II. Morphological Studies
a. Whole Plant
There is voluminous and fragmentary work concerning macro-morphological characteristics of some taxa of Caesalpinioideae. To cite but a few one can refer to the work of Taubert (1894), Britton & Rose (1930), Senna (1943), Ducke (1949), Burkart (1952), Leonard (1957) and Brenan (1967).
Moreover the morphological descriptions of caesalpinioid taxa are met within the texts of plant taxonomy ( Lawrence, 1951; Linnaeus, 1953; Benson, 1957; Engler, 1964; Willis, 1966; Hutchinson, 1967 & 1973; Smith, 1977 and Cronquist, 1981); the texts of cultivated plants ( Bailey, 1935 & 1949; Bricher, 1960 and Graf, 1981) and the texts of flora (Oliver, 1871; Brandis, 1911; Post, 1932; Motasir & Hassib, 1956; Täckholm, 1956 & 1974 and Boulos, 1999).
b. Leaf Architecture
Leaf architecture, as defined by Foster (1952) refers to the placement and form of those elements constituting the outward venation pattern, marginal configuration, leaf shape and gland position.
Ettingshausen (1861) made the first comprehensive effort to systematizes the description of the vegetative leaf architecture with his classification of venation patterns.
Subsequent publications were proposed by botanists and paleobotanists stressed the importance of this parameter in the solution of a number of taxonomic and phylogenetic querries, and different classifications of dicotyledonous leaf architecture. To cite but a few one can refer to Lesquereux (1878), Kerner (1895), Goebel (1905), Berry (1916), Lam (1925), Hollick (1936), Troll (1938), Hickey (1971b, 1973 & 1979) and Melville (1976).
Leaf architectural characters have provided valuable taxonomic and systematic evidences both in fossil and living plants (Dilcher, 1974; Hickey & Wolfe, 1975; Li & Hickey, 1988; Sun et al ., 1991; Zhou & Li, 1994; Zhou et al., 1995; Wang et al., 2001; Yan and Zhe, 2002 and Jesudass et al., 2003).
Foster and Gifford (1974) showed that very diverse shapes in vascular systems in leaves of higher plants viz. long parallel major veins interconnected by short secondary veins (monocotyledons), open branching (ferns) and reticulated networks (dicotyledons). The latter may have a robustness advantage against damage due to their redundancies even when physical damage occurs, fluids can use a different path so the whole leaf is still well supplied (Sack et al., 2003). This gives the leaves more resistance to herbivores and pathogens. The vascular system is formed by a self-assembly process involving complex interactions which are still not well understood. Nevertheless, it is well known that the plant hormone auxin is involved in this process (Jacobs, 1952; Mitchison, 1981; Uggla et al., 1996; Mattson et al., 1999; Sieburth, 1999; Avsian-Kretchmer et al., 2002; Aloni et al., 2003; Fukuda, 2004 and Donner & Scarpella, 2009).
Both the outline and venation system of a leaf are essential in the recognition of plant species. Various venation structures can be found in plant kingdom. It is believed that venation patterns correlate closely with the taxonomic groups of plants and the shapes of leaves (Shenglian et al., 2009). The principal characteristics of the leaf venation pattern of a species are, in general, genetically fixed. This provides the basis for using the leaf venation as a taxonomic tool (Roth et al., 2001).
However, the use of data sets from leaf architecture as a clue to solve taxonomic problems was generally neglected. This was mainly due to the lack of a detailed, standardized and unambiguous classification of these features (Hickey, 1973). In this respect, a relatively recent approach has been mainly centered on trying to identify systematically informative leaf features that allow species to be recognized on the basis of dispersed leaves (Hickey, 1973; Hickey & Wolfe, 1975; Hickey & Taylor, 1991 and LAWG*, 1999). The main use of leaf architectural criteria as an aid in the delimitation of genera and species were performed in palaeobotany (Mouton, 1966 and Dilcher, 1974), certain genera from different families as the Araceae, Fagaceae, Rubiaceae and Rosaceae (Merriell, 1978; Jensen, 1990; Ray, 1992; Loutfy, et al., 2005 and Pacheco et al., 2009), or even entire families as the Lauraceae (Klucking, 1987; Hyland, 1989; Yu & Chen, 1991 and Christophel & Rowett, 1996).
Concerning the studies on Caesalpinioideae, Dianxiang (1994) studied leaves architecture of 134 species of intraspecific taxa, representing almost all species or subsections of the five genera of the tribe Cercideae and twenty venation types have been described and concluded that like other morphological or palynological characters, leaf venation characters can be source of information for systematics, but it can only be used in systematics together with data from other aspects. Seetharam & Kotresha (1998) emphasized the taxonomic importance of venation and its usfullness in classification of Bauhinia L., leaf venation of 12 species of Bauhinia and monotypic genus Hardwickia was investigated and thus the link between the two genera was established. Calvillo et al. (2002) studied leaf impressions from two specimens of Cercideae (Caesalpinieae). The phenetic analysis of morphological observations suggested that morphological differences between the fossil and extant plants correspond to natural variation within the tribe. Although the fossil material closely resembles both Bauhinia and Cercis.
c. Leaf Epidermal Study
Stomatographic studies have shown that stomata can provide valuable taxonomic and systematic evidence in both living and fossil plants and also have played a significant role in framing hypotheses about early angiosperm evolution (Bailey & Nast, 1948; Stebbins & Khush, 1961; Stace, 1965; Van Cotthem, 1970; Wilkinson, 1979; Upchurch, 1995; Kong, 2001; Prabhakar, 2004 and Carpenter, 2005).
Some particular groups of plants or taxa seem to be characterized by specific type of epidermal features, which are the epidermis, stomata, gland and trichomes (Park, 1994, Hong & Oh, 1999 and Hong & Son, 2000). Many distinct patterns of stomata have been found in epidermis of different plants and have often been used as morphological markers for plant taxonomy (Van Cotthem, 1970a; Guyot, 1971; Leelavathi, 1980; Jelani et al., 1990; Ferzana et al., 1991 and Wang, 2006). The peculiar types of stomata in dicotyledons have been reported in various families such as in Acanthaceae (Paliwal, 1966), Leguminosae, Commelinaceae and Nyctaginaceae (Edeoga et al., 1996 & 2001) and Polygonaceae (Ayodele & Olowokudejo, 2006 and Yasmin et al., 2010).
Concerning the studies on Caesalpinioideae, Kadiri and Olowokudejo (2008) examined and compared the epidermal features of the leaves in six Afzelia species using LM & SEM and concluded that the inter-specific taxonomic relationships existing in Afzelia can be investigated by the use of some endo-qualitative and quantitative characteristics of the leaf such as epidermal surfaces, epicuticular wax, stomatal size, crystals and stomatal orientation. Many of these features are both specific and generic constant, and they can be used to facilitate the recognition of the species and help in solving problems of adulteration in the commercial species even when the leaf is fragmentary.
Zou et al. (2008) examined the leaf epidermal microcharacters of nine taxa of Cercis using SEM & LM, and then concluded that the interspecific differences are minor in the genus. So, these characters can be used to identify fossils of this confusing group of plants.
III. Molecular Studies
a. RAPD-PCR Study
Application of DNA based molecular markers is the solution for the need of clear discrimination between genotypes and cultivars. Different DNA marker systems have been used for genetic analysis and characterization, and valuable tools for the identification of plant varieties.
RAPD (Random Amplified Polymorphic DNA) is one of the most widely used techniques for genetic diversity studies (Gupta & Rustgi, 2004) and genetic mapping (Carlier et al., 2004). This technique allows the detection of differences at the DNA level using small amounts of genomic DNA and does not require any previous information on DNA sequences. RAPD markers have been reported to be as efficient as AFLP (Amplified Fragment Length Polymorphism) markers (Ipek et al., 2003), SSR (Simple Sequence Repeat), RFLP (Restriction Fragment Length Polymorphism) and ISSR (Inter Simple Sequence Repeat) markers (Martins et al., 2003 and Zahuang et al., 2004) for genetic analysis at different plant species.
During recent decades, the use of RAPD markers for resolving the genetic diversity in fruit trees have been remarkable (Gillies et al., 1997; Cardoso et al., 1998; Dawson & Powell, 1999; Heaton et al., 1999; Lowe et al., 2000; Degen et al., 2001; Bekessy et al., 2002; Lee et al., 2002; Belaj et al., 2004; Uma et al., 2004 and Yamagishi et al., 2005).
Concerning the studies on Caesalpinioideae, Whitty et al. (1994) adopted RAPD method for use as a phenetic tool on the legume tribe Cassiinae, three Cassia species, 12 Chamaecrista species and 13 Senna species using eight primers and showed the potential for separation of the nodulated nitrogen fixing genus Chamaecrista from the previously congeneric groups Cassia and Senna.
Diallo et al. (2007) studied 10 Tamarindus populations using markers RAPDs with the seeds collected from Asia (India and Thailand), Africa (Burkina Faso, Senegal, Kenya and Tanzania), from three Islands (Madagascar, Réunion and Guadeloupe). The results showed that T. indica has a high intra population genetic variability with a higher value obtained in the population from Cameroon. This high intra-population variability did not allow us to determinate the origin of the species. However, if we take into account the paleontological and anthropological results, we can assume that T. indica has an African origin.
Kumar et al. (2007) carried out RAPD analysis to assess the authentic identification as well as to solve the taxonomic problems between Senna surattensis and S. sulfurea. Amplification with 10-mer primers was performed along with S. occidentalis, S. siamea, S. tora and Cassia fistula. Out of sixty primers utilized, fifty four were successful in amplification and among them one was species-specific. The results demonstrate the ability of RAPD markers to reliably differentiate between S. surattensis and S. sulfurea.
b. Isozymes Study
During the past 20 years, enzyme electrophoresis has been used to describe the population genetic structure of over 700 plant taxa (Hamrick & Godt, 1989). This information has contributed greatly to an understanding of the evolutionary history of individual species and related group of species (Haufler, 1987). These studies have shown that angiosperms have high level of genetic variations. Variation is the basic resource to be explored for genetic improvement in any species and hence play a key role in plant improvement programmes (Hedegart, 1976; Zobel & Talbert, 1984 and Tiwari, 1992).
Many researchers have studied the genetic variability in inter- and intra-populations on natural ecosystems for the purposes of gene pool conservation e.g. Amaral (2001) and Lakshmikumaran et al. (2001). Morphological characteristics might themselves be insufficient to distinguish between pairs of closely related species, because not all-genetic differentiation results in morphological differentiation. Thus, a genetic characterization of natural resources is an essential step for a better understanding of genetic resources for the implementation of in-situ and ex-situ conservation activities (NBPGR*, 2000).
Analysis of isozyme variation has been used widely to study genetic diversity within and among populations of Neotropical tree species (Buckley et al., 1988; Hamrick & Murawski, 1991; Hall et al., 1994 and Chase et al., 1995). Such studies have been generally employed in response to tropical deforestation and its consequences, i.e., the loss of biodiversity and the potential loss of genetic diversity within a species. However, isozyme analysis has also been used to provide information on optimal seed sources for reforestation when species are introduced and cultivated as exotics. For example, studies of Leucaena. (Schifino-Wittmann & Schlegel, 1990 and Harris et al., 1994) and Gliricidia sepium (Chalmers et al., 1992 and Chamberlain et al., 1996) provide two cases of the increasing use of fast-growing legume trees that are native to the neotropics, but planted for a range of purposes elsewhere. Such introductions may often be accompanied by loss in genetic diversity if little is known about the original species and its genetic structure.
Biodiversity and its potential loss have also renewed interest in taxonomy and species delimitation. The emergence of the phylogenetic species concept has prompted the increased use of isozyme data in this area of systematics (Nixon & Wheeler, 1990; Davis & Nixon, 1992; Davis & Goldman, 1993; Elisens & Nelson, 1993 and Chamberlain et al., 1996).
Fixed differences can be used to provide unique character combinations that can distinguish groups of populations as distinct from one another. The attributes of isozymes, i.e., the identification of individual loci and alleles and the relative ease of assaying many individuals from diverse populations can render them useful in the search for fixed differences among populations (Gottlieb, 1977).
Isozymes are useful biochemical markers for assessing genetic variability. They have been used in taxonomic, genetic, evolutionary and ecological studies. Isozymes have also been used for the identification of cultivars and lines (Peirce & Brewbaker, 1973; Gorman & Kiang, 1977 and Cardy & Beversdorf, 1984).
Despite the use of DNA markers such as RAPDs, AFLPs and RFLPs, isozymes are still widely employed in species delimitation and conservation (Schifino-Wittmann & Lange, 1997; Booy & van Raamsdonk, 1998; Chamberlain, 1998; Lang & Schifino-Wittmann, 2000; Siva & Krishnamurthy, 2005 and Mohamed, 2006); assessment of genetic variability in species and populations (Harris et al., 1994; Schifino- Wittmann et al., 1996; Aradhya et al., 1998; Buso et al., 1998 and Sonnante et al., 1998); cultivar identification (Samec et al., 1998 and Sanchez-Escribano et al., 1998); gene flow (Gauthier et al., 1998) and evolutionary studies (Cronn et al., 1997; Jaaska, 1997 and Testolin & Ferguson, 1997).
Isozymes are especially useful when several taxa, accessions and individuals are to be compared, as the assumption of homology is more accurate than with some DNA markers. To cite but a few one can refer to the work of Klaas (1998).
Concerning Leguminosae, isoenzymes have been applied as molecular-genetic markers to study genetic diversity and phylogenetic affinities in populations of Gleditsia triacanthos (Schnabel & Hamrick, 1990); Cassia species (Nualkaew et al., 1998); Prosopis glandulosa & P. velutina (Bessega et al., 2000); some Vigna species, subgenera Sigmoidotropis & Lasiospron, in comparison with the two pantropical Vigna luteola & V. vexillata of the subgenera Vigna & Plectrotropis (Jaaska, 2001); two cogeneric wild invasive Macroptilium species and their relationships to the related cultivated Phaseolus vulgaris L. var. nepraska (Fawzy, 2004) and Cassia auriculata (Siva & Krishnamurthy, 2005).
IV. Numerical Taxonomy
Numerical analysis is a data management and analysis product measures the similarities and dissimilarities between genera and species. It can perform a variety of data analysis and presentation functions, including statistical analysis and graphical presentation of data.
The taxa in numerical analysis are usually referred to as OTU’s viz. Operational Taxonomic Units. In some studies, specifically in ecological field, the species may be studied from more than one specimen, so it is more convenient to refer to taxa as OTU. This term indicates that in this specific numerical operation each OTU is a distinct taxon. Characters in numerical taxonomy should be carefully defined. There are three main types of characters: qualitative or two state, multi-state and quantitative characters.
Concerning Leguminosae, several authors checked the current classification for different genera and species and analysed their results by using different numerical analysis programs. Abou El-Enain & Loutfy (1999) discussed the similarities between Delonix regia and Caesalpinia pulcherima. Larmarque & Fortunato (2003) used the numerical analysis to discuss the taxonomic placement of Acacia emiliona and its affinity within subgenus Aculeiferum. Concerning the work on Caesalpinioideae by using numerical analysis. El-Azab (2005) showed the similarities between some of different taxa of Mimosoideae. El-Gazzar et al. (2008) reached to computer-generated keys to the flora of Egypt (Mimosoideae & Caesalpinioideae). Abou El-Enain et al. (2007) delimeted the genus Cassia into two subgenera viz. Fistula and Senna based on the basis of morphological criteria and seed protein electrophoresis.
a. Whole Plant
There is voluminous and fragmentary work concerning macro-morphological characteristics of some taxa of Caesalpinioideae. To cite but a few one can refer to the work of Taubert (1894), Britton & Rose (1930), Senna (1943), Ducke (1949), Burkart (1952), Leonard (1957) and Brenan (1967).
Moreover the morphological descriptions of caesalpinioid taxa are met within the texts of plant taxonomy ( Lawrence, 1951; Linnaeus, 1953; Benson, 1957; Engler, 1964; Willis, 1966; Hutchinson, 1967 & 1973; Smith, 1977 and Cronquist, 1981); the texts of cultivated plants ( Bailey, 1935 & 1949; Bricher, 1960 and Graf, 1981) and the texts of flora (Oliver, 1871; Brandis, 1911; Post, 1932; Motasir & Hassib, 1956; Täckholm, 1956 & 1974 and Boulos, 1999).
b. Leaf Architecture
Leaf architecture, as defined by Foster (1952) refers to the placement and form of those elements constituting the outward venation pattern, marginal configuration, leaf shape and gland position.
Ettingshausen (1861) made the first comprehensive effort to systematizes the description of the vegetative leaf architecture with his classification of venation patterns.
Subsequent publications were proposed by botanists and paleobotanists stressed the importance of this parameter in the solution of a number of taxonomic and phylogenetic querries, and different classifications of dicotyledonous leaf architecture. To cite but a few one can refer to Lesquereux (1878), Kerner (1895), Goebel (1905), Berry (1916), Lam (1925), Hollick (1936), Troll (1938), Hickey (1971b, 1973 & 1979) and Melville (1976).
Leaf architectural characters have provided valuable taxonomic and systematic evidences both in fossil and living plants (Dilcher, 1974; Hickey & Wolfe, 1975; Li & Hickey, 1988; Sun et al ., 1991; Zhou & Li, 1994; Zhou et al., 1995; Wang et al., 2001; Yan and Zhe, 2002 and Jesudass et al., 2003).
Foster and Gifford (1974) showed that very diverse shapes in vascular systems in leaves of higher plants viz. long parallel major veins interconnected by short secondary veins (monocotyledons), open branching (ferns) and reticulated networks (dicotyledons). The latter may have a robustness advantage against damage due to their redundancies even when physical damage occurs, fluids can use a different path so the whole leaf is still well supplied (Sack et al., 2003). This gives the leaves more resistance to herbivores and pathogens. The vascular system is formed by a self-assembly process involving complex interactions which are still not well understood. Nevertheless, it is well known that the plant hormone auxin is involved in this process (Jacobs, 1952; Mitchison, 1981; Uggla et al., 1996; Mattson et al., 1999; Sieburth, 1999; Avsian-Kretchmer et al., 2002; Aloni et al., 2003; Fukuda, 2004 and Donner & Scarpella, 2009).
Both the outline and venation system of a leaf are essential in the recognition of plant species. Various venation structures can be found in plant kingdom. It is believed that venation patterns correlate closely with the taxonomic groups of plants and the shapes of leaves (Shenglian et al., 2009). The principal characteristics of the leaf venation pattern of a species are, in general, genetically fixed. This provides the basis for using the leaf venation as a taxonomic tool (Roth et al., 2001).
However, the use of data sets from leaf architecture as a clue to solve taxonomic problems was generally neglected. This was mainly due to the lack of a detailed, standardized and unambiguous classification of these features (Hickey, 1973). In this respect, a relatively recent approach has been mainly centered on trying to identify systematically informative leaf features that allow species to be recognized on the basis of dispersed leaves (Hickey, 1973; Hickey & Wolfe, 1975; Hickey & Taylor, 1991 and LAWG*, 1999). The main use of leaf architectural criteria as an aid in the delimitation of genera and species were performed in palaeobotany (Mouton, 1966 and Dilcher, 1974), certain genera from different families as the Araceae, Fagaceae, Rubiaceae and Rosaceae (Merriell, 1978; Jensen, 1990; Ray, 1992; Loutfy, et al., 2005 and Pacheco et al., 2009), or even entire families as the Lauraceae (Klucking, 1987; Hyland, 1989; Yu & Chen, 1991 and Christophel & Rowett, 1996).
Concerning the studies on Caesalpinioideae, Dianxiang (1994) studied leaves architecture of 134 species of intraspecific taxa, representing almost all species or subsections of the five genera of the tribe Cercideae and twenty venation types have been described and concluded that like other morphological or palynological characters, leaf venation characters can be source of information for systematics, but it can only be used in systematics together with data from other aspects. Seetharam & Kotresha (1998) emphasized the taxonomic importance of venation and its usfullness in classification of Bauhinia L., leaf venation of 12 species of Bauhinia and monotypic genus Hardwickia was investigated and thus the link between the two genera was established. Calvillo et al. (2002) studied leaf impressions from two specimens of Cercideae (Caesalpinieae). The phenetic analysis of morphological observations suggested that morphological differences between the fossil and extant plants correspond to natural variation within the tribe. Although the fossil material closely resembles both Bauhinia and Cercis.
c. Leaf Epidermal Study
Stomatographic studies have shown that stomata can provide valuable taxonomic and systematic evidence in both living and fossil plants and also have played a significant role in framing hypotheses about early angiosperm evolution (Bailey & Nast, 1948; Stebbins & Khush, 1961; Stace, 1965; Van Cotthem, 1970; Wilkinson, 1979; Upchurch, 1995; Kong, 2001; Prabhakar, 2004 and Carpenter, 2005).
Some particular groups of plants or taxa seem to be characterized by specific type of epidermal features, which are the epidermis, stomata, gland and trichomes (Park, 1994, Hong & Oh, 1999 and Hong & Son, 2000). Many distinct patterns of stomata have been found in epidermis of different plants and have often been used as morphological markers for plant taxonomy (Van Cotthem, 1970a; Guyot, 1971; Leelavathi, 1980; Jelani et al., 1990; Ferzana et al., 1991 and Wang, 2006). The peculiar types of stomata in dicotyledons have been reported in various families such as in Acanthaceae (Paliwal, 1966), Leguminosae, Commelinaceae and Nyctaginaceae (Edeoga et al., 1996 & 2001) and Polygonaceae (Ayodele & Olowokudejo, 2006 and Yasmin et al., 2010).
Concerning the studies on Caesalpinioideae, Kadiri and Olowokudejo (2008) examined and compared the epidermal features of the leaves in six Afzelia species using LM & SEM and concluded that the inter-specific taxonomic relationships existing in Afzelia can be investigated by the use of some endo-qualitative and quantitative characteristics of the leaf such as epidermal surfaces, epicuticular wax, stomatal size, crystals and stomatal orientation. Many of these features are both specific and generic constant, and they can be used to facilitate the recognition of the species and help in solving problems of adulteration in the commercial species even when the leaf is fragmentary.
Zou et al. (2008) examined the leaf epidermal microcharacters of nine taxa of Cercis using SEM & LM, and then concluded that the interspecific differences are minor in the genus. So, these characters can be used to identify fossils of this confusing group of plants.
III. Molecular Studies
a. RAPD-PCR Study
Application of DNA based molecular markers is the solution for the need of clear discrimination between genotypes and cultivars. Different DNA marker systems have been used for genetic analysis and characterization, and valuable tools for the identification of plant varieties.
RAPD (Random Amplified Polymorphic DNA) is one of the most widely used techniques for genetic diversity studies (Gupta & Rustgi, 2004) and genetic mapping (Carlier et al., 2004). This technique allows the detection of differences at the DNA level using small amounts of genomic DNA and does not require any previous information on DNA sequences. RAPD markers have been reported to be as efficient as AFLP (Amplified Fragment Length Polymorphism) markers (Ipek et al., 2003), SSR (Simple Sequence Repeat), RFLP (Restriction Fragment Length Polymorphism) and ISSR (Inter Simple Sequence Repeat) markers (Martins et al., 2003 and Zahuang et al., 2004) for genetic analysis at different plant species.
During recent decades, the use of RAPD markers for resolving the genetic diversity in fruit trees have been remarkable (Gillies et al., 1997; Cardoso et al., 1998; Dawson & Powell, 1999; Heaton et al., 1999; Lowe et al., 2000; Degen et al., 2001; Bekessy et al., 2002; Lee et al., 2002; Belaj et al., 2004; Uma et al., 2004 and Yamagishi et al., 2005).
Concerning the studies on Caesalpinioideae, Whitty et al. (1994) adopted RAPD method for use as a phenetic tool on the legume tribe Cassiinae, three Cassia species, 12 Chamaecrista species and 13 Senna species using eight primers and showed the potential for separation of the nodulated nitrogen fixing genus Chamaecrista from the previously congeneric groups Cassia and Senna.
Diallo et al. (2007) studied 10 Tamarindus populations using markers RAPDs with the seeds collected from Asia (India and Thailand), Africa (Burkina Faso, Senegal, Kenya and Tanzania), from three Islands (Madagascar, Réunion and Guadeloupe). The results showed that T. indica has a high intra population genetic variability with a higher value obtained in the population from Cameroon. This high intra-population variability did not allow us to determinate the origin of the species. However, if we take into account the paleontological and anthropological results, we can assume that T. indica has an African origin.
Kumar et al. (2007) carried out RAPD analysis to assess the authentic identification as well as to solve the taxonomic problems between Senna surattensis and S. sulfurea. Amplification with 10-mer primers was performed along with S. occidentalis, S. siamea, S. tora and Cassia fistula. Out of sixty primers utilized, fifty four were successful in amplification and among them one was species-specific. The results demonstrate the ability of RAPD markers to reliably differentiate between S. surattensis and S. sulfurea.
b. Isozymes Study
During the past 20 years, enzyme electrophoresis has been used to describe the population genetic structure of over 700 plant taxa (Hamrick & Godt, 1989). This information has contributed greatly to an understanding of the evolutionary history of individual species and related group of species (Haufler, 1987). These studies have shown that angiosperms have high level of genetic variations. Variation is the basic resource to be explored for genetic improvement in any species and hence play a key role in plant improvement programmes (Hedegart, 1976; Zobel & Talbert, 1984 and Tiwari, 1992).
Many researchers have studied the genetic variability in inter- and intra-populations on natural ecosystems for the purposes of gene pool conservation e.g. Amaral (2001) and Lakshmikumaran et al. (2001). Morphological characteristics might themselves be insufficient to distinguish between pairs of closely related species, because not all-genetic differentiation results in morphological differentiation. Thus, a genetic characterization of natural resources is an essential step for a better understanding of genetic resources for the implementation of in-situ and ex-situ conservation activities (NBPGR*, 2000).
Analysis of isozyme variation has been used widely to study genetic diversity within and among populations of Neotropical tree species (Buckley et al., 1988; Hamrick & Murawski, 1991; Hall et al., 1994 and Chase et al., 1995). Such studies have been generally employed in response to tropical deforestation and its consequences, i.e., the loss of biodiversity and the potential loss of genetic diversity within a species. However, isozyme analysis has also been used to provide information on optimal seed sources for reforestation when species are introduced and cultivated as exotics. For example, studies of Leucaena. (Schifino-Wittmann & Schlegel, 1990 and Harris et al., 1994) and Gliricidia sepium (Chalmers et al., 1992 and Chamberlain et al., 1996) provide two cases of the increasing use of fast-growing legume trees that are native to the neotropics, but planted for a range of purposes elsewhere. Such introductions may often be accompanied by loss in genetic diversity if little is known about the original species and its genetic structure.
Biodiversity and its potential loss have also renewed interest in taxonomy and species delimitation. The emergence of the phylogenetic species concept has prompted the increased use of isozyme data in this area of systematics (Nixon & Wheeler, 1990; Davis & Nixon, 1992; Davis & Goldman, 1993; Elisens & Nelson, 1993 and Chamberlain et al., 1996).
Fixed differences can be used to provide unique character combinations that can distinguish groups of populations as distinct from one another. The attributes of isozymes, i.e., the identification of individual loci and alleles and the relative ease of assaying many individuals from diverse populations can render them useful in the search for fixed differences among populations (Gottlieb, 1977).
Isozymes are useful biochemical markers for assessing genetic variability. They have been used in taxonomic, genetic, evolutionary and ecological studies. Isozymes have also been used for the identification of cultivars and lines (Peirce & Brewbaker, 1973; Gorman & Kiang, 1977 and Cardy & Beversdorf, 1984).
Despite the use of DNA markers such as RAPDs, AFLPs and RFLPs, isozymes are still widely employed in species delimitation and conservation (Schifino-Wittmann & Lange, 1997; Booy & van Raamsdonk, 1998; Chamberlain, 1998; Lang & Schifino-Wittmann, 2000; Siva & Krishnamurthy, 2005 and Mohamed, 2006); assessment of genetic variability in species and populations (Harris et al., 1994; Schifino- Wittmann et al., 1996; Aradhya et al., 1998; Buso et al., 1998 and Sonnante et al., 1998); cultivar identification (Samec et al., 1998 and Sanchez-Escribano et al., 1998); gene flow (Gauthier et al., 1998) and evolutionary studies (Cronn et al., 1997; Jaaska, 1997 and Testolin & Ferguson, 1997).
Isozymes are especially useful when several taxa, accessions and individuals are to be compared, as the assumption of homology is more accurate than with some DNA markers. To cite but a few one can refer to the work of Klaas (1998).
Concerning Leguminosae, isoenzymes have been applied as molecular-genetic markers to study genetic diversity and phylogenetic affinities in populations of Gleditsia triacanthos (Schnabel & Hamrick, 1990); Cassia species (Nualkaew et al., 1998); Prosopis glandulosa & P. velutina (Bessega et al., 2000); some Vigna species, subgenera Sigmoidotropis & Lasiospron, in comparison with the two pantropical Vigna luteola & V. vexillata of the subgenera Vigna & Plectrotropis (Jaaska, 2001); two cogeneric wild invasive Macroptilium species and their relationships to the related cultivated Phaseolus vulgaris L. var. nepraska (Fawzy, 2004) and Cassia auriculata (Siva & Krishnamurthy, 2005).
IV. Numerical Taxonomy
Numerical analysis is a data management and analysis product measures the similarities and dissimilarities between genera and species. It can perform a variety of data analysis and presentation functions, including statistical analysis and graphical presentation of data.
The taxa in numerical analysis are usually referred to as OTU’s viz. Operational Taxonomic Units. In some studies, specifically in ecological field, the species may be studied from more than one specimen, so it is more convenient to refer to taxa as OTU. This term indicates that in this specific numerical operation each OTU is a distinct taxon. Characters in numerical taxonomy should be carefully defined. There are three main types of characters: qualitative or two state, multi-state and quantitative characters.
Concerning Leguminosae, several authors checked the current classification for different genera and species and analysed their results by using different numerical analysis programs. Abou El-Enain & Loutfy (1999) discussed the similarities between Delonix regia and Caesalpinia pulcherima. Larmarque & Fortunato (2003) used the numerical analysis to discuss the taxonomic placement of Acacia emiliona and its affinity within subgenus Aculeiferum. Concerning the work on Caesalpinioideae by using numerical analysis. El-Azab (2005) showed the similarities between some of different taxa of Mimosoideae. El-Gazzar et al. (2008) reached to computer-generated keys to the flora of Egypt (Mimosoideae & Caesalpinioideae). Abou El-Enain et al. (2007) delimeted the genus Cassia into two subgenera viz. Fistula and Senna based on the basis of morphological criteria and seed protein electrophoresis.
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