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.