Diversity in the Stem Anatomy and Tissues of Several Species of Annona ( Annonaceae ) in Nigeria

Comparative study of the stem anatomy of Annona trees was carried out to facilitate identification of taxa when their reproductive parts are not available. Stem specimens of four out of the five species categorized as important under-utilized species were collected and subjected to anatomical studies. Macerated materials followed Schutze’s method of maceration and the transverse sections of the stem were cut using Reichert Sledge Microtome. Intrageneric characters observed are: epidermal layer uniseriate with round, oval to polygonal collenchyma cells. Wood is diffuse-porous, vessel elements are diffuse, vessels are of two types, that is, short/wide with simple pits and long/narrow with bordered pits. Diagnostic characters for the identification of each of the species are also reported.


Introduction
Annona is a genus of tropical fruit tree belonging to the family Annonaceae (Nakasone and Paull, 1998). It has approximately 119 species of which seven and one hybrid are grown for domestic/commercial use. Five species have been selected as important under-utilized species (ICUC, 2002).
Economically, Annona trees can be categorized into two groups. In the first group are A. reticulata (custard apple) and A. senegalensis (wild soursop) which are grown by subsistence farmers under small holder conditions, resulting in low income to their growers. The second group includes A. muricata (soursop) and A. squamosa (sugar apple) which are grown in commercial farms as highly remunerative crops for both small and medium scale farmers (Vogt, 1995). Annonas are generally consumed as fresh fruits, but are also widely used in semi-processed and processed products (Salunkhe and Desai, 1986). Based on their nutritional status, A. reticulata and A. squamosa have been reported to have more nutritional (protein) and shelf-life than A. muricata and A. senegalensis (Folorunso and Modupe, 2007).
Suffice to say that a number of studies had been carried out on the genus Annona, there had been no report on the diversity of the stem anatomy and tissues of Annona trees. Olatunji (1992) in his work on the wood anatomy of some species of Zanthoxylum in Nigeria reported that despite the close similarities observed among the species in many of their wood anatomical features, some of the species possess specific features which may be employed to delimit them. Studies of this nature might facilitate identification of taxa most importantly when their reproductive parts are not available. And of course, this would enable both taxonomists and the general public to have inkling of this plant and as such harness all possible means of conserving them. In the present paper, the stem anatomy and tissues of four species of Annona is described and compared.

Materials and methods
Stem samples of four out of the five species categorized as important under-utilized species were collected within the experimental garden of Obafemi Awolowo University Campus, Ile-Ife, Nigeria. They are : A. muricata (soursop), A. senegalensis (wild soursop), A. reticulata (custard apple) and A. squamosa (sugar apple). The stem samples of the species investigated were kept as voucher specimen in the Herbarium, Department of Botany, Obafemi Awolowo University, Ile-Ife (IFE).
Small blocks of wood, about 1 cm x 1 cm x 1 cm were removed from the trunk of the trees of about 1.5 metres from the ground. The blocks were macerated following Schutze's method of maceration. This method of maceration involves the addition of fuming concentrated trioxonitrate (v) (HNO 3 ) into test tubes to a level just above the contained match-stick size wood samples. Few crystals of solid Potassium Chromate (K 2 Cr 2 O 7 ) was added. This was then placed on a hot plate for about 4 minutes when the samples must have macerated. Small pieces of the macerated wood were stained in 1% Safranin 0 and mounted on slide using dilute glycerol as mountant. Anatomical measurements were made in ocular units at 40X objective power and converted to micrometers. Illustration of the wood anatomical features were done by camera lucida under x25 objective power of Leitz DIALUX research microscope.
with thin walls and simple pits. They have length of 205.2-406.8 µm and average width of 7.2-18.0 µm. Ray consists of both uniseriate and multiseriate rays. Ray parenchyma cells are homocellular and scarcely distributed with length of 190.8-367.2 µm and width of 43.2-57.6 µm. They have thin walls and simple pits.

Annona muricata
Epidermal layer uniseriate, collenchyma cells round, oval to polygonal; wood diffuse-porous. The vessels have thin walls and have simple intravascular pits or bordered pits with opposite pair pitting arrangement. The blocks of wood were boiled in water for about two hours to soften them. The transverse sections of the stem were cut on a Reichert Sledge microtome at varying thicknesses between 20-30 microns. The sections were stained in Alcian blue, counter stained with Toluidine blue (to enhance contrast) washed thoroughly in water, dehydrated through series of ethyl alcohol : 50, 70, 90, 95%, cleared in xylene and mounted in D. P. X. mountant. Photomicrographs of the transverse section were made from the photomicrographic outfit fitted to Wild M20 microscope.

Annona senegalensis
Epidermal layer uniseriate, collenchyma cells round, oval to polygonal; wood diffuse-porous. Vessels are of two types, short and wide type and the long and narrow type (Tab. 1). They have simple intravascular pits which occur in tiny circular sizes scattered throughout the vessels or some could possess bordered pits arranged in opposite pair. Most vessels that exhibit bordered pits are long with length 162.0-334.8 µm and width of 54.0-126.0 µm. Other simple pitted vessels are relatively short with length of 43.3-169.2 µm and width of 21.6-111.6 µm. Vessel elements are round, oval to elliptic in radial multiples of 2-5 pores. Vessel diameters are 30±1.0 µm. Fibres are non-septate. Fibre-tracheids are 133.2-655.2 µm long and 10.8-25.2 µm short, they have blunt ends to tapering ends with thin walls, simple pits which could be scattered to few cluster or slit-like pits which run in an alternate manner across the length of the fibre. Libriform fibres are 586.8-972.0 µm long with very thin width of 3.6-10.8 µm.

Annona reticulata
Epidermal layer uniseriate, collenchyma cells round, oval to polygonal; wood diffuse-porous. The vessel member comprises of both the wide and the narrow vessels. The vessels are less abundant than the fibre cells and are evenly distributed throughout the entire length of the wood. The vessels consist of a series of axially superimposed cells whose intervening end walls have been perforated. The vessels are relatively short in length 147.6 µm to 360 µm. The narrow vessels have width of 64 µm to 90 µm and the Vessel diameters are 35±1.5µm. Fibres are more abundant than any other cell types in the wood of Annona squamosa. The libriforms are thinner and longer with length ranging from 414 µm to 547.2 µm and has a width of 17 µm-36 µm. Fibre tracheids are shorter, their length ranges from 272 µm to 490 µm and has a width of 18 µm to 42 µm. Axial parenchyma abundant, confluent paratracheal, they are thick walled and are found in varying thickness; their length ranges from 184 µm to 240 µm. they have width with the range of 8 µm to 17 µm. They also have abundant pitting system arranged in opposite rows. Ray parenchyma cells are uniseriate except few that are multiseriate and they are less abundant in the wood, they could be as long as 60 µm to 78 µm with their width varying from 20 µm to 32 µm. There are many crystals of Calcium Oxalate scattered in the cells. Osteosclerids are in the pith.

Discussion
Evidence of common evolutionary origin in the Annona species had been reported (Folorunso and Olorode, 2006a). According to them, there are intrageneric relationships among the Annonas (Folorunso and Olorode, 2006a, b). In this work, more intrageneric characters are reported. These are epidermal layer uniseriate with round, oval to polygonal collenchyma cells. The wood is diffuseporous, vessel elements are diffuse, and the vessels are of two types, that is, short-wide with simple pits and the long-narrow with opposite pair pitting arrangements of bordered pits. All these characters are reportedly typical of the primitive angiosperms and as such would be useful in their identification. Also, the occurrence of solitary vessels which are more than grouped vessels in all the species of Annonaceae studied is an indication that the family is a primitive one.
The diagnostic characters for both A. reticulata and A. squamosa are the presence of axial parenchyma which is confluent paratracheal and the presence of osteosclerids in their piths. These two species are reported to have wide vessels on the other hand have width of 133.2 µm to 200 µm. The vessels have a very thin cell wall with the abundance of pits. The pits are either simple or bordered with opposite pitting. Some of the intervascular pits are alternate and bordered; the vessels observed do not have tails at their perforated ends. Vessel elements are round, oval to elliptic in radial multiples of 2-5 pores. Vessel diameters are 40±2.0 µm. The fibres are more abundant in the stem of Annona reticulata and are found throughout the entire length of the wood. The fibres, either fibre tracheids or libriform type are longer and narrower than their corresponding vessels and are all tapered towards their ends. Fibre-tracheids 540 µm-600 µm long and 43.2 µm to 79.2 µm short. They are characterised by their long, thin and tapered ends. They are mostly thin walled. Their pits are simple which could be scattered to few clusters or slitlike than runs in an alternating pattern across the length of the fibre. Libriform fibres are always longer and thinner than their corresponding fibre tracheids, the shortest has the dimension of 396 µm by 7.2 µm. Libriform fibres are very scanty in the species of A. reticulata and are generally characterised by their thin lumen and thick cell wall. They have few or no pits. The axial parenchyma cells are abundant and confluent paratracheal, they are 160 µm to 192 µm long and 21.6 µm to 36 µm short. They are thick walled and have abundance of simple pits scattered on their cell surfaces. The ray parenchyma cells are predominantly homocellular. Their length varying from 54 µm to 76 µm and with width of 24.8 µm to 30 µm. Osteosclerids are in the pith.

Annona squamosa
Epidermal layer uniseriate, collenchyma cells round, oval to polygonal; wood diffuse-porous. The vessels occur in horizontal rows, with opposite pitting, they are long with length range of 172.8 µm to 342 µm and are mostly wide with width range of 64.8 µm to 100.8 µm. The vessels are characterized by their plate-like perforated ends which are wide and almost taking the whole width of the vessel, the perforations are simple and are tailed. Vessel elements    (Folorunso and Modupe, 2007). It may therefore be concluded that the earlier mentioned diagnostic characters conferred more nutritional value on them. The character that separates the two species is the presence of more fibres in A. reticulata than in A. squamosa. This may suggest the arborescence habit of A. reticulata while A. squamosa is fructicose. The diagnostic feature for A. senegalensis is the occurrence of more vessels and the presence of axial parenchyma that is paratracheal. The higher number of vessels might be one of its diagnostic characteristics for adapting in the savanna. If one vessel is disabled by an air embolism due to water stress, neighbouring vessels may retain water columns and the conductive pathway remains intact.
In the present paper, wood anatomical characters of importance in the identification of the species of Annona have been reported and a taxonomic key for the identification of each of the species is hereby produced.