Abstract: Generally, biodiversity is being threatened globally by climate change as well as human activities and this has aroused concerns about the conservation status. This study was designed with the aim of searching for existing, new and unrecorded plant species of the family Sapindaceae in West Africa so as to better understand the extent of diversity and distribution of the species in the family remaining in existence and conserve them for maximum use. The methodology employed include: field sampling, preservation of voucher specimens in secured repositories and DNA conservation of the collected samples. It was observed that the family Sapindaceae are represented by 26 genera and 104 species in western Africa. The most species rich regions are Nigeria (47 taxa), Cameroon (45 taxa) and Ghana (25 taxa). Southern highlands of Nigeria have the highest number of species followed by western river banks of Ghana. Taxa shared are highest between Nigeria and Ghana and endemism is highest in the western regions with 9 species endemic to the mountains and coasts. High quality genomic DNA were obtained and deposited in the DNA bank at the Royal Botanic gardens Kew. This research can be seen as a key step in the conservation of the family Sapindaceae as it reveals that most of the plants are endangered mainly due to deforestation and agricultural practices in forest reserves across West Africa.
INTRODUCTION
Sapindaceae Juss. (Soapberry family) is a family of flowering plant belonging to the order Sapindales. It comprises about 1900 species with a predominantly pan-tropical distribution though some taxa occur in temperate areas (Buerki et al., 2009). It includes trees and shrubs and a few climbers. Many of the members of the family grow in the under storey of forests and are on the borderline between shrubs and trees. Members can be found in South America, Africa and Australia however, the majority of the species are native to Asia (APG, 2003). Members include maple, horse chestnut and lychee but the largest genera are Serjania, Paullinia, Acer and Allophylus. They serve as important sources of nuts, beverages (Paullinia cupana), oils (Schleichera trijuga) and drugs (Blighia sapida) as well timber trees. They are common ingredients in traditional herbal remedies in rural and urban Africa (Odugbemi and Akinsulire, 2006; Sofidiya et al., 2007, 2008; Adesegun et al., 2008; Antwi et al., 2009; Muanya and Odukoya, 2008; Ripa et al., 2010; Pendota et al., 2008). Several species contain saponins in their fruits, seeds and other tissues hence they serve as soap substitute (e.g., Sapindus saponaria). Many other members are grown for their edible fruits (e.g., Melicocca bijuga) and aril (e.g., Pometia pinnata) Blighia sapida, akee is a fruit from West Africa, with an edible aril but poisonous if not eaten at the correct stage of ripeness. In Africa, 25 genera are represented in the East with 61 species between them; 14 species are regarded as endemic to the area while about 18 (Hutchinson and Daziel, 1958) to 22 (Burkill, 2000) genera are represented in West with only about 13 genera found widely spread throughout Nigeria (Keay et al., 1964). Conservation through DNA technology constitutes a powerful tool for ex situ conservation programmes especially for species with reduced populations. Essentially, for long term persistence of species, it is important to minimize the probability of their being lost as well as maximize their representation in places where they are currently present (Pressey et al., 2004). Although, varieties of protocols have been developed for proficient means of extracting DNA of both higher quality and yield, the fundamentals of DNA extraction remains the same (Tiwari et al., 2012). According to the IUCN (2008), many members of the family Sapindaceae are categorised as threatened. Apart from over-exploitation by humans, droughts and land clearance for mining, dams and construction are other threats to the existence of members of Sapindaceae in Africa, hence the need for adequate conservation strategies. Although, DNA and tissue banks have been shown to have great potential as tools for biodiversity conservation, only few have been established (Savolaninen et al., 2006). Also, the need to study the germplasm of crops using molecular methods in addition to quantitative methods has been stressed by several authors including: Samal et al. (2003) and Taamalli et al. (2006) and there has been such studies as Kumar et al. (2006) on the family Sapindaceae however, there is no record of the genomic conservation of members of the family in West Africa. Therefore, this study was designed to search for existing, new and unrecorded plant species of the family Sapindaceae in West Africa and conserve their DNA for future use.
MATERIAL AND METHODS
Source of plant materials: Herbarium dried and fresh leaves were used for the study. Plant material used for DNA extraction was obtained from field, botanical garden, forest reserves sources in Cameroon, Ghana, Nigeria and Togo and this was complemented with herbarium samples. These were dried and stored in silica gel prior to DNA isolation. From herbarium specimens, 0.5 cm2 of plant tissue was removed and either stored in plastic tubes or used immediately.
Identification of the plant samples: Voucher specimens were prepared and sent to the Forestry Herbarium, Ibadan for authentication. These were then deposited at the University of Lagos Herbarium for reference purpose.
DNA extraction: Total genomic DNA was extracted using the 2-cetyltrimethylammonium bromide (CTAB) procedure of Doyle and Doyle (1987) with minor modifications followed by additional purification.
Gel electrophoresis: This involved quality check of the DNA samples on 1% agarose gel. The gel was run on 0.5X Tris Borate EDTA (TBE) buffer at 75 V for 1 h 30 min. The gel was visualized by staining with 10 mg mL-1 ethidium bromide under Ultraviolet (UV) light and photographed with the gel documentation system (UVitec).
Quantification of DNA samples: This involved the determination of the concentration and relative absorbance of each of the DNA samples using a biophotometer. It was achieved by measuring 55 μL of the diluent i.e., sterile water into a cuvette followed by 2 μL of the DNA sample. The cuvette was then placed in a biophotometer and readings were documented.
RESULTS
Sapindaceae is represented by a hundred and four species distributed in 26 genera i.e., Allophylus, Aporrhiza, Blighia, Cardiospermum, Chytranthus, Cupania, Deinbollia, Dodonaea, Eriocoelum, Glenniea, Harpullia, Haplocoelum, Laccodiscus, Lecaniodiscus, Lepisanthes, Lychnodiscus, Majidea, Melicoccus, Nephelium, Pancovia, Paullinia, Placodiscus, Radlkofera, Sapindus, Schleichera and Zanha. They are widely distributed throughout western Africa and the highest number of taxa was recorded in Nigeria (47 taxa). Other countries assessed include Cameroon (45 taxa), Ghana (25 taxa), Ivory Coast (23 taxa), Sierra Leone (19 taxa), Togo (13), Liberia (12 taxa) and Guinea (10 taxa). Taxa shared are highest between Nigeria and Ghana. In Tropical West Africa the southern highlands of Nigeria have the highest number of species followed by the Western and Eastern River banks in Ghana and Ivory Coast. Endemism was highest in the western regions of Nigeria and Ghana with 9 species endemic to the mountains and coasts. Deoxyribonucleic Acid (DNA) samples were extracted from all the samples collected and deposited in the DNA bank at the Royal Botanic Gardens Kew, London (Table 1).
| Table 1: | Sources of materials used for the study |
| Fig. 1: | Electropherogram of extracted DNA samples |
All the 104 samples had distinct bands showing the presence of DNA. The quality of extracted DNA samples was determined using agarose gel electrophoresis and this revealed that the cell constituents were properly released into the buffer despite the total exclusion of the use of liquid nitrogen. Subsequently, DNA isolated are of high molecular weight bands (Fig. 1).
| Fig. 2: | Relative absorbance ratio of DNA samples |
The DNA samples were quantified using spectrophotometry and this revealed that the concentration of the DNA samples ranges from 20-2261 ng μL-1. Also, purity of the DNA samples were measured at 260 and 280 nm and the absorbance ratio recorded (A260/280) ranged from 0.87-2.01 (Fig. 2).
DISCUSSION
All over the world, over-exploitation of living resources takes place in order to meet short term needs but more often than not the process destroys exactly those resources on which the welfare of the inhabitants depends on the long term. Although members of the family Sapindaceae have been recorded to be widely distributed in Africa, their occurrence is being threatened by high rate of deforestation and agricultural practices leading to loss of forest and threatened status of members of the family as recorded in the IUCN (2008). However, our sampling revealed that there are twenty six genera and a hundred and four species in West Africa in contrast to the twenty two genera recorded by Burkill (2000). The other four genera include Aphania, Ganophyllum, Haplocoelum and Laccodiscus.
Members of the family were largely found in the lowland forest region with a few taxa located in the highland and mountains (Allophylus bullatus, Schleichera trijuga, Sapindus saponaria). In West Africa, the most species rich regions are Nigeria, western Cameroon and Ghana with forty seven, forty five and twenty five taxa, respectively. Endemism and No. of taxa shared are highest between Nigeria and Ghana with nine species endemic to the mountains and the coast, respectively.
It was observed that, some of the species earlier recorded in some of the reserves were no longer found largely due to the high rate of deforestation and agricultural activities observed in the reserves. This could be attributed to the fact that the secondary forest remnants may lack key resources needed to maintain the species year-round and potentially the species has not been able to persist. Also, forest remnants in this area may be too few and too heavily impacted to retain the necessary resources to sustain the species over the long term.
A number of authors have shown the need to study the germplasm of crops using molecular methods in addition to quantitative methods including: Samal et al. (2003) and Taamalli et al. (2006). However, DNA extraction protocols are specific for different plant species though not always simple and reproducible (Pandey et al., 1996; Porebski et al., 1997). According to Vallejos (2007), preventing the oxidation of phenolic substances that can react with nucleic acids and proteins and eliminating polysaccharides that interfere with the enzymatic manipulations of DNA are some of the major challenges associated with DNA extraction protocols. This is further aggravated if green over matured tissues are used rather than etiolated leaves (Sharma et al., 2000). In view of the foregoing, a modification of the CTAB protocol was used in this research. Genomic DNA was successfully extracted from all the collected samples and 48% of the samples had an absorbance ratio of 1.40-2.0. The low absorbance ratio values recorded in some of the taxa can be attributed to highly degraded quality of some of the extracted DNA samples especially as herbarium materials were also employed in the study (Savolainen et al., 2000). No ultracentrifuge was used in this study rather a bench centrifuge of 4,000 rpm was used and the time of spinning was increased from 5-20 min however, genomic DNA was successfully extracted from all the collected samples. All the samples have been assigned DNA bank number and deposited at the Royal Botanic Gardens Kew.
CONCLUSION
This study has helped in enumerating the number of species belonging to the family Sapindaceae represented in West Africa. Also, genomic DNA of varying concentration and molecular weight were obtained and this can be used for further molecular studies on the family. Therefore, this experiment would serve as a basis upon which further researches on the family could be based and as a valuable tool for geneticist and plant breeders.
ACKNOWLEDGMENTS
Special thanks to the Explorer Club, USA for the Expedition grant and the School of Postgraduate Studies University of Lagos, for the Graduate fellowship award.