cone, travels, and fertilizes the ovules in the female cones to produce seeds. Each pollen grain consists of the two celled male haploid plant enclosed in a thickened wall. The casing that houses the male gametophyte has a very complex structure which reflect the specific species' functional adaptations. The exine is the outer layer of a living pollen grain; it composed of sporopollenin and consists of complex polymers with a basic formula [C90H142O36] n with small quantities of polysaccharides, (Birks and Birks, 1980). Sporopollenin is chemically stable and resistant to almost all kinds of environmental damage. Exine is equipped with apertures and is divided into two sub layers: the outermost sexine and the unsculptured underlying nexine. The sexine has surfaces that are sculptured in elaborate ways, with reticulately arranged perforations; which give the exine an amorphous granular appearance. The inner layer of a living pollen grain is called the intine. It is composed of cellulose and is similar in construction to ordinary plant cell walls. A layer called the endexine separates the sexine and intine. The endexine has a laminated appearance (Fig. 3). Pollen grains are generally classified according to their physical appearance. There are three criteria for classification:

1 1) The number and position of the apertures. 2) The shape of the pollen grain as a whole. 3) The fine elaborate structure on the sexine. Apertures are any missing parts of the exine, which are independent of the exine pattern. Apertures are big and they cut across the fine structure pattern on the surface of the pollen grain. There are two types of apertures: pori or pores are mostly isodiametric apertures, although, they can be slightly elongated with rounded ends, colpi or furrows are long and boat shaped with pointed ends. Colpi are thought to be more primitive. In living pollen grains these apertures are not actually open. Instead, a very thin layer of exine covers them. Grains with pori are called porate; those with colpi are called colpate, and those with both pori and colpi are called colporate. The number of apertures is also indicated by prefixes: mono- for one aperture, di- for two apertures; tri- for three apertures, and so on (Fig. 2). The shape of a pollen grain refers to the shape of their outline in polar and equatorial views. The shape of a grain can sometimes be useful in identifying pollen species. It may vary considerably within one grain type, and sometimes within one species (Fig. 1). The sculpture refers to the fine structure and pattern of the sexine. It is composed of small radially directed rods. If these rods support something (such as a plate or a small knob) they are called columnellae, if they do not support anything they are called baclate. The shape of the rods can further classify them. If they have club shaped they are called clavate, if they are sharply pointed they are called echinae, if they have swollen heads they are called pila; and if they are short and globular they are called gemmae (Moore et al. 1978). There are many more classifications for the shape of rods on the surface of the sexine, but these four are the most common (Fig. 4). Each plant species has specific shape, size, and structure of its pollen grain. This is used in

2 identifying plant species in the fossil remains found in deposits and other sites. Pollen analysis methods are usually used to prepare slides of different species.

Objective of the study: The aim of this study is to describe the pollens of plant species in Erkwit and to prepare reference slides of these species. Ecologically Erkwit is very important being of unique nature and special vegetation. It is hoped that the vegetation history of the area will be studied to see the effects of climate and soil. The description of pollen is very important since vegetation history can only be possible by using pollen analysis. Therefore reference slides of different pollen types are essential. The study covers a selected number of plants species and their pollen grains. This will provide additional characters to be used in plant taxonomy. Reference slides will be prepared for further examination and use in palaeoecological studies that are expected to be done in this area. The effect and different treatment in pollen grains size will also be studies. Correction factors are calculated when measuring the pollen size.

CHAPTER TWO LITERATURE REVIEW

2.1. Palynology and pollen analysis:

Palynology, a term coined by Hyde and Williams cited by Erdtman (1971), means the study of pollen and spores, generally focusing on the structure of the walls rather than living internal features. Pollen analysis was born in 1916 and in the beginning the use of the technique was limited to the study of Quaternary lake and bog deposits. Today pollen analysis is by far the most important method for the reconstruction of past vegetation and environments. Lennart von Post (1918) laid out the fundamental approach of quantitative pollen analysis, though the techniques and equipment have been developed and improved since then Faegri and lversen (1989). Erdtman (1934) published the acetolysis technique. The preparation, however, removes cellular content and the intine layer and some times produces unnatural shaped pollen grains. Palynology has proven to be useful in the study of taxonomic groups. The application of palyological data has proven to be of value in interpreting problems related to origin, migration, and evolution of flora as well as some of scientific studies such as palaeoecology, archeology, ethnobotany, distribution and reproductive biology.

Pollen analysis is a technique for reconstructing former vegetation by means of the pollen grains it produced. While palynology technically refers to the study of both pollen grains and spores, these will be referred to collectively as ‘pollen’ for the sake of convenience. Pollen analysis has been used to document long-term vegetation dynamics ever since the success of von Post’s pioneering experiments in 1916 (Birks, 1993). The basic assumption of the

4 technique is that the number of pollen grains deposited per unit time, at a given point, is directly related to the abundance of the associated species in the surrounding vegetation. However, pollen data are presented as proportions of a total pollen sum, rather than as discrete numbers (Davis, 1963). Therefore, difficulties with the representivity between and within species are experienced, as some taxa produce far greater quantities of pollen, which are more widely dispersed than others (Birks and Birks, 2005). In other words, pollen data require careful interpretation as the representivity of the pollen spectrum is shaped by differences in pollen productivity, dispersal and preservation (Faegri and Iverson, 1989). Pollen grains are well suited to analysis for a number of reasons: (1)They have extremely resilient exines, which allow for their survival in deposits where other fossil types have been destroyed. (2) They are abundantly produced. (3) They are widely and evenly dispersed. (4) Pollen data are easily quantified (Faegri and Iverson, 1989). It is useful to think of pollen analysis as a remote sensing instrument, which records the past and present composition of vegetation (Webb et al., 1978). It has certain response characteristics, which limit its application to certain contexts (Prentice, 1988). Pollen data have been used in a wide variety of Quaternary applications including; chronostratigraphic correlation, palaeoecology, palaeoclimatology and archaeology (Macdonald, 1988). Data derived from pollen studies can be used to provide an indication to the response of natural vegetation to human impacts through history, as well as to climatic and environmental change (Prentice, 1988; Edwards and Macdonald 1991). Birks (1981) attributes the importance of the pollen record as a source of palaeoclimatic information to both its length (102 - 105 years) and sample resolution (10-1000 years). At the largest spatial scale, pollen data have been used to reconstruct past changes of biomes, using pollen records from entire

5 modern biomes as a basis (Jolly et al., 1998; Elenga et al., 2000). In addition, Quaternary palynological data constitute a valuable quantitative record against which climatic models such as general circulation models (GCMs) can be validated; for studies of global change. These data strengthen predictions of how vegetation is likely to respond to future climatic conditions, there by providing an indication of the future agricultural and silvicultural potential of various regions (Huntley, 1990).

2.2. Pollen taxonomy:

Pollen taxonomy is essential for the identification of fossil pollen and spores to reconstruct past floras and palaeoenvironments. While acknowledging the limitations of relating 'actuo' pollen morphology to palaeopalynological studies (Joosten and de Klerk, 2002), palaeoenvironmental and plant history investigations still benefit from the knowledge of modern pollen for comparison. This assists the interpretation of pollen types and the taxa they are likely to represent. In the case of vegetation and environmental history studies it is particularly important to have an understanding of the variability, as well as the similarities, in the pollen flora to assist the interpretation of fossil pollen records. Superficially similar pollen types from the same family may represent genera or species with very different habits and/or habitat/environmental conditions (Erdtman, 1943). Pollen wall patterns are so diverse and often so characteristic of each species that they have long been used for taxonomic classification and even for forensic identifications (Szibor et al., 1998). In a study of pollen of the taxonomically complex Acacia genus in Australia, Guinet (1986) has found a progressive morphological series within the pollen following a north/south gradient in Australia, the results agree with current taxonomic views based both upon gross plant morphology and biochemistry.

6 Exine also varies in the number, distribution, and architecture of the apertures. Apertures are diverse across taxa, within families, within species, and even within a single plant (Mignot et al., 1994). Pollen from monocots characteristically has a single aperture, a trait considered to be ancestral. Most dicot pollen grains have three apertures.

2.3. Pollen morphology:

Pollen morphology supports the taxonomic suggestion to separate the species in a special section or genus, (Keri and Zetter, 1992). Also it supports a phylogenetic study of molecular and morphological data by Bradford and Barnes (2001) who placed Eucryphia in Cunoniaceae rather than Eucryphiaceae, and that was consistent with molecular studies. Zavada’s (1983) study found: (1) Wall structure and exine sculpturing were the best diagnostic pollen characteristics for determining subfamilial affinities in Ulmaceae. (2) The pollen morphological characteristics of the subfamily Celtidoideae (which includes Trema) are more like those of Moraceae than of subfamily Ulmoideae. (3) The pollen morphology supports family status for Celtidoideae, which would occupy a phylogenetically intermediate position between the Ulmaceae (Ulmoideae) and the Moraceae. Perveen and Qaiser (2002) investigated three species, namely P. laxmanni Pall., P. nubicola Wall. ex Royle and P. palustris, in Pakistan. Previous studies have suggested that Parnassia is a stenopalynous. The pollen grains of some Parnassia species are prolate to spheroidal and tricolporate; tetracolporate and syncolpate grains also occur in this genus (Simmons 2004). The work of Erdtman (1952) about the morphology and taxonomy of pollen grains is essential for those who work in this field. Shaw (1971) described the chemistry of sporopollenin. Keys and manuals for identifying

7 pollen grains are numerous, Hyde and Adams (1958), Moore et al. (1978) and Punt (1976). Previous works on pollen morphology are few in Sudan. El Amin (1972) recognized four pollen types, El Ghazali (1989 and 1993) described and made a key of more than three hundred Sudanese pollen types, Kordofani and Ingrouille (1992) described the pollen morphology of some Acacia species.

2.3.1. Shape:

The shape of pollen varies in different views (Fig. 1). The outline in polar view is circular, triangular, or in other geometrical shape, while in the equatorial view, the ratio between the polar and equatorial diameters multiplied by 100 gives an indication of the shape. Accordingly, the shape obtained from the formula:

P × 100 E P ≡ Polar axis diameter E ≡ Equatorial axis diameter

If the results is less than 50 it represents peroblate grains, 50-75 oblate grains, 75-88 suboblate grains, 88-100 oblate spheroidal grains, 100-114 prolate spheroidal grains, 114-133 subprolate grains, 133-200 prolate grains, over 200 perprolate grains (Erdtman, 1971).

8 P E P E

Fig. (1) Polar (P) and equatorial (E) shapes in pollen grains Fægri, and Iversen (1989).

9 2.3.2. Apertures:

Pollen grains are generally aperturate, the apertures being elongated or circular. The elongated apertures are termed as colpi and the circular ones as pori (Fig. 2). Apertures are diverse across taxa, within families, species, and even within a single plant (Mignot et al., 1994). Pollen from monocots characteristically has a single aperture, a trait considered to be an ancestral. Most dicot pollen grains have three apertures, although in both instances, aperture numbers have increased or decreased repeatedly and independently during the course of evolution. At the extremes, pollen exines from some species lack apertures entirely whereas, others are ‘‘omniaperturate,’’ lacking exine or coated everywhere with a very thin exine layer (Pacini and Franchi, 1991, Rowley et al., 1997). The positions of the apertures are set during the tetrad stage, when gaps in the exine form at sites of close position between the plasma membrane and the adjacent callose wall (Heslop-Harrison, 1968). The mechanism of aperture placement is still unclear, but it has been linked to microsporocyte cytokinesis (Hulskamp et al., 1997; Spielman et al., 1997 and Yang et al., 2003). Several apertures seem alike although, it has been difficult to link experimentally according to aperture position and number. However, there are logical functions for apertures: (1) sites for egress and ingress of water during pollen desiccation and hydration. (2) structural components that distribute stresses caused by grain shrinkage and swelling (termed harmomegathy). (3) portals for the exit of the pollen tube. First, although the exines of some pollen grains are perforated with microchannels (Rowley et al., 1959), others are impermeant to water and rely on apertures to regulate grain desiccation and hydration. Consequently, some

10 types of apertures are associated with certain plant types, especially with wet or dry stigmas. For instance, Heslop-Harrison (1979) discussed the association between multiple circular aperture (forate) exines and dry stigmas, hypothesizing that their adaptive value lies in their ability to focus water entry into the grain. Second, rigid walls are liabilities for pollen grains that undergo enormous volume decreases in the anther and subsequent increases on the stigma surface. In this context, apertures are analogous to architectural spacers in concrete, absorbing and permitting necessary shifts without breakage. The elasticity of the sporopollenin wall itself also is valuable for these deformations (Rowley et al., 2000). Third, it has long been recognized that pollen tubes exit through apertures in many pollens. Curiously, this behavior is by no means universal; some pollens lack apertures altogether, whereas others, such as Arabidopsis pollen, have apertures but do not regularly use them for tube emergence. A comparative study of tube emergence across taxa could yield insight into the evolution of the mechanisms necessary for different forms of tube emergence. Although, apertures provide an important means of communication between pollen and the outside world, they also are sites of vulnerability to excess desiccation and invasion by fungal hyphae (Bih et al., 1999).

Fig. (2) Different kinds of pollen grains apertures Fægri, and Iversen (1989).

12 2.3.3. Exine stratification:

The exine refers to the chemically resistant outer layer of the wall of pollen and spores, and is primarily composed of sporopollenin. Sporopollenin is a substance composed of oxidative copolymers of carotoid and carotenoid esters. It is an extremely durable substance and can be found in anaerobic sediments dating back hundreds of millions of years. Various terms have been used to define the layers that make up the exine. Following Erdtman (1971), the inner, non-sculptured layer is the nexine lying below the outer, usually sculptured sexine. The sexine is usually stratified, consisting of sculpture elements on the outer surface of a layer called the tectum which overlies a layer of columellae (rod-like elements supporting the tectum) or a combination of these three layers. Layers in the sexine, such as the tectum, may be complete (continuous), partially present (discontinuous) or completely absent, resulting in characteristic surface patterns (sculpturing types). When a columellae layer is present the exine can be described as columellate (Fig. 3).

Fig. (3) Exine Stratification of pollen grains Fægri, and Iversen (1989).

14 2.3.4. Sculpturing types (Ornamentation):

Differences in the exine structure, in the way various layers are structured and the elements arranged, are responsible for the different types of sculpture or ornamentation seen in pollen grains. The main terms used in this study to describe exine surface sculpturing follow Faegri and Iversen (1989) and Moore et al. (1978). This include smooth (or psilate), perforate (surface pitted with holes < 1 µm diam.), granulate (or scabrate), striate, rugulate and reticulate ornamentation. Reticulate are further defined as micro or macro when the lumina i.e. the spaces between the muri or walls, the reticulum are < 1 µm, 1–2 µm or > 2 µm wide, respectively. The ornamentation is microreticulate when the muri and lumina are smaller than 1 µm. When pollen grains have projections or spines, they may be: rod-shaped where the elements are of even thickness and longer than broad (baculate); wart-like where the elements are equal or broader than high and not constricted at the base (verrucate); club-shaped where the elements are longer than broad and narrowed towards their base (clavate); drumstick-shaped with a short shaft and swollen top (pilate); short, globular and basally constricted (gemmate); or sharply pointed (echinate). Echinae are referred to as spinules when they are ≤ 3 µm long or spines when > 3 µm long (Fig. 4). Schols et al. (2001) reported perforate, striate and micro reticulate sexine patterns in the family Dioscoreaceae. Ornamentation may not only be less distinct under light microscope (LM) but can appear different to the type of sculpture revealed by scanning electron microscope (SEM) analyses.

Fig. (4) Pollen grains Sculpturing types Fægri, and Iversen (1989).

16 2.3.5. Size:

A contrasting pattern of variation in pollen size among and within species implicates the action of strong selection promoting pollen performance. Among angiosperm species, pollen volume varies almost five orders of magnitude (Wodehouse, 1935; Muller, 1979), implying diverse opportunities for pollen-size evolution. However, within species pollen diameter commonly has a coefficient of variation less than 5%, Vonhof and Harder (1995), suggesting that particular reproductive conditions favour one specific pollen size. According to this interpretation, adaptive explanation of pollen-size variation in angiosperms lies in understanding how pollen size affects a plant’s siring success.

2.3.5.1. Effect of different treatments in pollen grain size:

It is common knowledge among palynologists that the size of a pollen grains is affected by both chemical treatment (Christensen, 1946) and mounting media (Andersen, 1960). As pointed out by Christensen (1946) the change in size of pollen grains under various conditions is of considerable interest because the dimensions of such grains can be used as a means of characterization and identification. Unfortunately such an identification based on the dimensions of a pollen grain may be erroneous if attention is not given to the method applied. Wenner (1947) compared the size of pollen grains caused by boiling in potassium hydroxide and by boiling in an acetolysis mixture. He found that in most cases acetolysis caused a significant increase in size when compared with the potassium-hydroxide treatment. Ting (1966) examined changes in size of pollen grains of Pinus under various conditions. According to his results acetolysed pollen grains produce the same mean size as untreated pollen grains mounted in glycerin jelly.

17 He stated "the cause of expansion is vaporization of latent moisture owing to intense heating, either directly applied or generated by chemical reaction". Cushing (1961) reported a relationship between the distance of the cover glass and the slide, which he called the thickness of the slide, and the swelling of pollen grains. The size was found to be directly proportional to the thickness of the slide, in other words to the amount of pressure on the grains by the cover glass. Pollen grains mounted in slides thicker than the grains did not show any swelling even after long storage. Faegri and Iverson (1989) measured 20 grains of corylus avellana for each preparation and averaged. The measurement taken were converted to the standard size by dividing the size measured (P, E, D) by a conversion factor (K).

CHAPTER THREE MATERIALS AND METHODS

3.1. The study area:

Erkwit, a deserted summer resort, lies at about 45 Km to the South West of Suakin on the Red Sea, and about 30 Km East of Sinkat on the railway line. It is a plateau about 1000-1200 meters above Sea level. The area lies a proximately at latitude180 48', longitude 370 10' in eastern Sudan, (Kassas, 1956). The area geographically is part of the semi desert, but due to its elevation and proximity to the Red Sea, it is strikingly different in climate and vegetation. The vegetation of Erkwit is rich and shows a great deal of diversity.

3.1.1. Topography and geology:

The Erkwit plateau lies at the edge of a steep escarpment dropping abruptly (2000 ft., c.600m.) to the Red Sea plains. At the northern boundary is Jabel Nakeet (3921 ft., c.1176m.) and Jebel Essit (3810 ft., c. 1143m.). The two Jebels drop to Khor Dahand which separates the Erkwit oasis from the barren hills on the other side of the Khor. At the eastern boundary is Jebel Sela (4244 ft., c.1273m.) which is the highest evergreen mountain of the district. At the southern boundary is Jebel Tatasi (3967 ft., c. 1190m.), Jebel Lagagribab (4030 ft., c. 1209m.) and Jebel Auliai(3970 ft., c. 1191m.) which are separated, by Khor Amat, from the barren mountains further south Jebel Erbab (5077 ft., c. 1523m.). At the western boundary are Jebel Hadast (3826 ft., c. 1147 m.) and Jebel Mashokriba (3710 ft., c.1113 m.) which drop to Khor that separates the Erkwit plateau from the desert plains to the east.

19 3.1.2. Rainfall:

Erkwit, receives a rainfall greater than the neighboring areas represented by the two towns: Suakin to the north-east and Sinkat to the west. Suakin, a Red Sea port, represents the coastal climate with winter rainfall. Sinkat, which lies on the inland plain, represents area with summer rainfall. Erkwit, lies in between (Fig.1) and receives both the summer and winter rainfall. As Tothill suggests,…، the climate of Erkwit may be due to the happy combination (1948) of three things : latitude, situation and elevation; its latitude (18o 46' N.) is close to latitude 19o which divides the Sudan into a desert region to the north and tropical continental region to the south (Ireland, 1948). During the winter months, the Erkwit plateau is frequently swathed in clouds for weeks. This entails considerable dew precipitation which is more marked the higher the elevation, and which supplies the vegetation with a valuable water resource.

3.1.3. Vegetation:

Andrews (1947) suggests a tri-zonal pattern of Erkwit vegetation: namely, an arid zone, a transitional zone and a moist zone, without attempting a detailed survey of the features of these zones. The vegetation has been studied by Kassas (1956), who divided the area into zones that show different elevations and distances from the sea, namely: zone I, this zone faces directly the water-laden winds and sea mists as they roll inshore. Mosses and lichens are commonly found on trunks and twigs of trees and shrubs. Maytenus senegalensis is dominant and is characteristic of the zone. Zone II lies in between the previously described moist zone I and the Euphorbia sp. dominated zone III, Maytenus senegalensis and Euphorbia abyssinica are dominant as is characteristic of this zone. Zone III occupies the middle part of the Erkwit oasis. The outstanding feature is

20 the dominance of Euphorbia abyssinica. Zone IV lies on the south west boundary of the area and hence receives the sea mists and water-laden winds only after they have lost the greater part of their moisture. Mosses and lichens are extremely scarce; Maytenus senegalensis and Euphorbia abyssinica are dominant. Zone V to the west of zone IV and separating it from the desert plain that extends west of Erkwit, is a fringing zone where Euphorbia thi is most common. ZoneVI lies outside of Erkwit oasis, Aloe sp. is the most common plant in the zone.

Map (1) Erkwit showing main Khors and zones after Kassas (1956).

22 3.2. Chemicals: All general chemicals were of analar grade or equivalent. Some chemicals were from BDH chemicals (England) and Sigma Chemical Company (U.K). 3.2.1. 10 % Potassium Hydroxide: Potassium Hydroxide (100 g) pellets were dissolving in 900 ml distilled water. 3.2.2. Acetolysis mixture: The solution was prepared by mixing 9:1 acetic anhydride and conc. sulfuric acid (94-96%) mixed just prior to use. 3.2.3. Glacial acetic acid: Concentration of the glacial acetic acid used was 97-98 %. 3.2.4. Basic fuchsin: The dye was prepared by dissolved 0.01 g of basic fuchsin in 200 ml distilled water. 3.2.5. Glycerin: Concentration of the glycerin used was 85%. 3.2.6. 10 % Hydrochloric Acid: Distilled water 900 ml plus 100 ml conc. hydrochloric acid. Measure with graduated cylinder; pour acid slowly into water. 3.2.7. Lycopodium sp. spores tablets. 3.3. Equipment: Fume hood: All reactions were done in a fume hood. Centrifuge, device LAB EUGF 1 model KOLB, scientific technical supplies D-6072 Dreleich-West Germany (x1000 rev/min). Electric hot plate. Water bath. Test-tube rack. Acid, base, and solvent storage.

23 Conical centrifuge tubes. Glassware: Graduated cylinder (50 ml and 1000 ml). Heavy duty beakers (25 m, 250 ml and 1000 ml). Several bottles for storing solutions. Adjustable pipette. Pasteur pipettes (short, disposable) for taking sample check material. Microscope slides and cover slips for checking samples. Sieves (ca. 0.2 mm). Stir sticks: Glass stir rods. Vials: to keep final samples. Nail polish.

3.4. Sampling:

This work involves study of 30 species belong to 17 families collected either as fresh material in the field from Erkwit area in the period from February to March 2006, or taken from Herbarium of Khartoum University Faculty of Science Botany Department. Flowers or pollen bearing plant parts were collected just at the point of their opening to avoid pollen contamination and that the anthers were not emptied.

3.5. The Pollen Preparation:

In this study pollen morphology of about 30 plant species have been investigated. Collection include fresh polliniferous samples of dominant families which were obtained from different sites in Erkwit area in the period from February to March 2006, as well as specimens from University of Khartoum herbarium at Botany department.

24 For each species, three samples were studied, pollen grains were fully described and different measurements were taken, families and species were arranged alphabetically. After collection of the flowers bearing the pollen grains, the samples were acetolysed according to the standard acetolysis method for recent pollen grains Erdtman (1934 and 1960); Faegri and Iversen (1989) modified by El Ghazali (1989), to remove all the cellulose as well as other cell constituents that may be present and that obscure the exine characters. Each sample was put in a beaker then subjected to the following treatments: 1- One marker pollen tablet (Lycopodium sp. spores) was added to each sample and five ml of 10% HCL was added to remove carbonates that covered the marker pollen tablets. 2- 10% KOH was added and gently stirred then transferred to a 900 C water bath for 10 minutes, then removed from heat and washed with distilled water through a sieve (ca. 0.2 mm). 3- Sample was then centrifuged at 30.000 rpm for three minutes then decanted. Three ml of conc. glacial acetic acid were added to remove water from sample; mixture was gently stirred, centrifuged at 30.000 rpm for three minutes then decanted. 4- After that the sample undergoes acetolysis to remove some organic matter, clean surface of grains, and stain them with golden brown color. Acetolysis mixture was prepared fresh (1part concentrated sulfuric acid to 9 parts acetic anhydride) while the samples were centrifuged after glacial acetic acid was added; 3-5 ml of mixture were added to each sample slowly since the reaction is exothermic, then transferred to a 900 C water bath for three minutes, centrifuged at 30.000 rpm for three minutes then decanted. Addition glacial acetic acid was repeated and samples were washed with distilled water and boiled with 10% KOH for five minutes.

25 After the final centrifugation and decantation, the samples were washed three times with distilled water, samples were stained with basic fuchsin, stored in small stock vials then put on electric hot plate to evaporate water. After eva- poration glycerin was added. Samples of the prepared material were mounted in glycerin on slides, covered with cover-slips and sealed with colourless nail polish. Light microscopic (LM) examinations were done with a Zeiss microscope using phase contrast (ph) objectives ×100 magnification. then LM photographs were taken with a Zeiss axiomat. Automatic Camera attachment model E 4500 was used.

3.6. Measurements:

The grains were measured with seven replicates for each species from each site under ×100 magnifications using a calibrated micrometer. The measurements include dimensions of polar axis (P) and equatorial axis (E) or diameters (D) of all the pollen grains examined. The measurements were taken from different parts of the slide since smaller grains tend to move towards the edges when the cover slip is on (Brookes and Thomas, 1967). Pollen grains were affected by both chemical treatment (Christensen, 1946) and mounting media (Andersirn, 1960), the measurements taken were converted to "standard size". This was achieved by multiplying the sizes measured (P, E, D) by a correction factor (L).This correction factor (L), is the comparison of the measured size of Lycopodium sp. for each preparation with the standard mean size of Lycopodium sp. (= 40.6). Accordingly measures were corrected for each sample according to the following equation:

Correction factor (L) =

Standard lycopodium sp. size – sample Lycopodium sp. size Standard lycopodium sp. Size

P/E is multiplied by 100 for obtaining general shape of a pollen grain from the table previously set by Erdtman (1947, 1952).

3.7. Species description and terminology:

For all the specimens examined a uniform system of pollen morphological description was followed. The definitions and terminology, listed in this study, follow various authors: Erdtman (1952, 1947) for shape classes, Faegri and Iversen(1989) for pollen classes, Faegri and Iversen (1989) and Praglowski and Punt (1973) for sculpturing. In addition to pollen morphological description, the synonym (s), vernacular name(s), habit, distribution were also given. The nomenclature, synonym(s), habit and distribution were after Andrews (1950, 1952 and 1956) and Hassan (1974).

27 CHAPTER FOUR RESULTS

Pollen descriptions are provided in alphabetical order by family, genus and species. Abbreviations and measures are put between brackets.

Family: Anacardiaceae

Genus: Rhus L.

Scientific Name: R. abyssinica Hochst. (F).

-Vern. Name: (Ar.) Sumub.

Shape Class: prolate. LM polar view Dimensions: P = 28.9µm (28.4 – 29.4). Ε = 21.4 µm (20.6 − 22.3) , P/E = 1.35, L = 0.19. Pollen Class: tricolporate. Sculpturing: striate. Habit: small tree. Leaves trifoliate. Flower brownish-white, Fruit small, brown. LM equatorial view

Distribution in Erkwit: Jebel Dahand .

Family: Apocynaceae

Genus: Carissa L. Scientific Name: C. edulis Forssk. (Vahl.) 1790 (F). -Vern. Name : (Ar.) Alalli.

Shape Class: oblate spheroidale. Dimensions: P = 42.7µm (45.2 – 40.2)

Ε = 43.7 µm (46.1 − 41.3) , P/E = 0.97, L = 0.14. LM polar view Pollen Class: tricolporate. Sculpturing: psilate.

Habit: the plant is spiny evergreen shrub or small tree to 5 m. The bark is grey, smooth with straight woody spines to 5cm, often in pairs, rarely branching. Milky latex.

Distribution in Erkwit: zone 1 and zone 2.

Family: Arecaceae

Subfamily: Polemonioideae Genus: Phoenix L. Scientific Name: P. dactylifera L. 1753 (F). - Syn.Palma major Garsault; Phoenix cycadifolia hort., Attens ex Regel. -Vern. Name: (Ar.) Nakhl (Nakhal), Tamar, Temer; (Engl.) Date palm.

Shape Class: prolate. Dimensions: P = 28.9µm (28.4 – 29.4), E = 17.7µm (17.3 –18.2), P/E = 1.63, L = 0.19. Pollen Class: monocolporate. Sculpturing: reticulate. LM polar view

Habit: monoecious. This tall evergreen, unbranched. The trunk is covered with fibers, is surrounded from the ground upward with the base of earlier formed leaves. The end of the leaf fronds are needle sharp. The fruit is a drupe with one seed.

Distribution in Erkwit: cultivated in Erkwit Zone III.

Family: Asclepiadaceae

Genus: Calotropis L. 1770, nom. cons. Scientific Name: C. procera (Ait.) Ait.f. (F). -Vern. Name: (Ar.) Oushar, (Engl.) Sodom Apple, giant milkweed.

Form: pollinia. Shape: oblanceolate. Dimensions: maximum length: 1.89 mm (1.97 - 1.81), Maximum breadth: 0.75 mm (0.92 – 0.57), L = 0.01. Pollen Class: inaperturate. LM horizontal view Sculpturing: psilate.

Habit: small tree to 4 m, exuding copious milky sap when cut or broken; leaves opposite, grey-green, with a pointed tip, flowers waxy white, petals 5, purple-tipped inside and with a central purplish crown, carried in stalked clusters at the ends of the branches, fruit grey-green.

Distribution in Erkwit: widespread.

Family: Balanitaceae (Simarubaceae)

Genus: Balanites. Scientific Name: B.aegyptiaca (L.) Del. (F). - Syn. Ximenia aegyptiaca L., Agialida senegalensis vin Tiegh., Balanites ziziphoides Milbr. -Vern. Name: (Ar.) Hijlij, (Engl.) Desert Date.

Shape Class: subprolate. LM polar view Dimensions: P = 41.3µm (43.1 – 39.4), E = 35µm (37.3 –32.8), P/E = 1.18, L = 0.19. Pollen Class: tricolporate. Sculpturing: striate.

Habit: spiny shrub or tree up to l0 m tall. Crown spherical, in one or several distinct masses. LM equatorial view Trunk short and often branching from near the base. Bark dark brown to grey. Flowers in fascicles in the leaf axils. Fruit sweet and edible.

Distribution in Erkwit: Khor Aiet.

Family: Capparidaceae

Genus: Cadaba Forssk, Atilde and yen;l, 1775 Scientific Name: C. farinose Forssk (F). - Syn. C. apiculata Gilg & Bened. - Vern. Name: (Ar.) Al_saraha.

Shape Class: subprolate. LM polar view Dimensions: P = 28.9µm (30.9 – 26.9), E = 23.3µm (26.2 – 20.4), P/E = 1.24, L = o.o8. Pollen Class: tricolporate. Sculpturing: microechinate.

LM equatorial view

Habit: slender shrub with a strongly furrowed stem, rarely straight with a yellowish grey barks. Young twigs densely covered with sessile or subsessile scales, sometimes mixed with stiff glandular and eglandular hairs. Leaves small.

Distribution in Erkwit: Khor Aiet, Khor Abent.

Genus: Capparis Linnaeus, 1753 Scientific Name: C. decidua Forssk. (F). -Syn. Sadada decidua Forssk; Capparis aphylla Heyne ex Roth. -Vern. Name: (Ar.) Tundub, Humbuk.

Shape Class: subprolate. LM polar view Dimensions: P = 21.9µm (19.6 – 24), E = 16.8 µm (18.9 – 14.6), P/E = 1.30, L = 0.11. Pollen Class: tricolporate. Sculpturing: striate.

LM equatorial view

Habit: small tree with many green Vine-like apparently leafless branches, hanging in bundles. Bark turns whitish-grey colour with age, but most branches and twigs are a glossy dark green. Small, light brown spines.

Distribution in Erkwit: Zone 4.

Genus: Maerua Scientific Name: M. angolensis DC (F). -Syn. M. tomentosa Pax (1891). , M. bukobensis Gilg & Bened (1915)

-Vern. name: (Ar.) Shagar elzaraf. LM polar view

Shape Class: subprolate. Dimensions: P = 20.5µm (22 – 19.1), E = 17.3µm (19.5 – 15.2), P/E = 1.18, L = 0.05. Pollen Class: tricolporate.

Sculpturing: scabrate. LM equatorial view

Habit: tree of up to 20m heights. Growing in bush and rocky areas. Leaves are small; seeds are born in chained pods. Produces abundant leaves in the dry season. The stem is white in color.

Distribution in Erkwit: Gebel Dambobei, Khor Handoub.

Family: Ebenaceae

Genus: Diospyros Linnaeus, 1753 Scientific Name: D. mespiliformis Hochst. (F). - Syn. Maba abyssinica Hiern. -Vern. name: (Ar.) El Jogan, (Engl.) Jackalberry

Tree, Jakkalbessie, African Ebony. LM polar view

Shape Class: prolate. Dimensions: P = 48.8µm (51 – 46.6), E = 33.9µm (36.1 – 31.7), P/E = 1.44, L = 0.12. Pollen Class: tricolporate. Sculpturing: psilate. LM equatorial view

Habit: tall, upright tree that can reach a height of 25 m. The bark is black to grey; the fresh inner skin of the bark is reddish. Flowers are cream-colored and bell-shaped. Male flowers are arranged in stalked bunches and female flowers are solitary. The fruit is a fleshy berry.

Distribution in Erkwit: Zone 1 and zone 2.

Family: Euphorbiaceae

Genus: Euphorbia L Scientific Name: E. abyssinica J.F. Gmel. (F). - Syn. E. erythraeae (Berger) N.E.Br. - Vern. Name: (Ar.) Shajar alsim; (Engl.) Desert Candle.

Shape Class: subprolate. Dimensions: P = 54.6µm (60.5 – 48.7), E = 43.4µm (48.4 –38.4), P/E = 1.26, L = 0.12. LM polar view Pollen Class: tricolporate. Sculpturing: reticulate.

Habit: arbores cent species from Ethiopia forming a tree. The erect- angled branches have very deep angles and are edged with a border of closely packed, paired spines to 1cm long.

Distribution in Erkwit: widespread.

Subfamily: Crotonoideae Genus: Jatropha Linnaeus, 1753 Scientific Name: J. gossypifolia L. (F). - Syn. Adenoropium gossypiifolium (L.) Pohl. -Vern. name: (Engl.) Bellyache Bush.

Shape Class: circular Dimensions: D = 52.1µm (54- 50.2), L = 0.03. Pollen Class: inaperturate. Sculpturing: clavate.

LM polar view

Habit: evergreen shrub, erects monoecious shrubs with conspicuous glandular hairs and young foliage dark red to purple, up to 4 m high, fruits and foliage are toxic to humans and animals.

Distribution in Erkwit: Khor Ashat.

Subfamily: Crotonoideae Genus: Jatropha Scientific Name: J. hastata Griseb (F). -Syn. Adenoropium hastatum (Jacq.) Britton and Wilson, Jatropha integerrima Jacq. var. hastata (Jacq.) Fosberg. -Vern. Name: (Engl.) Physic nut, Spicy Jatropha.

Shape Class: circular. Dimensions: D = 62.6µm (56.5- 68.7), L = 0.15. Pollen Class: inaperturate. Sculpturing: clavate. LM polar view Habit: small trees to 20 feet, with red flowers. The stems are smooth or slightly hairy. It has thin, often greenish bark. Fruit globular to 3 angled small capsule-like.

Distribution in Erkwit: Khor Gwob.

Subfamily: Crotonoideae. Genus: Jatropha L. 1753. Botanical Name: J. integerrima Jacq. (F). - Syn. Jatropha pandurifolia Andr. -Vern. Name: (Engl.) Coral Plant, Peregrina, Physic Nut, Rose - Flowered Jatropha, Spicy Jatropha.

Shape Class: circular. Dimensions: D = 66.4µm (62.6- 70.2), L = 0.04. Pollen Class: inaperturate. Sculpturing: clavate.

LM polar view

Habit: evergreen shrub or small tree with glossy leaves and clusters of star shaped bright scarlet or vermilion flowers. The plant has a rounded or narrow domed form and gets up to 15 ft (4.6 m) tall.

Distribution in Erkwit: Khor Ashat and Khor Gowb.

Subfamily: Crotonoideae Genus: Jatropha L'HÃ©ritier ex W. Aiton, 1789 Scientific Name: J. podagrica Hook. (F). - Vern. Name: (Engl.) Bottleplant Shrub, Buddha Belly Plant, Gatemala Rhubarb, Gout Plant, Gout Stick.

Shape Class: circular. Dimensions: D = 66.8µm (62.6- 71), L = 0.12. Pollen Class: inaperturate. Sculpturing:clavate.

LM polar view

Habit: shrub, to about 18 inches, stems swollen and knobby, ith bristled scars; leaves orbicular-ovate, peltate, long-petioled, to 12 inches across, deeply 3-5 lobed with obtuse sinuses; cymes terminal, long-peduncled, pedicels red; flowers small, coral-red.

Distribution in Erkwit: Khor Gowb.

41 Family: Fabaceae (Leguminosae)

Subfamily: Caesalpinioideae Genus: Tamarindus Linnaeus, 1753 (F). Scientific Name: T. indicus L -Vern. Name: (Ar.) Aradaib, (Engl.) Tamarind.

Shape Class: subolate. Dimensions: P = 30 µm (33 – 28.5), E = 35µm (35 - 32), P/E = 0.86, L = 0.027. Pollen Class: tricolporate.

Sculpturing: striate. LM polar view

Habit: Evergreen tree. Leaves abruptly pinnate. Flower with three yellow petals and four reddish sepals. Bark grey, stongly fissurated and scaly. Pod pendulous, curved.

Distribution in Erkwit: Jebel Dambobei.

Subfamily: Mimosoideae Genus: Acacia. Scientific Name: A. mellifera (Vahl) Benth. (F). - Syn. A. senega L.; Mimosa mellifera Vahl. -Vern. Name: (Ar.) Kitr. LM horizontal view Form: 16-celled. Shape: compressed biconvex disc, characteristically differentiated into a central part with wedge-shaped cells and a peripheral part with square to rectangular cells. Dimensions: D = 57.6µm (54.7- 60.4), L = 0.16. Pollen Class: polyad. Sculpturing: psilate.

Habit: tall rounded shrub or small tree with ball-shaped crown, Branches covered with very sharp recurved thorns.

Distribution in Erkwit: Zone IV.

Subfamily: Mimosoideae Genus: Acacia P. Miller, 1754 Scientific Name: A. nilotica (L.)Delile (F). - Syn. Mimosa nilotica L. ; Acacia arabica (Lam.) Willd; Acacia subalata Vatke. -Vern. Name: (Ar.) Sunut. LM horizontal view Form: 16-celled. Shape: compressed biconvex disc characteristically differentiated into a central part with wedge-shaped cells and a peripheral part with square to rectangular cells. Dimensions: D = 42µm (39.8- 44.3), L = 0.13 Pollen Class: polyad. Sculpturing: psilate.

Habit: tree 5-20 m high with a dense spheric crown, stems and branchlets usually dark to black coloured, fissured bark, grey-pinkish slash, exuding a reddish low quality gum. Thin, straight, light, grey spines in axillary pairs.

Distribution in Erkwit: Zone IV.

Subfamily: Mimosoideae Genus: Acacia P. Miller, 1754. Scientific Name: A. tortilis (Forssk.)Hayne. (F). - Syn A. fasciculata Guill. and Perrott. LM horizontal view A. raddiana Savi , A. spirocarpa Hochst. ex A. Rich

Form: 16-celled. Shape: compressed biconvex disc characteristically differentiated into a central part with wedge-shaped cells and a peripheral part with square to rectangular cells. Dimensions: D = 52µm (48.8- 55.2), L = 0.14. Pollen Class: polyad. Sculpturing: psilate.

Habit : gregarious, wide-spreading, flat-topped or umbrella-shaped tree, up to 4 m high; branchlets pubescent, red-brown; spines mixed, some white, straight, slender, and up to 7.5 cm long, others grey with black or brown tips, sharply curved, very small. Pinnae in three to ten pairs.

Distribution in Erkwit: Zone III, zone V and zone IV.

Family: Moraceae

Genus: Ficus L. Scientific Name: F. glumosa Delile (H). - Syn. F. barbata Warb; F. congensis Eng; Urostigma glumosum Miq. -Vern. Name: (Engl.) African rock fig, Mountain rock fig.

Shape Class: subprolate. LM polar view Dimensions: P = 18.65µm (21.4 – 15.9), E = 14.9µm (17.2 –12.6), P/E = 1.25, L = -0.01. Pollen Class: biporate. Sculpturing: psilate.

Habit: trees up to 10 m. tall or shrubs, terrestrial, branches spreading. Leafy twigs 2–6 mm. thick, indumentums dense, of short white hairs intermixed with much longer yellow to whitish hairs especially on the nodes.

Distribution in Erkwit: Jebel Dambobei.

46 Genus: Ficus L. Scientific Name: F. platyphylla Delile. (H). -Vern. Name: (Engl.) broadleaf fig.

Shape Class: subprolate. Dimensions: P = 27.3 µm (30 – 24.5), E = 23.8µm (24.8–22.8), P/E = 1.14, L = 016. Pollen Class: biporate. LM polar view Sculpturing: psilate.

Habit: tree. Leaves broadly oblong, cordate. Lateral nerves red when young, prominent.Figs many in clusters of two to five towards the top of the branchlets.

Distribution in Erkwit: Jebel Dambobei also reported widespread.

Genus: Ficus L. Scientific Name: F. sycomorus L. (F). - Syn. F. chanas Forssk; F. damarensis Engl; Sycomorus antiquorum Gasp. -Vern. Name: (Ar.) gomiz, (Engl.) figuier sycomore ; Sykomore

Shape Class: subprolate. Dimensions: P = 12.8µm (9.8 – 15.7), E = 10.6µm (8.6 –12.5), P/E = 1.21, L = 0.02. Pollen Class: biporate. Sculpturing: psilate. LM polar view

Habit: thick-branched tree often buttressed with branches rising from near the ground; produces cluster of edible but inferior figs on short leafless twigs.

Distribution in Erkwit: widespread.

Family: Olacaceae

Genus: Ximenia. Scientific Name: X. americana L. (F). -Vern. Name: (Engl.) Monkey Plum, Tallow Nut; Seaside Plum.

Shape Class: subprolate. Dimensions: P = 19.3µm (22 – 16.5) Ε = 16.1µm (19.2 − 13) , P/E = 1.19, L = 0.04. Pollen Class: tricolporate. Sculpturing: psilate.

LM polar view

Habit: shrub or small tree. Pale grey bark, purple-red branches with waxy bloom, yellowish-green/whitish, yellow. Fruit yellow plum- like edible.

Distribution in Erkwit: Zone I and Zone II.

Family: Oleaceae

Subfamily: Jasminoideae Genus: Jasminum Linnaeus, 1753 Scientific Name: J. abyssinicum R.Br. (F). - Vern. Name: (Ar.) Monkey Plum, Tallow Nut; Seaside Plum.

Shape Class: subprolate. Dimensions: P = 56.7µm (58.2 – 55.1) Ε = 46.9 µm (49.5 − 43.7) , P/E = 1.21, L = 0.11. Pollen Class: tricolporate. Sculpturing: clavet.

LM polar view

Habit: climber with long flexuous twining stems attaining 5 m. or more; nodes of main branches well spaced. Young shoots and petioles smooth and glabrous or minutely puberulous, bark of older branches becoming rough. Leaves trifoliolate.

Distribution in Erkwit: Jebel Dambobei also Karora Hills.

Family: Rhamnaceae

Genus: Ziziphus. Scientific Name: Z. spina-christi (L.) Willd. (F). - Syn. Rhamuns spina-christiL., Ziziphus africana Mill. - Vern. name: (Ar.) Sidr, Nabbag; (Engl.) Christs Thorn, Jujube.

Shape Class: oblate spheroidale. Dimensions: P = 26.8µm (25.6 – 28), E = 27.6µm (25.2 –30.1), P/E = 0.97, L = 0.06. Pollen Class: tricolporate. Sculpturing: psilate.

LM polar view

Habit: spiny tree, distributed in the warm-temperate. The fruit is an edible yellow-brown, red, or black, oblong, 1-5 cm long, often very sweet and sugary.

Distribution in Erkwit: Khor Abent.

51 Family: Salvadoraceae

Genus: Salvadora Linnaeus, 1753 dora Linnaeus, 1753 Scientific Name: S. persica Wall. (F). -Vern. Name: (Ar.) El Arak., (Engl.) Saltbush, mustard tree, tooth- brush tree.

Shape Class: subprolate. Dimensions: P = 20.5µm (18.8 – 22.3), E = 17.3µm (16.8 –18.2), P/E = 1.18, L = 0.14. Pollen Class: tricolporate. Sculpturing: psilate view.

. LM polar view

Habit: much-branched small tree with white branchlets. Leaves opposite, oblong. Flowers tetramerous. Fruit red-purple, one-seeded. Suitable for arid regions and sandy soils.

Distribution in Erkwit: Zone III.

52 Family: Sapindaceae

Subfamily: Rubioideae. Genus: Dodonaea J.R. Forster and J.G.A. Forster, 1775. (F). Scientific Name: D. viscosa (L.) Jacq. (F0. -Vern. name: (Ar.) Erkwit.

Shape Class: subprolate. LM polar view Dimensions: P = 35 µm (40.6 – 29.5), E = 27.5µm (29.2 –25.8), P/E = 1.27, L = 0.06. Pollen Class: tricolporate. Sculpturing: psilate.

LM equatorial view

Habit: small tree. Branchlets angular. Leaves simple, Flowers apetalous, unisexual or polygamous. Fruit winged recticulate.

Distribution in Erkwit: Zone I and Zone II.

Family: Tamaricaceae

Genus: Tamarix Scientific Name: T. aphylla (L.) Karst. (F). - Syn.T. orientalis Forsk., T. articulate Vahl. - Vern. name: (Ar.)Tarfa.

Shape Class: prolate spheroidal. Dimensions: P = 19.6µm (21.5 – 17.6), E =17.4µm (20 –14.5), P/E = 1.13, L = 0.06.

Pollen Class: tricolporate. LM polar view Sculpturing: reticulate.

Habit: tree. Leaves feathery to minute triangular teeth from a sheathing base. Flowers in long spikes.

Distribution in Erkwit: Zone III.

Family: Tiliaceae

Subfamily: Grewioideae. Genus: Grewia. Scientific Name: G. asiatica (L.) Phalsa. (F). - Syn G. subinaequalis DC G. vestita Wall. -Vern. Name: (Engl.) phalsa Shape Class: subprolate. LM polar view Dimensions: P = 58.8µm (62.1 – 55.4), E =44.3µm (50.1 –38.6), P/E = 1.33, L = 0.14. Pollen Class: tricolporate. Sculpturing: reticulate.

Habit: small deciduous tree or large straggling shrub, up to 4.5 m tall; bark rough, grey; branches long, slender, drooping, young ones densely coated with stellate hairs. Leaves are alternate, simple. Fruit a globose drupe. Red or purple.

Distribution in Erkwit: Jebel Wad Nobao.

Subfamily: Grewioideae. Genus: Grewia. Scientific Name: G. flavescens Juss (F). -Vern. name: (Ar.) Basham. (Engl.) donkey berry.

Shape Class: subprolate.

Dimensions: P = 48.5µm (51.2 – 45.8), E = 41.1µm LM polar view (44.8 –37.3), P/E = 1.18, L = -0.03. Pollen Class: tricolporate. Sculpturing: reticulate. Habit: small bushy shrub native to Central Africa. Fruits are small, brown, reportedly edible, related to the phalsa.

Distribution in Erkwit: Jebel Wad Nobao .

Subfamily: Grewioideae. Genus: Grewia. Botanical Name: G. tenax Forssk. (F). - Syn. G. betulaejolia Juss. -Vern. Name: (A.) Gaddeim. Shape Class: subprolate. LM polar view

Dimensions: P = 53.2µm (54.5 – 51.9), E = 44.2µm (45.6 –42.8), P/E = 1.20, L = 0.38. Pollen Class: tricolporate. Sculpturing: reticulate.

Habit: multistemmed shrub up to 2 m tall usually rounded but generally battered and untidy due to browsing. Bark smooth, grey, very fibrous so that twigs are hard to break, Fruits are small edible.

Distribution in Erkwit: Jebel Wad Nobao and Khor Gobob.

57 Corrector Family Samples P E Lycopodium Corrector P P/E*100 Shape E Anacardiaceae Rhus abyssinica 28.9 21.4 48.44 5.58068966 4.13241379 135.0467 Prolate Apocynaceae Carissa edulis 42.56 43.68 46.2 5.87034483 6.02482759 97.4359 Oblate spheroidale Fabaceae Tamarindus indicus 32.8 32 41.7 0.88866995 0.86699507 102.5 Prolate spheroidal Capparidaceae Cadaba farinosa 28.9 23.3 43.68 2.19241379 1.76758621 124.0343 Subprolate Capparidaceae Capparis decidua 21.9 16.8 44.8 2.26551724 1.73793103 130.3571 Subprolate Capparidaceae Crateva adansonii 29.68 23.8 50 6.87172414 5.51034483 124.7059 Subprolate Capparidaceae Maerua angolensis 20.5 17.27 42.8 1.11083744 0.93581281 118.703 Subprolate Ebenaceae Diospyros mespiliformis 48.8 33.96 45.5 5.88965517 4.09862069 143.6985 Prolate Euphorbiaceae Euphorbia abyssinica 54.6 43.4 45.5 6.58965517 5.23793103 125.8065 Subprolate Moraceae Ficus glumosa 18.67 14.93 40.13 -0.2161305 -0.172835 125.0502 Subprolate Moraceae Ficus platyphylla 27.3 23.8 47.13 4.39086207 3.82793103 114.7059 Subprolate Moraceae Ficus sycomorus 12.8 10.6 41.43 0.26167488 0.21669951 120.7547 Subprolate Olacaceae Ximenia americana 19.32 16.1 42 0.6662069 0.55517241 120 Subprolate Oleaceae Jasminum abyssinicum 56.7 46.9 44.8 5.86551724 4.85172414 120.8955 Subprolate Arecaceae Phoenix dactylifera 28.9 17.7 48.7 5.76576355 3.53128079 163.2768 Prolate Rhamnaceae Ziziphus spina-christi 26.6 27.6 43.24 1.72965517 1.7946798 96.37681 Oblate spheroidale Salvadoraceae Salvadora persica 20.5 17.3 46.2 2.82758621 2.3862069 118.4971 Subprolate Sapindaceae Dodonaea viscosa 35 27.5 43.2 2.24137931 1.76108374 127.2727 Subprolate Simarubaceae Balanites aegyptiaca 41.3 35 48.5 8.0362069 6.81034483 118 Subprolate Tamaricaceae Tamarix aphylla 19.6 17.36 43.12 1.21655172 1.07751724 112.9032 Prolate spheroidal Tiliaceae Grewia asiatica 58.8 44.3 46.2 8.11034483 6.11034483 132.7314 Subprolate Tiliaceae Grewia flavescens 48.53 41.07 39.6 -1.1953202 -1.0115764 118.1641 Subprolate Tiliaceae Grewia tenax 53.2 44.24 56 20.1793103 16.7806897 120.2532 Subprolate

Table: (1) Pollen shapes using the correction factor

58 Chapter Five Discussion

The present study deals with the pollen morphology of 18 families which comprised 30 species distributed in Erkwit area. It represents a first attempt to provide a description of pollen types and illustrations of the area. Comparing the description of the studied species with the previous description, the differences outlined were connected with the number of apertures, types of sculpturing, shape classes and size. According to El Ghazali (1989); Bonnefille and Riollet (1980) and Rao and Tain (1974), some of the differences might be accounted for by methodologies, including: pollen preparation, e.g. chemical treatment (Christensen, 1946) or mounting medium (Andersen, 1960) (Fig. 6) which may affect the size and shape of pollen. Light microscope versus scanning electron microscope (SEM) or Transmission Electron Microscope (TEM) observations, SEM is particularly useful when studying external features such as surface sculpturing and ornamentation for taxonomic research; degree of magnification which influences the type of surface sculpturing or other features revealed; different geographical localities of the same species and different environmental condition. Differences observed in the sculpturing and shape classes when compared with the previous work of El Ghazali (1989) can be related to misinterpretation due to the use of scanning electron microscope (SEM) by El Ghazali and LM in the present work because SEM can give fine details more than LM. In this study all species examined showed aperturate grains except that of the genus Jatropha sp. L.; while the monocolpate type of apertures is only found in Phoenix dactylifera L.; pollinia (monads or tetrads in irregular geometric arrangements. (Faegri and Iversen, 1989)) is found in species

59 Calotropis procera (Ait.) Ait.f. while pollen class polyad (monads united in groups of more than 4 grains in regular geometric arrangement. (Faegri and Iversen, 1989)) is found in subfamily Mimosoideae. The tricolporate type of apertures dominated over all other types within the species belonging to the studied families of Erkwit area. In general the shape of pollen grains does not change as the result of various treatments This applies to both acetolysed and non-acetolysed pollen grains, but it does not apply to grains treated according to the method described by Erdtman (1960), and Schoch-Bodmer (1936) pointed out that living, untreated pollen grains shrink under dry and expand under wet conditions and that this process is reversible. Consequently, this process is a physical one. It seems evident that the amount of shrinkage depends on the solidity of the exine It follows from the above that physically preserved pollen grains can be expected to shrink. Acetolysis for a long time gives rise to a sudden size increase of the pollen grain (Fig. 5) this increase cannot be compared with the expansion caused by the above-mentioned expansion solutions. The shape of the acetolysed pollen grains depends on the shape before treatment with acetolysls mixture this might be due to a chemical reaction of the acetolysls mixture with the pollen wall. i e. the sporopollenine. On the other hand expansion could also be the result of a physical reaction of the following expansion solutions with the cell contents 10% potassium hydroxide and this belief contradicts the opinion of Ting (1966) who stated " the cause for expansion in both cases is vaporization of latent moisture, owing to intense heating" Moreover he stated "Little or no expansion will occur by overheating an acetolysed sample mounted in glycerin jelly, since the response to acetolysed heat has already been achieved " This statement is also contrary to the results of the present study, which shows that treatment of acetolysed pollen grains with hot glycerin causes a significant increase in size.

60 Acetolysls by itself, causes an increase in the size of pollen grains without reaching a maximum. It seems likely that the exine retains its elasticity after the process of acetolysis. This is confirmed by the work of Andersen (1960) who reported size changes in acetolysed pollen grains when transferred to another chemical medium it is also substantiated by the size decrease on prolonged acetolysis since the chemical composition of the exine is not exactly known. It is impossible to explain the cause of the decrease in size during acetolysis the acetolysis mixture also changes the colour of the exine. The exine of untreated pollen grains shows a faint colour, however, after acetolysis the colour of the exine becomes yellow-brown (Bjork, 1967) (Fig. 7). Not all pollen grains show the same colour after acetolysis, this may be explained by the following hypotheses (1) There are different types of lignin-fractions. (2) There are varying amounts of the lignin-fractions. (3) The permeability of the protecting layers is variable. (4) There is a combination of the three possibilities (Heslop-Harrison, 1968). Differences in pollen descriptions of the same taxon will also result from natural differences in samples (particularly size) and possibly due to the different interpretations and terminologies of investigators.

(a) (b) Fig. (5) Size of grain before acetolysls (a), Size of grain after long time of acetolysls (b)

Fig. (6) Effect of mounting in glycerin jelly

(a) (b)

Fig. (7) Colour of grains after acetolysis (a), Colour of grains after staining (b).

63 Conclusions

Pollen morphology supports the taxonomic suggestion to separate the species in a special section or genus. Also it supports a phylogenetic study of molecular and morphological data. The tricolporate type of apertures dominated over all other types within the species belonging to the studied families of Erkwit area. Dicots pollen was mostly tricolporate while those of monocots are monoporate. Differences observed in the sculpturing and shape classes in pollen grains when compared with other previous studies could be attributed to the misinterpretation due to the use of scanning electron microscope (SEM), different geographical localities of the same species and different environmental condition. The pollen characteristics among species in the same family are similar to each other. The results indicate that pollen characters may be significant in taxonomical studies. Acetolysis has an affect on the pollen grain size. The effect of various chemicals on the size of pollen grains was studied. The size of acetolysed grains was affected by the treatment prior to acetolysis, and the measurements taken were corrected by a correction factor (L).

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