PHYLOGENETIC STUDIES OF THREE SPECIES OF THE GENUS ANNONA

ABSTRACT

 

Karyotype, phytochemical, palynological, molecular, morphological, anatomical analysis and growth studies were carried out on three species of Annona (A. muricata L. (soursop), A. squamosa L. (sweetsop) and A. senegalensis Pers (wildsop) obtained from the Uturu and Umuahia in Abia State to assess phylogenetic relationship among them. Karyotype analysis was carried out using the conventional squashing protocol in aceto–orcein of the root tissues after heating was adopted. Photomicrograph of the tissues were taken. All the species studied were diploid with somatic chromosome number of 2n = 2X = 14. The total length of short arm ranged from 1.234 µm in sweetsop to 1.344 µm in soursop. The total length of long arm varied from 1.789 µm in the soursop to 1.834 µm in sweetsop.  Centrometric index of the species varied from 1.906 µm in sweetsop to 2.095 µm in soursop. The highest arm ratio of 1.486 µm was obtained in sweetsop while soursop had the shortest arm ratio of 1.331 µm. Phytochemical studies of ethanol leaf extacts revealed the presence of alkaloid, tannins, flavoniod, phenol, saponin and steroid/triterpenes. Gas-chromatography-mass spectrometry (GC-MS) analysis revealed fifty-six compounds with their fragmentation pattern, molecular formular and molecular weight. Compounds identified includes Aminopyrrolidial, longifolenaldehyde, Ntegerrimine, Dodecannoic acid, Neophytadiene, etc. Palynological study was carried out using acetolysis treatment. Soursop had the highest pollen diameter of 229 μm and the monad diameter of 111 μm followed by wildsop with the diameter of 130 μm and the monad diameter of 72 μm while sweetsop pollen had the diameter of 99 μm and the monad diameter of 50 μm. In all the species, the pollen were tetrads and inaperturate, tetragonal, acalyme except for soursop which was rhomboidal tetrahedral. The exine sculpture of sweetsop and wildsop was rugulate while forsoursop it was recticulate. The differences and similarities in pollen morphology of the three investigated species showed significant evidences which are of diagnostic importance to complement plant identification. Cross-species amplification of all the SSR markers used, successfully amplified in soursop and sweetsop but did not in wildsop. RAPD markers showed cross species-amplification. Polymorphic information content (PIC) value ranged from 0.3457 (OPT05) to 0.5926 (OPH04) for RAPD markers with an average of 0.5103 indicating existence of sufficient genetic diversity among the three species genotypes studied. The PIC for SSR markers were uniform (0.6250). Phylogenetic tree showed genetic relatedness of the three species studied. SSR markers indicated that soursop and sweetsop are monophyletic and are more closely related than they are to wildsop, while RAPD markers indicated that wildsop and soursop are more related than they are to sweetsop. Growth parameters were taken at interval of two weeks and measurements were made with a meter rule. Plant materials were sectioned and stained for anatomical studies. The intrageneric characters they share in common includes 2n=2x=14 chromosome number, tetrad and inaperturate pollen grains, uniseriate collenchymatous stem epidermal cells, hypostomatic stomatal occurrence, 6 yellow petals, entire leaf margin, alternate leaf arrangement, syncarpous fruit, eucamptodromous venation etc. All these characters are useful in their identification and classification. Observations made in the similarities of characters studied, supports the present-day classification of these species in the same genus.

 

TABLE OF CONTENTS

 

Title page                                                                                                                    i

Declaration                                                                                                                             ii

Certification                                                                                                                            iii

Dedication                                                                                                                               iv

Acknowledgements                                                                                                                v

Table of contents                                                                                                        vi

List of Tables                                                                                                              ix

List of Figure                                                                                                              x

List of Plates                                                                                                               xi

Abstract                                                                                                                       xiii

 

CHAPTER 1

1.0       INTRODUCTION                                                                                        1

1.1       Statement of Problem                                                                                     6

1.2       Objectives and Scope of the Study                                                                 7

1.3       Significance of the Study                                                                               7

1.4       Taxonomic Classification of A. muricata, A. senegalensis and

            A. squamosa.                                                                                                   8

1.5       The Genus Annona                                                                                         9

1.5.1    Annona muricata L. (soursop)                                                                       9

1.5.11 Distribution                                                                                                     11

1.5.12 History of introduction and spread                                                                 12

1.5.13  Habitat                                                                                                            12

1.5.14 Genetics                                                                                                          12

1.5.15 Reproductive biology                                                                         12

1.5.16 Growth and development                                                                               12

1.5.17 Environmental requirements                                                                          14

1.5.18 Uses                                                                                                                15

1.5.19  Invasiveness                                                                                                    17

1.5.20 Introduction's danger                                                                                       17

1.5.2    Annona squamosa L. (sweetsop)                                                                    17

1.5.21  Plant type                                                                                                        18

1.5.22 Distribution                                                                                                     19

1.5.23 Danger of introduction                                                                                   19

1.5.24 Habitat                                                                                                            19

1.5.25 Genetics                                                                                                          20

1.5.26  Biology of reproduction                                                                                21

1.5.27 Physiology and phenology                                                                             21

1.5.28  Ecological prerequisites                                                                                 22

1.5.29 Natural enemies                                                                                              23

1.5.30  Invasiveness                                                                                                    24

1.5.31  Uses                                                                                                                24

1.5.3    Annona senegalensis Pers. (wildsop).                                                            25

1.5.31 Habitat                                                                                                            27

1.5.32 Distribution                                                                                                     27

1.5.33 Uses                                                                                                                27

 

 

CHAPTER 2

2.0       LITERATURE REVIEW

2.1       Phytochemicals                                                                                               29

2.1.1    Classification of phytochemicals                                                                    30

2.1.2    Phenols                                                                                                           30

2.1.3    Flavoniods                                                                                                      31

2.1.4    Tannins                                                                                                           32

2.1.5    Alkaloids                                                                                                         33

2.1.6    Terpenoids /triterpenes                                                                                   34

2.1.7    Saponin                                                                                                           36

2.2       Palynology                                                                                                      37

2.3       Karyotyping                                                                                                    39

2.4       Plant Anatomy                                                                                                42

2.5       Morphology                                                                                                    43

2.6       Molecular Genetic Studies                                                                             44

 

 

CHAPTER 3

MATERIALS AND METHODS

3.1       Species identification                                                                                    45

3.2       Fruit collection                                                                                              47

3.3       Seed extraction                                                                                              49

3.4       Experimental site                                                                                           51

3.5       Sowing of seeds                                                                                             51

3.6       Potting of seedlings                                                                                       51

3.7       Experimental Design                                                                                     53

3.8       Seedlings Management                                                                                  53

3.9       Germination Studies                                                                                      53

3.10     Growth Parameters                                                                                         53

3.10.1  Plant height (cm)                                                                                           53

3.10.2  Stem girth (cm)                                                                                              53

3.10.3  Number of leaves                                                                                           53

3.10.4  Leaf area (cm²)                                                                                               53

3.11 Morphological studies                                                                                        53

3.11.1  Leaf apex                                                                                                       54

3.11.2  Base of leaf                                                                                                    54

3.11.3 Shape of leaf                                                                                                   54

3.11.4  Leaf vein                                                                                                        54

3.11.5  Petiole length                                                                                                 54

3.11.6  Nodal length                                                                                                  54

3.11.7  Abaxial and adaxial surface                                                                           54

3.11.8 Width of leaf                                                                                                    54

3.12     Anatomical Studies                                                                                         54

3.12.1  Stem and petiole anatomy                                                                             54

3.12.2  Epidermal peels                                                                                             55

3.12.3  Photomicrography                                                                                         55

3.13     Genome studies                                                                                              55

3.13.1  Genomic DNA extraction                                                                               56

3.13.2  Agarose gel electrophoresis                                                                           56

3.13.3  DNA amplification                                                                                        57

3.13.4  Molecular data analysis                                                                                 57

3.14     GC-MS Analysis of Leaf Extracts                                                                  59

3.15     Qualitative Phytochemical Test                                                                      59

3.15.1 Collection and identification of plant materials                                             59

3.15.2  Sample preparation                                                                                         59

3.15.3  Phytochemical studies                                                                                    60

3.15.4  Preparation of reagents                                                                                   60

3.15.5  Wagner's reagent                                                                                            60

3.15.6  30% Ferric chloride solution                                                                          60

3.15.7  10% Acetic acid in ethanol                                                                             60

3.15.8  20% Sodium hydroxide                                                                                  60

3.15.9  20% Aqueous ethanol                                                                                     61

3.16     Phytochemical tests                                                                                        61

3.16.1  Test for flavonoid                                                                                           61

3.16.2  Test for terpenoid (salkowski test)                                                                 61

3.16.3  Test for tannin                                                                                                61

3.16.4  Test for alkaloid                                                                                              61

3.17.1  Karyotype analysis                                                                                         61

3.17.2  Determination of karyotype in Annona species                                             64

3.17.3  Statistical analysis of data                                                                              66

3.17.4  Significance of the processes used for karyotyping                                       66

3.18     Pollen Extraction                                                                                            66

3.18.1 Acetolysis treatment (palynological analysis)                                                66

3.18.2 Microscopic examination                                                                               67

 

 

CHAPTER 4

4.0       RESULTS AND DISSCUSION

4.1       Morphological Studies                                                                                   68

4.1.1    Leaf apex                                                                                                        68

4.1.2    Leaf base                                                                                                        68

4.1.3    Leaf shape                                                                                                      68

4.1.4    Leaf vein                                                                                                        68

4.1.5    Petiole length                                                                                                 70

4.1.6    Nodal length                                                                                                  70

4.1.7    Flower                                                                                                             70

4.2       Growth Studies                                                                                               74

4.2.1    Leaf area of the three species of Annona assessed at 2 weeks

             interval for 48 weeks                                                                                     74

4.2.2    Number of leaves of the three species of Annona assessed at 2 weeks

             interval for 48 weeks                                                                                     74

4.2.3    Petiole length of the three species of Annona assessed at 2 weeks

             interval for 48 weeks                                                                                     74

4.2.4    Plant height                                                                                                     74

4.2.5    Stem girth                                                                                                       75

4.2.6    Leaf length                                                                                                      75

4.2.7    Leaf width                                                                                                       75

4.3       Anatomical Studies                                                                                         83

4.3.1    Epidermal peel anatomy of Annona muricata, Annona squamosa

            and Annona senegalensis                                                                                83

4.3.2    Midrib of Annona muricata, Annona squamosa                                            86

            and Annona senegalensis

4.3.3    Stem anatomy of Annona muricata, Annona squamosa                                89

            and Annona senegalensis

4.3.4    Root anatomy of Annona muricata, Annona squamosa                                 91

            and Annona senegalensis

4.4       Phytochemical Studies                                                                                   94

4.4.1    Qualitative phytochemical tests                                                                     94

4.4.2    Quantitative phytochemical tests                                                                   95

4.4.2.1 GC-MS analysis of ethanol leaf extracts of Annona muricata,                         95

            Annona squamosa and Annona senegalensis

4.5       Palynological Studies                                                                                     99

4.5.1    Pollen attributes of the Annona species studied                                             99

4.6       Karyotype Studies                                                                                          104

4.6.1    Mitotic Indices of Annona species studied                                                     104

4.6.2    Chromosomal attributes of the three Annona species studied                        109

4.7       Genomic and Molecular Studies                                                                    116

4.8       Discussion                                                                                                       127

 

CHAPTER 5

5.0       CONCLUSION AND RECOMMENDATION                                          138

 

References

Appendix

 

 

 

 

 

 

 

LIST OF TABLES

 

4.1	Folia Morphological Characters	71
4.2	Important Floral and Fruit Morphological Characters	72
4.3	Leaf Area (cm2) at WAP (Weeks After Planting)	76
4.4	Number of Leaves at WAP (Weeks After Planting)	77
4.5	Petiole Length (cm) at WAP (Weeks After Planting)	78
4.6	Plant height (cm) at WAP (Weeks After Planting)	79
4.7	Stem girth (cm) at WAP (Weeks After Planting)	80
4.8	Leaf length (cm) at WAP (Weeks After Planting)	81
4.9	Leaf Width (cm) at WAP (Weeks After Planting)	82
4.10	Epidermal features on the leaves of the plants studied.	85
4.11	Anatomical characteristics of the midrib of the Annona species studied	88
4.12	Anatomical Characters in stem and root of the three species studied	93
4.13	Qualitative Test of Phytochemical Compounds present in the Ethanolic leaf extracts of A. muricata, A. senegalensis and A. squamosa.	94
4.14	Measurement of some attributes of the tetrad pollen grains of the three test plants	103
4.15	Mitotic index of three Annona species	105
4.16	Short arm (S), long arm (L), total length (S+L), arm ratio (L/S), relative value (S/L) and centrometric index (S/L + S) of three Annona species Short arm Long arm Total length Arm Ratio R- Value    Centrometric index	108
4.17	Coefficient of variation (CV), total form (TF), disparity index (DI), Intrachromosomal index (A1), interchromosomal index (A2) and Karyotype formula (KF) of three Annona species.	111
4.18	Scored sheet for statistical analysis	118
4.19	Genetic diversity and Polymorphic Information Content	119
4.20	Scored sheet for statistical analysis	124
4.21	Genetic diversity and Polymorphic Information Content	125

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

4.1	GC-MS Chromatograph of A. senegalensis	96
4.2	GC-MS Chromatograph of A. squamosa	97
4.3	GC-MS Chromatograph of A. muricata	98
4.4	Karyotype of three Annona species.	115
4.5	Phylogenetic Tree of the samples for SSR markers	120
4.6	Phylogenetic Tree of the samples for RAPD markers	126

 

 

 

 

 

 

 

 

 

LIST OF PLATES

1	Annona squamosa (sugar apple) fruit and leaves	46
2	Annona muricata (soursop) fruit and leaves	46
3	Annona senegalensis, (wildsop) fruit and leaves	46
4	A. muricata fruit	48
5	A. squamosa fruit	48
6	A. senegalensis fruit and seeds	48
7	A. muricata seeds	50
8	A. senegalensis seeds	50
9	A. squamosa seeds	50
10	Potting of seedlings	52
11	A. senegalensis leaves	69
12	Annona muricata leaves	69
13	Annona squamosal leaves	69
14	A. squamosa flower	73
15	A.muricata flower	73
16	A. senegalensis flower	73
17	Epidermal leaf peel of adaxial and abaxial leaf surfaces of three Annona species (x400). Anomocytic stomatal type and hypostomatic stomatal occurrence of the abaxial leaf surface of  A. senegalensis (A), A. squamosa (C) and A. muricata (E) sinuous and pol ygonal epidermal leaf cell shape	84
18	Leaf midrib anatomy of Annona species parenchyma cells round the midrib region in A. senegalensis x40(A), A. squamosa x40(C) and A. muricata x40 (E). Absence of pith in A. senegalensis x40(A). A continuous layer of vascular bundles interrupted by parenchyma cells in A. senegalensis (x100) B. A thick layer of vascular bundles in the abaxial region A. squamosa (x100) D. A thick layer of vascular bundles around the abaxial and adaxial region in A. muricata (x100) F.	87
19	Stem anatomy of Annona species (A, C, E x40 showing the collateral vascular bundles (a), cambium, cortex (b), epidermis and presence of pith (c) in A. senegalensis, A. squamosa and A. muricata respectively. B x100 showing biseriate medullary pith rays (a), sclerenchymatous cells (b) and dialated phloem rays (c). D x100 showing protoxylem (a) and metaxylem (b). E x40 indicating protoxylem and metaxylem. F x100 showing sclerenchymatous cells (a), cambium (b) and uniseriate pith rays (c).	90
20	Root anatomy of Annona species (A, C, E x40 showing absence of pith in A. senegalensis, A. squamosa and A. muricata respectively and vascular rays with large intercellular air spaces in A. senegalensis and A. squamosa while bundle sheets are present with small air spaces in A. muricata. D x100 showing the epidermis and cortex cells.)	92
21	Pollen of  Annona senegalensis (A, C, F, H x1000 showing the type of sculpture /ornamentation “rugulate”on the exine; B, D, E and G x100  showing the tetrad acalymmee, tetragonal)	100
22	Pollen of Annona squamosa A, B, C, D, E x1000 and F, G, x100 (tetrad acalymmee tetragonal with rugulate sculpture).	101
23	Pollen of Annona muricata (A, F x100 showing tetrad rhomboidal tetrahedral symmetry and B, C, D, E, G, H x1000 showing the sculptural pattern. B and C clearly showing the reticulate (network- like) sculpture of exine).	102
24	A. muricata Chromosomes (A showing chromosomes at x1000. B showing chromosomes at x400. C metaphase chromosomes showing the long arm and short arm).	112
25	A. squamosa Chromosomes (A showing chromosomes at x1000. B showing chromosomes at x400. C metaphase chromosomes showing the long arm and short arm)	113
26	A. senegalensis Chromosomes (A and B showing chromosomes at x1000. C metaphase chromosomes showing the long arm and short arm)	114
27	Genomic DNA loaded on 1% agarose gel	117
28	Gel image of PCR amplicons on 2% agarose gel after polymerase chain reaction.	117
29	Genomic DNA loaded on 1% agarose gel	121
30	Gel images of PCR amplicons on 2% agarose gel after polymerase chain reaction.	122
31	Gel images of PCR amplicons on 2% agarose gel after polymerase chain reaction.	123

 

 

 

 

 

 

 

 

 

CHAPTER 1

1.0     BACKGROUND OF STUDY

Phylogenetics is the systematic study of reconstructing the past evolutionary history of extant species or taxa, based on present-day data, such as morphologies or molecular information (sequence data) (Jarvis et al., 2017). Phylogenetic relationships are depicted by branching diagrams called cladograms, or phylogenetic tree. Cladogram show relative affinities of group of organisms called taxa. Such groups of organisms have some genealogical unity, and are given a taxonomic rank such as species, genera, families, or orders.

Phylogenetic studies already have direct applications in many disciplines. In the age of genomics, the power of these applications will increase an interpretation of phylogenies helps to identify those taxa for which genomic treatments will answer fundamental and in some cases long-standing questions about metabolic and regulatory networks in all the evolutionary corners of the plant kingdom (Ohlrogge and Bennings, 2000).

The impact of molecular data on the field of plant systematic can hardly be overstated. In combination with explicit methods for phylogenetic analysis, molecular data have reshaped concepts of relationships and circumscriptions at all levels of the taxanomic hierarchy (Crawford, 2000). Phylogenetic tree provide not only the basis for classification, but also studies of character evolution (Schultheis and Baldwin, 1999), hybridization (Hughes et al., 2002; Linder and Rieseberg 2004), polyploidy (Doyle et al., 2003b; 2004) biogeography ( Pennington et al., 2004), origins domestication (Nesbitt and Tanksley, 2002) and speciation and species diversification (Barraclough and Vogler, 2000; Barraclough and Nee, 2001).

Evaluating genetic variation allows accessions to be ranked for use in breeding programs. There have been few attempts to identify different Annonas germplasm. Because of protogynous basis of cross-pollination, the Annona species have a lot of variation. Morphological characters have previously been used to characterize unexplored potential of germplasm for producing high-yielding genotypes of improved fruit content (Folorunso and Modupe, 2007). Traditional morphological markers, on the other hand, are considered to be greatly influenced by edaphic and climatic factors, and therefore are unreliable due to this high level of environmental impact (Kumar et al., 2014). As a result, using molecular markers to analyze variation is preferable.

 In Annonas, there are just a few studies on the use of molecular markers for diversity research. Few of these markers used are random amplified polymorphic marker (RAPD) markers (Bharad et al., 2009; Ronning et al.,1995), amplified fragment duration polymorphism (AFLP) markers (Rahman et al., 1998 and Zhichang et al., 2011), and simple sequence repeat (SSR) markers (Bharad et al., 2009; Kwapata et al., 2007; Ronning et al., 1995). Knowledge on nutritional value, on the other hand, is critical for selecting the desired genotype for domestication in the area of adaptation. There is also little research on proximate study of Annona fruits (Onimawo, 2002; Boake et al., 2014). Keeping in view about the scarcity of information on Annona, efforts have been made to investigate the genetic diversity using RAPD and SSR molecular markers, as well as proximate analysis of the fruit of specified accessions.

To exploit plant genetic resources for any crop improvement programmes, a proper understanding of the diversity present within the plant species, which can be assessed using morphological, biochemical or molecular markers (Goncalves et al., 2009), is quite essential for rational utilization of the germplasm (Piyasundara et al., 2009). Until recently, the characterization of plant species was based on morphological traits, which was considered very useful in germplasm management for purposes of proper utilization in any breeding programmes (Piyasundara et al., 2009).

Phytochemicals are biologically active, naturally existing chemical substances in plants that have therapeutic and nutritive properties for humans (Hasler and Blumberg, 1999). They protect plants from disease and injury, as well as contributing to the color, fragrance, and flavour of the plant. Phytochemicals are plant chemicals that contribute to protecting plants from ecological threats such as pollution, stress, drought, UV exposure and pathogenic attack (Gibson et al., 1998; Mathai, 2000). In recent times, there has been a gradual resurgence of interest in the use of medicinal plants in developing countries, owing to reports that herbal medicines are healthy and have no adverse side effects, particularly as compared to synthetic drugs. As a result, looking for innovative drugs with safer and inexpensive plant-based alternatives is a natural option. The therapeutic importance of these plants is due to a number of chemical compounds that have a specific physiological effect on the human body (Edeoga et al., 2005). World Health Organization, reports shows that more than 60% of global population is using the traditional medicine system to overcome several health related issues. This percentage goes up in the developing countries where, rural and tribal population is higher because of its low cost (Mehra et al., 2014; Bajpai et al., 2016). The World Health Organization (WHO) estimated that about 80% of the African continent depend on medicinal herbs for their main healthcare. Since the advent of medicine, natural products, especially those obtained from plants, have been used to help mankind maintain its wellbeing. Plant phytochemicals have provided critical platform for pharmaceutical research for the past century. The importance of the active ingredients of plants in agriculture and medicine have stimulated significant scientific interest in the biological activities of these substances. Phytochemicals and active constituents in plants have played a pivotal role in pharmaceutical discovery during the last century. The role of plant bioactive materials in medicine and agriculture has sparked a lot of interest in bioactivities of substances (Moghadamtousi et al., 2013). Despite the limited research on a number of plant species, all existing knowledge about their underlying position in nature are relatively inadequate. Therefore, the rational production of natural products necessitates a comprehensive study of these plants' bioactivities and essential phytochemicals (Moghadamtousi et al., 2014). Plants with a long history in ethno-medicine are avast resources of active phytoconstituents that provide medicinal and nutritional effects against a variety of ailments and diseases in the pharmaceutical sector (Moghadamtousi et al., 2015).

Palynology is the science that studies contemporary and fossil palynomorphs, including pollen, spore, dinoflagelate cysts, acritarchs, chitinozoans and scolecodonts, together with particulate organic matter and kerogen found in sedimentary rocks and sediments (Walker, 1976).

 Palynology is a method employed by a range of disciplines all concerned with the environment. Palynology is an interdisciplinary science and is a branch of earth science (geology or geological science) and biological science (biology), especially plant science (botany). Stratigraphical palynology is a branch of micropalaeontology and paleobotany that studies fossil palynomorphs from the Precambrian to Holocene. Many scientists have recently developed interest in the palynological characteristics of plants. Nyananyo (1985), for instance, found that palynology is useful for intrageneric classification of broad genera based on pollen morphology of Talineae. Seed coat morphology and other palynological features of Talinum and Calandrinia were also used by Nyananyo (1987) and Nyananyo and Olowokudejo (1986) to generate a more acceptable classification of the species in these taxa. Pollen is produced in the microsporangia in the male cone of a conifer or other gymnosperm, or in the anthers of an angiosperm flower. On the exine of pollen grains, there are many morphological characters that are of diagnostic importance to complement plant identification (Edeoga et al., 1998; Mbagwu and Edeoga, 2006; Mbagwu et al., 2009). Pollens therefore are used to identify the source of a plant during the analysis of palynological samples in fields such as biostratigraphy, climatology, medicine-alleviation of pollinosis (hay fever-allergenic disease), forensic studies, mellisopalynology, plant advancement, taxonomy and ecological reinstatement practices.

Karyotyping is the process by which cytogeneticists take photographs of chromosomes in order to determine an individual's chromosome complement, including the number of chromosomes and any anomalies. The term may also refer to the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number.

Karyotyping is the process of determining an organism's chromosome count and how the chromosomes appear under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics. The analysis of whole chromosome sets is sometimes referred to as karyology. The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size.

The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated by 2n. Chromosome numbers of Annona are 2n = 2x = 14 and 16, except for A. glabra, which is a tetraploid species (2n = 4x = 28) (Hirdayesh et al., 2016)

So, in normal diploid organisms, autosomal chromosomes are present in two copies. There may, or may not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. The study of karyotypes is important for cell biology and genetics, and the results may be used in evolutionary biology (karyosystematics) and medicine. Karyotypes can be used for many purposes; such as to study chromosomal aberrations, cellular function, taxonomic relationships, and to gather information about past evolutionary events.

JUSTIFICATION

Plant systematics develops evolutionary relationship among the different groups of plants. It also has great importance in agriculture, forestry and herbal medicine (Sharma, 1993). Plant biodiversity is remarkably important in social relations and conservation of the cultural heritage of Nigeria. Production and over consumption are important causes of biodiversity loss. In Nigeria, threat to biodiversity can be accessed from the list of 484 plant species in 12 families, which are now endangered with extermination (Aju and Ezeibekwe, 2010). Preservation of biodiversity could be achieved through various strategies such as creation of awareness on the need to reserve biological reserves or gene bank, use farmer’s indigenous knowledge and research based strategies (Emma-Okafor et al., 2010). Annona species has received little systematic research attention. Molecular studies of the three species have not been carried out together to ascertain its ancestry.  It is urgent to increase biosystematics work on Annona species because of the continued neglect and under-utilization of the African species A. senegalensis. Owing to modernization of agricultural practices and land use shifts, the genetic resources and plant variety of outcrossing tropical tree types such as Annonas are eroding.

1.1       STATEMENT OF PROBLEM

The scientific study of the types and diversity of species, as well as any and all relationship within them, is known as systematics (Simpson, 1961). It is important in providing a foundation of information about the tremendous diversity of life (Silva, 2006). It is largely concerned with morphological, anatomical, genetical, cytological, chemical and palynological aspects. Biosystematics may therefore be considered as the taxonomic application of these types of experimental disciplines which definitely is costly to carry out. Various research studies have been done on Annona species in Obafemi Awolowo University, Nigeria. There is no complete systematic study available on genus Annona in Nigeria since they are mostly exotic. The execution of this research is necessary to boost the knowledge reserves on the genus Annona and to add to few phylogenetic studies yet done on the indigenous and exotic species of Annona in Nigeria.

1.2       OBJECTIVES AND SCOPE OF THE STUDY

The objectives of this research work are to:

1.              Examine morphological characteristics of A. muricata, A. squamosa and A. senegalensis.

2.              Identify the anatomical characters of A. muricata, A. squamosa and A. senegalensis.

3.              Investigate the genome variation within A. muricata, A. squamosa and A. senegalensis to assess the genetic diversity of the resultant seedlings as well as to ascertain their phylogeny.

4.              To determine qualitative phytochemical constituents of A. muricata, A. squamosa and A. senegalensis.

5.              Examine the phytochemical constituent of the matured leaves of A. muricata, A. squamosa and A. senegalensis using GC-MS analysis.

6.              Study the karyotype of A. muricata, A. squamosa and A. senegalensis.

7.              Carry out the palynological studies on A. muricata, A. squamosa and A. senegalensis.

1.3       SIGNIFICANCE OF THE STUDY

This study would help to highlight the morphological, anatomical, cytological, phytochemical, palynological and molecular variation on the selected species and to give a better understanding of relationships at generic level within the selected species of Annona. It would be useful for classification, phylogeny, and comparative studies between species. It will equally add to existing knowledge of systematics in Annona and provide useful taxonomic data that would give further insight into and identification.

1.4     TAXONOMIC CLASSIFICATION OF ANNONA MURICATA, ANNONA       SENEGALENSIS AND ANNONA SQUAMOSA.

Based on Integrated Taxonomic Information System (ITIS), A. muricata, A. senegalensis and A. squamosa are classified as follows:

 Kingdom:                   Plantae

 Sub-kingdom:            Tracheobionta

 Superdivision:            Spermatophyta

 Division:                    Magnoliophyta

Class:                          Magnoliopsida

Subclass:                     Magnoliidae

Order:                          Magnoliales

 Family:                       Annonaceae 

Genus:                         Annona L.

 Species:                      A. muricata

                                    A. senegalensis

                                    A. squamosa

 

1.5       THE GENUS ANNONA

Annona, belongs to the family Annonaceae. This family is one of the largest tropical and subtropical families of trees, shrubs, and lianas with about 135 genera and 2500 species widely distributed in this world (Escribano et al., 2007). The genus Annona, commonly known as the custard-apple, consists of some 125 species with some species widely cultivated for their edible fruits and often becoming naturalized beyond their native range of tropical America and Africa (Wagner et al., 2014). There are six species in the genus Annona that produces edible fruit. They are A. squamosa L (widely cultivated), A. reticulata, A. cherimola, A. muricata, A. atemoya (a natural hybrid of A. squamosa and A. cherimola) and A. diversifolia. Majority of the Annona species are believed to have originated from South America and the Antilles. However, soursop or prickly custard apple (Annona muricata) and Annona senegalensis (wild soursop) is believed to be of African origin (Pinto et al., 2005a). The current range of essential species spans almost all continents, with soursop and sugar apple A. squamosa having the most widespread distribution, mostly in tropical areas. A. squamosa is thought to have originated in India as a secondary source.

 Annonas pulp is rich in minerals and vitamins, as well as a possible source of dietary fiber (up to 50% w/w dry basis) (Gyamfi et al., 2011). Annona seeds, especially A. squamosa, contain a significant amount of oil that can be used for industrial purposes (Mariod et al., 2010). Furthermore, the genus Annona the leaves, stems, barks, buds, and seeds have been regarded as possible sources of medicinally valuable compounds (Pinto et al., 2005a). Annona, a versatile plant with many uses, is hardy and deciduous in nature, very simple to grow with minimal inputs. It needs comparatively little maintenance, and do not suffer from serious pests and diseases attacks.

Fruits of A. squamosa and A. muricata have a bright future in the fruit market industry today, thanks to high demand from the manufacturing companies (Santos et al. 2011). However, domestication of their production is only in its early stages (Zonneveld et al., 2012). Due to rapid development of cultivation and land use act, the genetic resources and plant variety of out-crossing tropical tree species such as Annonas are eroding. As a result, edible Annonas genetic facilities are only found in situ, i.e. on farms, in home gardens/orchards, and/or in natural populations.

 

1.5.1    Annona muricata L. (soursop)

Wagner et al. (2014) gave the following description of A. muricata. A. muricata is a small tree 7.5-9 m tall, new growth puberulent with reddish brown hairs. Leaves distichous, petiolate, blade narrowly obovate, narrowly elliptic or obovate-elliptic, 6.5-20 cm long, 2.5-6.5 cm wide, base acute to rounded, apex acuminate, shiny and glabrous above, strigillose along costa and secondary veins beneath, with barbate somatia in secondary vein axils, secondary veins 8-12 pairs, petioles thick, 5-8 mm long. Inflorescences cauliflorous or ramiferous, sometimes in fascicles on knobby outgrowths of trunk, or solitary and leaf-opposed, pedicels stout, 15-20 mm long, 2-2.5 mm in diameter, obconical, rusty puberulent, bracteolate; sepals 3, valvate, broadly triangular, 3-4 mm long, 5-6 mm wide, thick, apex acute, puberulent externally, petals thick, fleshy, glabrous, greenish-yellow to yellow, 3 outer petals 30 mm long, 22 mm wide, ovate, base cordate, apex acuminate, 3 inner petals 20-22 mm long, 10-15 mm wide, elliptic, cucullate, base acute to attenuate, apex obtuse; stamens numerous, 4 mm long, 1 mm in diam., clavate with thickened apex; ovary ca. 5 mm long, 8 mm wide, broadly conical, carpels densely velutinous. Fruit a large, fleshy syncarp to 30 cm long, 15 cm long, ovoid to oblong, often somewhat curved, when fresh green without, the surface bearing regularly well-spaced, soft, conical, curved spines 2-3 mm long, the flesh white, juicy and with cotton-like fibers, surrounding the numerous seeds. Seeds light to dark brown, 13-17 mm long, 9-10 mm wide, ellipsoid, compressed, with low marginal ridge; endosperm ruminate. 

This small, evergreen tree may be slender and upright or low branching and bushy; it often becomes straggly and untidy with age. The dark green leaves emit a strong odour when crushed. The flowers, which have a peculiar smell, are hermaphrodite and are often produced singly or in small clusters on old wood. Normal fruits are generally heart-shaped to oval, but if there is poor pollination, unfertilized ovules fail to develop and the resulting fruit assumes distorted irregular shapes and is usually undersized. The dark green skin has many recurved, soft spines 0.5-1.3 cm apart. There is often a constriction like a fault on the side of the fruit, where the skin has not swollen and the spines are much closer together. The fruit stalk is about 3-8 cm long and woody. The ripe pulp, which adheres to the skin but is easily separated into segments (which were the separate ovaries), has an agreeable subacid flavour with a distinct aroma (Janick and Paull, 2008).

1.5.11 Distribution

A. muricata is of tropical American origin although its exact origin is unknown (PIER, 2014 and Wagner et al. 2014); it is probably native to Central America and northern South America (Hanelt et al., 2001). It is known to be cultivated in Africa (mainly the warm lowlands of eastern and western Africa), temperate and tropical Asia, Australasia, North America, the south-central Pacific Islands, the Caribbean, and Mesoamerica (USDA-ARS, 2014). Some differences were found between sources as to where the species is native. It is reported by some authorities as exotic to the Caribbean and West Indies including Puerto Rico (Acevedo-Rodriguez and Strong, 2012; Randall, 2012), but it was listed as native to Puerto Rico by USDA-NRCS (2014) and as native to Caribbean territories by ITIS (2014). Hanelt et al. (2001) reports the species to be “found wild and cultivated from sea level to 1000 m in the Antilles and from south Mexico to Peru and north Argentina”, although the species is reported as exotic in the Lesser Antilles (Acevedo-Rodriguez and Strong, 2012).

1.5.12  History of introduction and spread

A. muricata is of tropical American origin (PIER, 2014; Wagner et al., 2014a), and it is probably native to Central America and northern South America (Hanelt et al., 2001; Acevedo-Rodriguez and Strong, 2012). Archaeological evidence indicates both A. muricata and A. cherimola were present in pre-Hispanic Peru rather than an introduction by Spaniards, as was thought by 17th-century records (Bonavia et al., 2004). It was one of the first fruit trees carried from America to the Old World Tropics (Morton, 1987). Both A. cherimola and A. muricata were introduced to Asia by way of the Spanish galleon trade at an early date, and A. muricata is now widely cultivated across the Asia Pacific region for its edible fruit (Koesriharti, 1991; PIER, 2014). Date of introduction of A. muricata to the West Indies and Caribbean region is uncertain, but it was observed growing in Jamaica in the early 16^th century by Sir Hans Sloane, who collected a specimen that is now in the Sloane herbarium of the UK Natural History Museum in London (specimen BM000594141). Morton (1987) states that Oviedo described the fruit as abundant in the West Indies and in northern South America in 1526. The species has been present in Puerto Rico since before the 1880’s, (Bello, 1883; Britton and Wilson, 1924). It has been grown in Florida since probably the 1870s (Morton, 1987).

1.5.13 Habitat

A. muricata L., although widely cultivated, occurs in thickets, hillsides, mountain woodlands, and shaded ravines in Puerto Rico (Britton and Wilson, 1924; Liogier and Martorell, 2000) and in humid premontane forests in Colombia (Vascular Plants of Antioquia, 2014). In Bolivia, the species occurs in lowland rainforests (Bolivia Checklist, 2014). It is also cultivated in coastal island, and Amazonian parts of Ecuador, where the species is native (Vascular Plants of Ecuador, 2014). The species has also been reported to occur in disturbed lowland areas (Peru Checklist, 2014). 

1.5.14  Genetics

The basic chromosome number is 2n = 14 and n = 7 (IPCN Chromosome Reports, 2014).

1.5.15  Reproductive biology

Trees may be cultivated clonally, via different budding and grafting techniques on seedling stocks, as practiced in certain areas of the United States (e.g. Columbia, Venezuela). The genus is, nevertheless, generally grown from seed (Morton, 1987).

1.5.16  Growth and development

A. muricata branches freely through the emergence of sylleptic shoots. Extension growth can occur at any time of the year and proceeds fairly steadily; there are no prominent flushes. The emergence of flower buds follows extension growth. The position of the flowers - mainly terminal on short shoots and anywhere along the axis of long shoots - suggests that they are initiated terminally, the meristem being pushed to a lateral position as extension growth of the shoot is resumed. Dry season imposes synchronous shoot growth and flowering, leading to a harvest peak three months later, but the synchronization is gradually lost in the course of the rainy season. Annona species generally require 27-35 days for flower bud development from initiation to anthesis. The soursop plant produces fruit throughout the year, but peak production in most areas comes during summer and early autumn, sometimes with a secondary peak during early spring. No photoperiod responses have been reported (Janick and Paull, 2008). Natural pollination in soursop is complex and in most cases results in very low fruit set and yields, with wind- and self-pollination being low (1.5 %). The flowers are protandrous, the pollen is shed as the outer petals open towards the evening. The inner petals open much later and only very slightly, admitting small insects attracted by the fragrance of the flowers. Presumably, these insects effect cross-pollination, though rather inadequately, for few flowers set fruit and many fruits are misshapen as numerous ovules are not fertilized. Nitidulid beetles (Carpophilus and Uroporus spp.) are considered important pollinators, although no significant effect has been observed from their presence in some cases. These beetles breed very fast in the remains of fruit, so it is recommended to retain rotting fruit as an attractant. Some reports have indicated that the presence of three nitidulid beetles per flower can increase fruit set by 25 %. Inadequate pollination appears to be the main factor limiting yield and hand pollination is often recommended for commercial production. However, it is feasible only where there is a definite flowering period. Often, very efficient hand pollination can result in significant economic returns from higher fruit set and larger and more symmetrical fruit. Success in hand pollination is sometimes variable, being less successful on very humid overcast days and with young, vigorous trees. About 150 flowers can be pollinated by a skilled labourer in 1 hour with a success rate of 80-100 % (Paull and Duarte, 2012).

Fruit growth shows the typical sigmoidal curve with maturation occurring in 16-24 weeks. Low humidity <60 % RH) and temperature <13 °C) near fruit maturity can increase the severity of fruit skin russeting as well as delaying maturation.

1.5.17  Environmental requirements

Annona muricata requires a warm and humid tropical climate, prefers well-drained and loose, fairly rich, deep loamy soil with a pH range of 5 - 6.5, is intolerant of waterlogged soil, and can be stunted or killed by cold spells or light frost as the tree is shallow-rooted (Koesriharti, 1991; Orwa et al., 2009). Defoliation and an interruption of fruiting occur when the temperature drops to near freezing. However, soursops are capable of growing in a wide range of soil types from sandy to clay loams provided that the soil has good drainage. Although the tree is commonly grown on slightly acid soils with optimum pH at 5 - 6.5, it also grows on the porous, oolitic limestone of south Florida and the Bahamas. Higher and more consistent yields are obtained on trees grown on well-drained sandy to sandy loam soils. Waterlogging is a major cause of floral abscission and root rot such as bacterial wilt caused by Pseudomonas spp. Soursop cannot tolerate standing water for any length of time but will tolerate dry soil conditions. Dry season enhances leaf fall and synchronizes extension growth and flowering to some extent. Poor pollination is a frequent problem and occurs at high temperature (30 °C) and low humidity (30 % relative humidity (RH)), even with hand pollination. Lower temperature (25 °C) and high humidity (80 % RH) greatly improves pollination (Janick and Paull, 2008).

The soft wood of the trees makes them susceptible to wind damage and limb breakage. Wind may also be partially responsible for the penetration of collar rot organisms. Fruit productivity is improved by the provision of windbreaks.

Its altitudinal range is reportedly 0-2000 m; in Bolivia and Peru, Annona muricata has been reported growing at 0-500 m (Bolivia Checklist, 2014; Peru Checklist, 2014), while in Colombia and Nicaragua, the species is cultivated at elevations of 0-1000 m (Flora of Nicaragua, 2014; Vascular Plants of Antioquia, 2014), and in Panama it reportedly grows at elevations up to 2000 m (Panama Checklist, 2014).

1.5.18  Uses

The edible fruits of A. muricata are the most well-known feature of the plant. The fruit of the species may be "eaten pure, or combined with sugar and water, to which wine and nutmeg are often added (Macfadyen, 1837). It is enjoyed by horses, pigs, hogs, and even other types of livestock.” In Java, Cuba, and parts of America, the fruit pulp is used to make sherbets, ice cream, jellies, and other sweets, as well as a cocktail (Brown, 1950; Koesriharti, 1991 and Flora of Pakistan, 2014). Fruit pulp is boiled and combined with sugar to produce sweetcake ('dodolsirsak') in Indonesia, and young fruits are consumed as a food in the Philippines (Koesriharti, 1991). Since “yields are usually low: one to two dozen fruit per tree per year, each fruit weighing more than 1 kg on average” and “mature, firm fruit ripen 3—5 days after harvest and can be stored just 2—3 days after harvest, even if cooled.” Report have it that the species is difficult to cultivate for large-scale, global commercial purposes. As a result, collected fruit are sent to remote markets as soon as possible. Fruit should be treated with extreme caution due to their delicate skin”; “production of soursop is too dispersed to supply a significant manufacturing company due to volatile yield and poor shelf life.” This is probably why “soursop output in Southeast Asia has stalled in recent years” and “international exchange is mostly restricted to imported products” (Koesriharti, 1991). The seeds and several plant sections of A. muricata are used in herbal medicine in addition to being used as a food crop (Hanelt et al., 2001; Orwa et al., 2009 and USDA-ARS, 2014). Badrie and Schauss (2010) describe its use in indigenous Indian medicine as well as in Jamaica, Haiti, Brazil, and the Peruvian Amazon for the treatment of kidney problems, fever, nervousness, ulcers, and wounds, with antispasmodic, antidysenteric, and parasiticidal activity, its leaves for fever, its bark as tonic, roots as antispasmodic and parasiticidal, and its flowers as bechic (relieving coughs). Ayurvedic medication uses the herb as a bitter, tonic, abortifacient, febrifuge, scorpion bite treatment, high blood pressure treatment, and respiratory depressant. It's been common as a cancer remedy, and although there's no data from clinical studies to back this up, some recent laboratory studies indicate that leaf extracts can kill cancer cells (Pieme et al., 2014; Yang et al., 2015). Sadly, the fruit and seeds also produce annonacin, which has been shown to be detrimental to dopaminergic and other neurons in vitro and in vivo, and intake of the fruit has been linked to an elevated risk of human atypical parkinsonism (Lannuzel et al., 2006; Badrie and Schauss, 2010).

Hidalgo (2003) documented the usage of this species' bark for the treatment of liver disorders in typical riverine communities in the Brazilian Amazon. In addition, people along Colombia's Pacific coast apply leaves to the sick person's forehead to relieve headaches induced by malaria (Blair and Madrigal, 2005). Decoctions made from fresh or dried leaves of A. muricata are taken in Colombia to relieve fevers caused by malaria (Gómez-Estrada et al., 2011). Fresh leaves of this plant are combined with Ocimum americanum and Ocimum gratissimum (Lamiaceae) 1for the treatment of malaria in various African populations (Kaou et al., 2008).

 Ann0na muricata has also been used as an intercrop species in agriculture. Being a tiny and early-bearing plant, the soursop is reported be planted as an intercrop between larger fruit trees such as mango, avocado, and santol, (Koesriharti, 1991). The soursop trees are grubbed out when the main crop needs the space.” The  seeds have insecticidal properties and have been used for this function in the past against pests (Hanelt et al., 2001; Flora of Pakistan, 2014).

 

1.5.19  Invasiveness

A. muricata is a small American tree, perhaps native to Mexico, Central America, and South America, and also occuring in the West Indies and Caribbean. It is considered an invasive species in CenBIO (Acevedo-Rodriguez and Strong, 2012). The species is tolerant of poor soils, is propagated by both seed and cuttings, and has a history of repeated, intentional introductions in places beyond its native range (Pier, 2014). The species received a low risk score of -3 in an assessment prepared for Pier (2014), indicating it is not currently a major threat, but it is included in the Global Compendium of Weeds as an “agricultural weed, cultivation escape, environmental weed, naturalised, weed” (Randall, 2012), and is cited as invasive in some Pacific islands by references listed by PIER (2014). In the Asia Pacific region it is known to be occasionally adventive or sparingly naturalized in parts of French Polynesia (Wagner et al., 2014), and has been reported to be “moderately invasive on Nauru” and cited by PIER (2014) as invasive in parts of Tonga, Hawaii and the Galapagos Islands.

 

1.5.20 Danger of introduction

Despite the fact that A. muricata is a recognized farm weed that has escaped planting in certain areas (Randall, 2012), it earned a low risk score of -3 in a PIER risk evaluation of 2014. The risk of A. muricata being introduced is presently minimal, although it has shown some invasive characteristics which may create issues in areas where the species has become prevalent.

1.5.2    Annona squamosa L. (sweetsop)

The species Annona squamosa L. is commonly known as ‘sugar apple’ or ‘sweetsop’ in English. In Northern Nigeria, it is called ‘fasadabur’ in Hausa.

A. squamosa, a small deciduous tree about 3-5(-6) m tall, produces its first branches near the base of the trunk. The branches are irregularly spreading and the young growth is densely pubescent (Carangal et al., 1961). The leaves are alternate, ovate-oblong or elliptic-oblong, thin, sparsely downy, dark green above, 8-15 cm long and 2-5 cm wide. When young, leaves are pubescent and give a peculiar smell when crushed (Troup, 1975). The petiole is about 1.0-1.5 cm long (Coronel, 1983). The small, pendulous flowers occur singly or in pairs in the leaf axils of young shoots or opposite the leaves. The pedicel is 1.5-2.5 cm long and hairy. The three sepals are short, deciduous, densely or thinly pubescent, and 0.2-0.3 cm long. The 6 petals are biseriate. The three outer petals are lanceolate, thick, fleshy, trigonous, finely pubescent, and yellowish-green on the outside, yellowish-white inside, 2.0-2.5 cm long and 0.5-1.0 cm wide. The three inner petals alternate with the outer ones and are minute, sometimes absent, ovate and never more than 0.1 cm long. The stamens are numerous, yellowish-white in many rows on the glabrous, raised receptacle (torus), 0.12-0.15 cm long and crowded in a whorl around the gynoecium. The pistils are also numerous, dark violet, finely pubescent, and are found above the stamens. The stigmas are sessile, stuck together, and deciduous. The stamens and pistils form a cone-shaped structure at the centre of the flower (Coronel, 1983). The fruit is a syncarp developed from the fusion of numerous ovaries. It is irregularly heart-shaped, about 5-20 cm in diameter. The exterior is marked by polygonal tubercles which correspond to the fused carpels from which the fruit is formed. The ripe fruit is light yellowish-green or purple and the exterior readily separates along the lines between the tubercles. The flesh is white, soft and juicy with a mild, agreeable flavour. The numerous seeds are obovoid or elliptic, dark brown or black, shiny, slightly compressed, 1.0-1.5 cm long and 0.5-0.8 cm wide, and each is enclosed in the edible pulp.

1.5.21  Plant type

A. squamosa is a broadleaved, woody perennial shrubby tree.

 

1.5.22  Distribution

According to Wester (1912), A. squamosa is native to tropical South America and the West Indies; however, Pinto et al. (2005) states that the sugar apple originated in lowland Central America where it is indigenous, and was spread across Mexico and tropical America from there. The more restricted natural distribution of mainland Central America is included in this datasheet, since it is more probable that it was spread in prehistory. It is found in a naturalized or wild condition in Mexico's lowlands, and it is grown from Central America to northern South America, reaching as far as northeastern Brazil, where it is one of the most common fruits. It's a native species in the West Indies, where it's been around since 1689. (UK Natural History Museum, 2015).

1.5.23  Danger of  introduction

The risk of this species being introduced is minimal, though not negligible. In a risk evaluation prepared for Hawaii (PIER, 2015), the species achieved a low risk score of -2, but it is reported to have escaped from agriculture in Australia and Costa Rica (Morton, 1987; Randall, 2012), and it is known to be invasive in non-native environments such as French Polynesia, Nauru, and Mayotte (PIER, 2015). It is expected to be launched as a commercial fruit in the future.

1.5.24  Habitat

A. squamosa is a lowland tropical or mildly subtropical plant native to Central America's hottest and driest areas, rising between latitudes 23 °N and S, but it also thrives in humid climates and has been recorded in cultivation in semi-arid climates like Northeastern Brazil (Pinto et al., 2005). The species is said to be found in “dryish, sandy substrates and dry hammocks” in North America (Flora of North America Editorial Committee, 2015) and “in thickets, on roadsides, and in valleys, in the southern districts” in Puerto Rico (Liogier and Martorell, 2000); on Saint John, US Virgin Island, it is naturalized and found along roadsides and secondary forests (Acevedo-Rodrguez, 1996). It was allegedly emerging in scrublands in the Bahamas (Britton and Millspaugh, 1920), and it is now found wild in pastures, woodland, and along roadside in dry regions of North Queensland, Australia (Morton, 1987). The species can be found in Colombia's wet tropical forests (Vascular Plants of Antioquia, 2015) and Ecuador's coastal regions (Vascular Plants of Ecuador, 2015). In India, hillocks, gravelly soils, and waste ground are the most common habitats for wild A. squamosa. It can be found at lower elevations in dry regions. The species may be the most adaptable of all Annona spp., thriving in low, sandy, or limestone soil as long as it is well drained, and tolerating more dryness and wind than other Annona spp.

 

1.5.25  Genetics

There are a few well-known sugar apple varieties, the majority of which are found in India, and their names serve as introductions to their origins: ‘Mammoth,' ‘Barbados,' ‘British Guinea,' ‘Balondegar,' ‘Red Sitaphal,' and ‘Sindhan,' the latter of which is native to Gujarat, as well as a dwarf cultivar called ‘LalSitiphal' (Pinto et al., 2005). In the Philippines, there are three types of A. squamosa: a seeded green-fruited form grown throughout the world, a purple-fruited seeded form allegedly imported from India (Galang, 1955), and a seedless green-fruited form whose origin is unknown. Collection of superior strains is recommended from the green-fruited seeded type (Coronel, 1983); however, there has been very little selection of superior seedlings to date. PJ Webster hybridized sugar apple (A. squamosa) and cherimoya (A. cherimola) in 1907 in Florida, USA, yielding a new fruit known as atemoya (Pinto et al., 2005a). The hybrid is naturally produced in Australia (where it is confusingly named custard apple), the United States, Israel, South Africa, the Philippines, and various areas of Central and South America, where it occurred spontaneously in the field in 1850 and again in Palestine in 1930. This hybrid is chosen because pollination issues do not seem to be an issue (Pinto et al., 2005a).

1.5.26  Biology of reproduction

By seed or asexual proliferation, A. squamosa may regenerate rapidly. If the seeds are freshly planted, 90-95 percent germination occurs 20-30 days after sowing (Miraflores, 1915; Paguirigan, 1951; Galang, 1955). During the stagnant season stem cuttings of good matured wood may be used to cultivate A. squamosa (Noonan, 1953; Ochse et al., 1961). The fruitfulness of A. squamosa in the Philippines, according to Wester (1912), is attributed to the existence of some types of coleopterous insects that exist there and pollinate the flowers. A. squamosa bears fruit 2-4 years after planting, but the production per tree is typically poor, with just 40-75 fruits per year on average (Cabbab and Soliven, 1938). When the skin between the segments turns from greenish-yellow to creamy-yellow, the fruit is called ripe. Birds and bats consume ripe fruit left on the tree, and ripe fruit has a propensity to break open on the tree if left to ripen too long. The fruits ripen at sporadic periods over a three-month cycle and are picked when they ripen. Fully mature fruits ripen 2-5 days after harvest, and since they are perishable, consumption is recommended as soon as possible (Gonzalez, 1934; Ochse et al., 1961; Vinas, 1972).

1.5.27  Physiology and phenology

A. squamosa grows in the open or in partly shaded areas. When young, it requires shaded areas and at a later stage full overhead light is neede for vigorous growth. It can withstand drought as it has deciduous growth characteristics during dry periods.

In India, fruit set takes place after the commencement of the rainy season (Thakur and Singh, 1965, 1967). Those fruits that are set during the summer will generally dry up from high temperatures and low relative humidity. Fruit set during summer may be increased by irrigating the trees and providing shade. Fruit growth of green-fruited and red-fruited A. squamosa is rapid from August to mid-September. Afterwards it gradually slows down in the red-fruited form. The growth rate of the green-fruited form, however, still increases into October, after which it slows down and ceases by mid-November (Thakur and Singh, 1965).

In Florida, the fruiting season begins in mid-summer (wet season) with irregular ripening lasting 3 months. In the Philippines, fruiting occurs during the beginning of the rainy season (summer) and in India, fruiting also occurs in the wet season (August to mid-September) and can occur from October to November (the end of the wet season) (Pinto et al., 2005). In Mexico, flowering occurs at the end of the dry season (March to May) with fruiting at the end of the wet season (September to November). In Brazil, flowering occurs at the end of the dry season (March to May) and fruiting in the wet season (December to January) (Pinto et al., 2005).

 

 

1.5.28  Ecological prerequisites

A. squamosa grows well in warm, humid tropical and subtropical lowlands below 1000-1200 m altitude (Paguirigan, 1951; Galang, 1955 and Vinas, 1972). It is drought-tolerant, but does not fruit well in high rainfall regimes (Paguirigan, 1951; Galang, 1955, and Vinas, 1972). Though it is less sensitive than some of its kin, it cannot withstand freezing temperatures or sustained cold (Noonan, 1953). On the other hand, can survive drought and be produced successfully in areas with a long dry season (Verkataratanam and Satyanaranaswamy, 1956; Cantillang, 1976). Since it does not have leaves for the rest of the dry season, its deciduous growth pattern adds to its drought tolerance. While it seems that dry conditions during the flowering season is preferred, fruits are often placed at the start of the rainy season (Noonan, 1953; Ochse et al., 1961). A. squamosa is possibly the most adaptable of the Annona genus in terms of soil conditions, since it can withstand wind and dry climates as well as infertile, dusty, and limestone soil as long as it is well-drained (Paraguay Checklist, 2015). It can not stand flooding for long periods of time. Sands, sandy or silty loams, and clays are ideal for A. squamosa. Trees grown on sandy loams, on the other hand, tend to produce consistently higher yields (Coronel, 1983; Verheij and Coronel, 1991). Since the root system is small, it does not take very deep soil. It favors mildly acidic soil environments with a pH of 5.5-6.5, as do most other fruit crops, and can withstand moderate salinity (Coronel, 1983). (Pinto et al., 2005). The species' elevation range is normally restricted. It has been recorded to grow between 0-50 m (Flora of North America Editorial Committee, 2015) and 0-100 m in Panama (Panama Checklist, 2014), while it grows between 0-500 m in Bolivia's lowland rainforests (Bolivia Checklist, 2015), and between 0-500 m along Ecuador's coastal regions (Vascular Plants of Ecuador, 2015). In Nicaragua, the species is grown in lowland areas of the Pacific region between 30 and 145 meters (Flora of Nicaragua, 2015). The species has been found in moist tropical forest at significantly higher altitudes of 500-1000 m in Colombia (Vascular Plants of Antioquia, 2015).

1.5.29  Natural enemies

Some Thoughts Several pests target A. squamosa, feeding on its roots, branches, leaves, and fruits (Gabriel, 1975). The moth borer Annona epestisbengalella is the most damaging insect in the Philippines (Leon, 1917; Estalilla, 1921; Galang, 1955), with larvae that feed and tunnel into the interior of the fruits (de Leon, 1917; Estalilla, 1921; Galang, 1955). Frasses are seen on the surface of attacked grapes, which are bound together by fine thread. The eggs are laid singly in fruit sutures and stems (rarely on leaves) and hatch in 4 - 5 days. The larvae immediately bore into the fruits after hatching, often creating fresh tunnels. The most significant harm is done by the time they hit their third molt. The fruit can fail to grow completely and, in extreme cases, fall off the tree. The larvae pupate in cocoons made near the skin. After 12 days of pupation, the moths emerge.

 Another parasite of A. squamosa is the root grub (Anomala sp.), which attacks the roots and causes abrupt wilting at the plant's base in advanced stages (Vinas, 1972; Gabriel, 1975). The grey mealybug (Ferrisia virgata) and cottony cushion mealybug (Planococcus lilacinus) are also typical pests of A. squamosa, sucking the sap from young leaves and fruits, inducing leaves to turn yellow and wither (Coronel, 1983). The larvae of the coffee carpenter moth (Zeuzera coffeae) destroy the stem by boring into the heart of the wood to eat and mature (Vinas, 1972; Gabriel, 1975). A. squamosa is susceptible to inflorescence rust, pink plague, and rhizoctonia thread blight.

1.5.30  Invasiveness

The plant has been shown to be invasive outside of its native range and dominant in its natural range and is known to be highly adaptable to various habitats. It is highly mobile locally and fast growing. It has high reproductive capacity and has propagules that can stay viable for more than one year. It reproduces asexually and has high genetic diversity.

1.5.31 Uses

A. squamosa's fruit is normally consumed raw. It is a source of carbohydrates, vitamins, and proteins, and comprises 50-61 percent edible matter (Pinto et al., 2005a). Commercially, the fruit is used to flavor ice cream and may even be rendered into sherbet. After the seeds have been removed, the pulp may be strained or homogenized to produce a tasty and soothing cocktail. A. squamosa's leaves, bark, stems, nuts, and berries have a variety of medicinal properties. The green fruit and seed are used as astringents in diarrhoea and dysentery and have powerful vermicidal and insecticidal properties. The seeds produce 45 percent of a yellow, non-drying oil that irritates lice and kills them. Ulcers and malignant sores may be effectively treated with crushed plants. For dyspepsia, a poultice made from fresh leaves is used, and when combined with oil, it is used for scalp diseases. In cases of fainting spells, crushed fresh leaves are added to the respiratory area. A root decoction is used as a harsh purgative (Coronel, 1983). The alkaloid anonaine is found in the astringent bark, leaves, unripe fruit, and seed (Troup, 1975). Its leaves is used in the management of rheumatism and sore spleen, it is also used as insecticidal and antispasmodic agents. Customarily, the plant is said to have analgesic, anti-inflammatory, anti-pyretic, anti-ulcer, antiseptic, and abortifacient properties. Several researchers looked at its use as an insecticide, and derivatives from the seeds were used in numerous phytochemical, pharmacological, anti-bacterial, and anti-ovulatory tests. Studies with the seed extracts of A. squamosa revealed post-cortical anti-fertility behavior (Chavan et al., 2010). Seeds, fruits and leaves were found to be effective as an insecticide, fish poison, and as a powerful irritant of the conjunctiva. The roots were found to be effective as a severe purgative and in the acute dysentery (Rahman et al., 2005). The hot aqueous extract of Annona squamosa leaves was found to have a significant hypoglycemic and anti-diabetic activity and its fruit has much higher nutritional value with the biological activity of lowering blood glucose level in experimental animals (Rajesh et al., 2008). Annona squamosa Vell is astringent and was established to be useful for the treatment of chronic diarrhoea and estomatic disease and also useful as an insecticide (Dos Santos and Sant’Ana, 2001). A. squamosa is commonly cultivated as a fruit tree in the backyard and as part of agroforestry systems. Its fruit is edible, and the flowers are used in beekeeping. Because of its appealing fruit color, A. squamosa is often planted as a shade and ornamental tree in parks or plazas (Coronel, 1983).

1.5.3    Annona senegalenses Pers. (wildsop).

Wildsop is commonly known as “Wild Custard Apple” in Nigeria and is a shrub or small tree commonly distributed in Africa (Adzu et al., 2005 and Ogbadoyi et al., 2007). It has aromatic flowers that are used to add flavour to food. The ripe fruit is yellow in colour and has a sweet edible jelly with pleasant odour. In Nigeria, A. senegalensis is known as “Gwandar daji” in Hausa, “Abo” in Yoruba, “Uburu ocha” in Ibo, and “Ikpokpo” among the Idoma speaking people in the Middle Belt region of Nigeria. It is widespread in the Savannah area and near streams and enjoys great reputation for its immense medicinal value and hence, ethno- medicinal uses.  

Annona senegalensis Pers. takes the form of either a shrub or small tree, growing to the height of between two and six meters. Occasionally, it may become as tall as 11 m. The plant is not generally cultivated, but often grows in the wild (Uphof, 1959). The fruit is sometimes sold in local markets (Ruffo et al., 2002). The plant is said to have the potential for domestication (Ruffo et al., 2002). Its bark is smooth or coarse in texture and is a gray-silver or gray-brown in colour. It is leaf-scarred, with nearly round flaking, showing lighter-hued spaces of under bark. Branches have thick, gray, brown or yellow tomentum when new, but this is later shed with age. Its green to blue-green leaves are alternate, simple, oblong to ovate to elliptic, measuring 6–18.5 long by 2.5–11.5 cm wide, with upper sides nearly hairless, but often hairy on the undersides with green to reddish, aracnose veins on both surfaces, with rounded to slightly notched apices. The leaf base is squared or barely lobeliar. The leaf margin is entire. Stout petioles are 0.5–2.5 cm long. Mature flowers are up to 3 cm in diameter, on 2 cm stalks, either singular, or two to four, ascending from the leaf axils. Six thick, creamy or xanthate petals are displayed in double whorls, and green on the outside, but either creamy or sanguine within; each is roughly 0.8–1.5 by 0.9–1.1 centimeters (0.31– 0.59 by 0.35–0.43 in), hairless or somewhat fuzzy. Petals' inner whorls curve over its stamens and ovary; three loose sepals are ovalish, and smaller than the petals (3–4 by 4–5 mm). The stamens range from 1.7 to 2.5 millimeters (0.067 to 0.098 inch) long. The plant flowers from April through June. Its pollen is shed as permanent tetrads (Walker, 1971). Fruits are formed of numerous fused, fleshy, bumpy, ovaform or globular carpels about 2.5–5 by 2.5–4 centimeters (0.98–1.97 by 0.98–1.57 inches). They are green when young, ripening to yellow, and eventually to orange, packed with many burnt-orange-colored, oblong, cylindrical seeds. The fruit stalk is 1.5–5 centimeters (0.59–1.97 inches) long. Annona senegalensis is generally pollinated by several species of beetle, but can be hand pollinated when grown as a crop plant. Its seed viability usually lasts no more than six months.

1.5.31 Habitat

Annona senegalensis grows in semiarid to subhumid coastal areas, mostly, but not always, on coral-based rocks with mainly sandy, loamy soils, from sea level to 2400 meters, at mean temperatures of 17 to 30 degrees Celsius and mean rainfall of 700 to 2,500 millimeters (28 and 98 inches). They are mostly solitary plants in the understory of woodland savannahs, but they may also be found in swamp forests, riverbanks, and on former cropland that has been left fallow for a long time.

1.5.32 Distribution

Tropical east and northeast, west and west-central, and southern Africa, as well as southern subtropical Africa and islands in the western Indian Ocean, are all home to this species. It's only present in South Africa, in the provinces of KwaZulu-Natal, Limpopo, and Mpumalanga. In certain areas of India, Annona senegalensis has been naturalized. It can also be found on the Maldivian islands

1.5.33  Uses

This flexible plant is mostly valued for its fruits, although it has applications in a wide range of human endeavors, and each section of the plant has its own set of properties and uses. Flowers, leaves, and fruit are edible and culinary, and the white fruit pulp has a faint pineapple taste. Flowers are used to season or garnish meals (Facciola, 1998); leaves are consumed as vegetables by humans or grazed by animals (Facciola, 1998). The West African giraffe often eats leaves as part of its diet (Mariama, 2008). The leaves may even be used to make a general health tonic, to cure influenza, and are also known to be used to stuff mattresses and pillows (Von, 1990). Leaves are boiled and used in the production of perfume in Sudan. Bark may be transformed into a yellow-brown stain, insecticide (Von, 1990), or medication for a range of infections, such as worms parasitic on the intestines or flesh (particularly guinea worms), diarrhea (Ruffo et al., 2002), gastroenteritis, respiratory problems, toothaches, and even snakebites (Ruffo et al., 2002). The bark's natural gum is used to seal open wounds (Ruffo et al., 2002). Roots are also used to cure a variety of ailments, including dizziness and indigestion, as well as chest colds and sexual infections Suckering shoots have binding fabrics, and the malleable, light brown to white wood is carved into tool handles and sticks. Wood ash is used as a solvent in soap manufacture and as an admixture in chewing tobacco and snuff. The organic chemical constituents of the essential oils in the fruits and leaves are valued: car-3-ene (in the fruit) and linalool (in the leaves). Annona senegalensis extracts are used to treat skin and eye problems. Many South Africans claim that the origins should be used to treat mental illness. Some Mozambicans offer them to babies to help them wean them off their mothers' breasts.

 In Northern Nigeria, the herb decoction is said to be trypanocidal (Ogbadoyi et al., 2007), analgesic, anti-inflammatory (Adzu et al., 2003), anti-arthritic, anti-trypanasomisic (Atawodi et al., 2003) and also effective againsts rheumatism (Audu, 1989), intestinal and guinea- worms (Alawa et al., 2003).

Yellow fever, measles, small pox, snakebites, hernia, necrotizing venoms, erectile dysfunction, trouble swallowing, contagious diseases, gastritis, male sexual dysfunction, and diabetes were all recorded to be treated with all sections of A. senegalensis (Okhale et al., 2016). These popular uses are consistent with the numerous pharmacological properties of the plant including anti-convulsant, anti-bacterial, anti-helminthique, anti-drepanocytary, anti-inflammatory, anti-venous anti-tripanosomal and anti-hyperglycemic activities.

 

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