AS AN AID TO TAXONOMIC CLASSIFICATION
EHAB ABDEL-RAZIK KAMEL ALY
Department of Biological & Geological Sciences,
Faculty of Education, Ain Shams University
The discovery of chromosomes in the last quarter of the nineteenth century and the subsequent demonstration that they are the carriers of genetic information provided a physical foundation for the experimental studies on which are based our knowledge of such taxonomically important matters as variation within plant species (Moore, 1978).
During these years, the important role of the chromosomes in aiding taxonomic understanding became evident through classical and much quoted studies on Cruciferae, Gramineae, Agavaceae and later, incisive monographs of, for example, Crepis, Datura, Nicotiana, Clarkia, Tragopogon and pteridophytes. Biosystematics (Camp, 1951) or experimental taxonomy, is concerned largely with data derived from studies of the chromosomes. The number, size and shape of chromosomes were used to characterize the karyotypes of plants and define the taxonomic differences between them. The pairing behaviour of the meiotic chromosomes in natural or artificial hybrids between plants of different populations indicated the extent to which they had differentiated and in some cases, showed how genomes had been combined like building blocks by polyploidy to provide new species. This information gives support for ecospecies, coenspecies and a hierarchy of precisely defined taxonomic units (Moore, 1978 and Briggs & Walters, 1997).
In several instances, studies of karyotype morphology have led the way to a new and fuller understanding of the systematic relationships within a major group of plants and to a complete reorganization of the taxonomic system of the group. The most outstanding example is the Gramineae. Avdulov (1931) published a study, which remains today as the most thorough analysis of generic relationships within a major plant family from the cytological point of view, which has ever been made (Stebbins, 1956).
In addition to karyotype, Avdulov studied leaf anatomy and histology, seedling development, structure of starch grains and geographic distribution. He found that evidence from all of these characteristics pointed toward a necessary revision of tribe Gramineae, especially the highly heterogeneous tribes Festuceae, Agrostideae and Phalaridea as then recognized. Avdulov’s results were fully confirmed by other authors, who added further evidence to support his system from embryo morphology, vegetative anatomy, morphology of lodicules and caryopses, resistance to herbicides and amino acid composition of seed proteins (Fig. 1) (Stebbins, 1956).
Fig. 2 presents the resulting system, which is widely accepted now. The tribe Festuceae as reconized by taxonomists of the nineteenth and early twentieth century has been broken up into three tribes (Festuceae, Eragroteae and Arundineae). These tribes are distributed in three different subfamilies, while similar, though less drastic, revisions have been made on former tribes Aveneae, Agrostideae and Phalarideae.
Fig. 1: Chart showing the differences between the largest subdivisions of the grass family with respect to diagnostic characters of seedling leaves, history of adult leaves and chromosomes.
A second example of the usefulness of karyotype in clarifying taxonomic relationships is the family Ranunculaceae. The traditional way of subdividing this family has been to place it into three tribes; Helleboreae includes genera having few carples with more than one seed per carple. The Anemoneae and Clematideae include those genera having several or many carples, which are one-seeded and indehiscent. In respect to karyotype morphology, however, the family was divided into two tribes in a completely different way by Gregory (1941). One group of genera has small chromosomes with prominent heterochromatic chromocentres. This group includes Coptis Salisb., Xanthorrhiza Marshall, Hydrastis Ellis ex L., Isopyrum Adans. and Aquilegia L. of the Helleboreae plus Anemonella Spach, Thalictrum L. and Trautvetteria Fisch. et Mey. of the Anemoneae. The remaining nine genera were assigned to the Helleboreae and five usually placed in the Anemoneae and the Clematideae, have large chromosomes and irregularly distributed heterochromatic regions. The first group has principally the basic number x = 7, while the predominant number in the second group is x = 8, although x = 7 and x = 6 are also found in it.
In respect to vegetative growth and overall appearance of the plants, a classification based upon karyotype similarity place plants, which look alike close to each other more consistently than does the classical system, which, is based upon characters of the carpels and fruits. For instance, Isopyrum Adans and Anemonella Spach are strikingly similar to each other in all characters except the number of seeds per carpel and the similarity is equally great between Caltha L., Trollius L. and Ranunculus L. This example certainly deserves further study using additional characteristics.
These examples illustrate that the karyotype morphology can be a most useful guide to taxonomic relationships. By itself, however, it can never be regarded as of overriding importance. In some genera, such as
Fig. 2: Diagram showing the evolutionary inter-relationships between the principal subfamilies and tribes of the Gramineae, based partly upon cytological and upon morphological characteristics.
Colchicum (Feinbrun, 1958), a great variety of karyotypes exist in a genus, which is morphologically and ecologically very homogeneous and distinctive. This is in marked contrast to the situation just described in the Ranunculaceae, in which similar karyotypes exist in highly diverse and heterogeneous groups of genera. This contrast should warn us that we can easily go astray if we use either karyology or external morphology uncritically, without careful consideration of the other characteristics of the plants in question (Stebbins, 1971).
Types of chromosomal variations
Cytologists refer to five different kinds of variations regarding chromosomes of related species. These variations are:
1- Variations in absolute chromosome size:
Chromosome size and the total DNA content of the nucleus may vary as much as 20-fold between genera of the same family having the same or similar basic chromosome number (Fig. 3). Plant chromosomes can be categorized into two types based on size; the large type and the small type. The small chromosome type is represented by species in such genera as Oryza L., Glycine L. and Brassica L. which is around 1 to 3 μm in length at metaphase. The large type is represented by species in such genera as Lilium L., Trillium L. and Vicia L., which have length of about 10 μm or more at metaphase.
Characteristics of Large and Small Type Chromosomes
Length at metaphase
8 – 10 μm or more
2 – 3 μm or less
Fig. 3: Contrasting chromosome sizes in two species belonging to the same family (Fabaceae) and having the same basic number, x = 6: Lotus tenuis and Vicia faba.
2- Variations in staining properties:
Cytologists are familiar with the fact that after similar fixation techniques and exposure to the same stains at the same concentrations, the chromosomes of different species vary greatly in their ability to be stained. Chromosomes of most Liliaceae and Gramineae are notable for the ease with which they can be stained; those of Malvaceae and Onagraceae stain with much greater difficulty.
3- Variations in chromosome morphology:
Chromosome morphology is usually studied at the metaphase of mitosis. At this stage chromosomes have become contracted to the maximum amount and they are most easily stained. The principal landmarks, which may be seen at this stage, are the centromere (sometimes known as the kinetochore), to which the spindle fibres are attached and in many chromosomes one or more secondary constrictions. In addition, one, two and rarely a larger number of chromosome pairs in the somatic complement of a species, bear at one end a satellite. This usually appears as a single small spherical body or a pair of such bodies, attached to the remainder of the chromosome by a slender thread. These chromosomal landmarks are illustrated in Fig. 4.
In most chromosomes, the centromere is localized in one particular region of the chromosome. According to its position, chromosomes are designated as follow (Fig. 5):
Telocentric: the centromere is at one end of the chromosome, so that the chromosome consists of a single arm.
Acrocentric: the centromere is near one end of the chromosome, so that it contains one long arm and one very short arm, which is nearly or quite isodiametric.
Fig. 4: Diagrams showing the landmarks or distinguishing features of a somatic chromosome at early prophase, pro-metaphase and early anaphase of mitosis.
Fig. 5: Diagrams showing the appearance of somatic chromosomes having centromeres at different positions.
Sub-metacentric: the centromere is nearer to one end of the chromosome than the other, so that the two arms are distinctly unequal, but less so than in acrocentric chromosomes.
Metacentric: the centromere is at or near the middle of the chromosome, so that its arms are nearly or quite equal in length.
Chromosomes in a few groups of plants have no centromere and were once known as acentrics (Stace, 2000). However, they behave regularly with the spindle and the separate parts of a fragmented chromosome all retain spindle affinity. They are now described more accurately as Holocentrics and their centromeres as diffuse rather than as lacking. They are well known in Carex and some other genera of Cyperaceae and in Luzula (Juncaceae), but it is wrong to extrapolate the latter genus to include the whole family as has been done in some publications. The evidence from Juncus is that its chromosomes are not holocentrics (Stace, 2000).
Diffused centromeres are very rare in Angiosperms (although highly scattered throughout Dicots and monocots) and their existence in Cyperaceae and Juncaceae, along with other embryological features in common, suggests that these two families are more closely related than most modern classifications.
4- Variations in relative chromosome size:
The chromosomal complements or karyotypes of most species of plants consist of chromosomes, which are comparable to each other in size. There are, however, many complements, which contain chromosomes of two contrasting sizes, large and small (Fig. 6).
The most important morphological variable in chromosomes is the variation in size between the chromosomes of one genome. This aspect is
Fig. 6: Somatic mitotic metaphase chromosomes of barley after aceto-carmine staining showing 2n = 14 chromosomes.
more significant than overall measurements because artificial variation in size is likely to apply equally to all the chromosomes in a cell. The chromosomes in a genome can vary from being virtually all identical in size to exhibiting a size difference ratio of five more (Stace, 2000).
5- Aneuploid variations in chromosome number:
The realisation that most species possess a constant chromosome number came well over a century ago (Stace, 2000). The earliest chromosome studies were not taxonomic investigations, but were carried out in order to understand more fully chromosome functions in embryology, development, cell division and live cycles. Today chromosome number reporting is still fashionable, but three main developments are apparent. Firstly, there is an emphasis on plant accessions from known wild localities; secondly, there is a requirement that a voucher specimen be deposited in a designated herbarium and thirdly, there is a desire for counts to be based on several cells and plants in each population. Many anomalous counts in the literature are the result of misidentifications, abnormal cells or tissues or unusual cultivated mutants, etc. and the above three developments, as well as improvements in cytological techniques, will surely reduce the frequency of such ambiguities. Chromosome numbers have been reported for only about 25% of angiosperms (Bennett, 1998).
Variations in chromosome number are of two very different kinds, aneuploid and polyploid or heteroploid.
Chromosome number is a product of two variables; the base-number (x) and the ploidy level (n). It must be emphasised that base-number is a relative, not an absolute, concept, i.e., it is meaningless out of context, having significance only in relation to a defined taxon. Hence the base-number of a genus might be different from that of the family to which it belongs, which can have a different base-number from that of the order or class to which that family is in turn assigned. In some cases the base-number of, say, a genus is thought to be the same as the lowest known haploid number in that genus, but subsequently new evidence (such as a lower haploid number) emerges that changes this view. For example the base-number of Zea (Poaceae) was considered to be x = 10 and all the taxa were believed to be diploids. Recently, despite all known taxa having 2n = 20, molecular evidence has indicated that Zea is in fact a tetraploid and therefore the base-number is x = 5 (Moore et al., 1995).
Chromosomes base-numbers are frequently of great evolutionary significance and taxonomic value. In the Poaceae, for example, the subfamilies are characterized by different base-numbers: Bambuusoideae have mostly x = 12; Arundinoideae mostly x = 9 or 12; Chloridoideae mostly x = 9 or 10; Panicoideae mostly x = 5, 9 or 10 and Pooideae mostly x = 7. However, within Pooideae some tribes, e. g., Glycerieae with x = 9 or 10, or genera, e. g., Anthoxanthum (tribe Phalarideae) with x = 5, deviate, the base-numbers in such cases providing important diagnostic characters. In the rather small genus Brachypodium (tribe Brachypodieae) (with probably fewer than 20 species) base-numbers of x = 5, 7, 8 and 9 occur and at least three species exhibit both 7 and 9 (Khan and Stace, 1999).
1- Preparation of small chromosome samples:
Enzymatic maceration / Air-drying method (EMA):
1. Root tips of around 0.5 to 1 cm are excised from fresh roots. Roots of 1 to 2 cm in length from germinated seeds are most suitable.
2. The root tips are fixed in fixative for at least 1 h and then washed thoroughly by dipping them into water for 30 to 60 min.
3. The root tips are macerated by dipping them into the enzyme cocktail in Eppendorf tube. Between 100 and 200 μl of the cocktail is enough for the maceration of around 10 to 20 roots tips. Then the root tips are incubated at 37oC for 30 to 60 min.
4. The macerated root tips are removed gently from the enzyme cocktail into a Petri dish. They are then washed with water and stir gently to remove enzymes around the root tips and left in water for 10 to 20 min. The macerated root tips are loaded with the minimal amount of water on a glass slide cleaned with ethanol and dried prior to use.
5. The root tips are tapped with the tip of a pair of fine forceps into invisible particles using a fresh drop of fixative. Visible cell debris should be pinched away from the surface of the glass slide. If needed, the fixative is blown away using a blower or a spray of nitrogen gas. Without blowing, the fixative may dry quickly but some may remain at the fringes of the spread area, which should then be removed by the cut blotting paper. The slide is then air-dried for at least 1 h.
6. The dry slides are dipped into a Coplin jar containing freshly staining solution.
7. After suitable staining for 10 to 40 min, the glass slides are withdrawn from the staining solution and dipped into water for washing for 1 to 10 min. The surplus water is removed from the glass slide by blotting paper, blow the excess away using a blower or a nitrogen gas spray and then dry them completely.
8. For microscopy, it is recommended to use a cover slip over the samples to avoid accidental damage. Use a drop of xylene as the mounting solution.
2- Preparation of large chromosome samples:
1. Pre-treatment of the root tips is sometimes performed for species of the large chromosome type to obtain appropriately condensed chromosomes and a higher mitotic index. The root tips are dipped into ice-cold water overnight. There are basically two types of pre-treatment, chemical treatment and low-temperature treatment. The low-temperature treatment is suitable for biennial plants, but takes a longer time than the chemical treatment methods. The method involves dipping the excised root tips into cold water (0 to 8oC) for 8 to 16 h, the temperature used being highly species dependent. For chemical pre-treatment, 0.1% colchicine, 2 mM 8-hydroxyquinoline and saturated α-bromonaphthalene solutions are the most popular chemical agents, although there is a report of structural modifications of chromosomes by colchicine (Iijima and Fukui, 1991). The duration of chemical treatment varies from 3 to 6 h at room temperature.
2. The root tips are fixed in Farmer’ s fluid for at least 1 h; 70% ethanol is used for preservation of the root tips at room temperature for a longer period.
3. The root tips are softened either in 45% acetic acid (either hot or at room temperature) or in 1 N HCl at 60oC for a few minutes. Either 2% aceto-orcein or 1% aceto-carmine with 10% 1 N HCl can be used to perform softening and staining at the same time.
4. The meristematic portion of the root tips is placed using a small knife with a drop of 45% acetic acid.
5. The cover slip (18 X 18-mm) is placed onto the meristematic tissue, adding a drop of 45?etic acid or staining solution. The cover slip is tapped slightly with a toothpick to spread the cells evenly under the cover slip, while firmly holding the cover slip with fingers of the other hand. Blotting paper is used to remove surplus 45% acetic acid or staining solution from around the edges of the cover slip. Then press the cover slip firmly without sliding the cover slip.
6. The cover slips are sealed with nail varnish or paraffin for temporary mounts and observes the sample under a microscope.
7. For permanent mounting or for other uses of the chromosome samples, the glass slides are placed in freezer, preferably at –80oC or dip them in liquid nitrogen or onto dry ice to freeze completely the 45% acetic acid. The cover slip is flicked away using a blade, ensuring that the cover slip does not move on the glass slide so as to break the chromosome samples.
8. The glass slides are dipped into 70, 90 and 100% ethanol and then xylene for 10 min each and air-dry.
3- Preparation of chromosome from woody plants:
1. Seeds are scattered on blotting paper in a Petri dish after sterilization, which involves dipping the seeds in 70% ethanol for several seconds and then in 1% sodium hypochlorite solution with 0.01% triton X for 10 min and finally washing the seeds thoroughly in sterilized water. The germination period depends very much on the species, with some species requiring special treatment for germination.
2. As soon as the root emerges from the seed, the tip is taken of the root using a fine knife.
3. The pre-treatment and fixation methods presented in the previous method is followed.
4. The root tips is macerated for 1 h with an enzyme cocktail enriched with Pectolyase Y-23. The enriched cocktail is composed of 4% Cellulase Onozuka RS; 1.5% Pectolyase Y-23; 0.3% Macerozyme R200; and 1 mM EDTA, pH 4.2.
5. After enzymatic maceration and washing in water as in the previous method, the root tips are placed on the glass slide and dissect out the meristematic tissues, removing the root cap cells and other tissues. The tissues are tapped with fine forceps, add a drop of fresh fixative and air-dry.
6. The cover slip is removed by the methods previously described and dry the glass slide either through an ethanol series of 70, 90, 100% for 10 min each or just air-dry.
7. The glass slide is stained either with Giemsa or Wright’ s solution.
4- Preparation of chromosome from flowers and leaves:
1. Pinch small (about 1 to 5 mm) young leaves close to the shoot tips or young buds and fix them with Farmer’ s fluid in an Eppendorf tube until the colour of the leaves or buds is removed. Replace with a fresh fixative if it is coloured by chlorophyll or anthocyanin.
2. Discard the fixative and dispense cold 1 N HCl into the Eppendorf tube. Float the tube in a water bath at 60oC for 1 to 10 min; then replace the warm 1 N HCl with ice-cold 1 N HCl. Wash the organs three to four times with distilled water for 5 min each. Then replace the water with 1 ml of Schiff, s reagent and stain for 30 min to 1 h. After staining, wash the organs twice with a 0.5% sulfurous acid solution for 10 min each.
3. Place an organ onto the glass slide and cut into small pieces with a drop of 45% acetic acid. Cover with cover slip and tap gently several times with a toothpick until the organ spreads evenly into an almost invisible layer under the cover slip. Press the cover slip firmly with a finger without moving the cover slip.
4. Seal the cover slip with nail varnish or paraffin and observe under a microscope.
5- Preparation on permanent chromosome samples:
1. Use completely air-dried glass. To ensure dryness. Dip the glass in an ethanol series of 70, 90 and 100% for 10 min each and then in xylene for 10 min using Coplin jars.
2. The slides are placed on blotting paper and before complete drying put a drop of resin onto the middle area of the sample spreads. Place a cover slip (18 X 18 mm, 24 X 32 mm, etc.) onto the resin, avoiding the inclusion of air bubbles. Put a weight on the cover slip and allow to dry for the duration recommended by the supplier.
3. Store the sealed glass slides in the slide racks of a slide container.
The most important morphological variable in chromosomes is the variation in size between the chromosomes of one genome. This aspect is more significant than overall measurements because artificial variation in size is likely to apply equally to all the chromosomes in a cell. The chromosomes in a genome can vary from being virtually all identical in size to exhibiting a size difference ratio of five or more.
Three terms, namely, karyotype, karyogram and idiogram, are often referred to in the identification of chromosomes. Karyotype is the exact haploid chromosome set of an organism. Karyogram is the physical measurement of the chromosomes from a photomicrograph where chromosomes are arranged in the descending order (longest → shortest). An idiogram represents a diagrammatic sketch (interpretive drawing) of the karyogram (Fig. 7) (Singh, 1993).
Differences in karyotype have aided many taxonomic decisions and have also provided telling clues in unravelling evolution, e.g., in tracing the parentage of hybrids or the origin of genomes in polyploids (Stace, 2000).
analysis of chromosome information
1- Manual Drawing of Karyotype:
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P. S. To obtain an accurate measurement of the magnification of drawings the scale of a stage-micrometer should be drawn under the same conditions as those of the chromosome drawings.
For cytological investigations, the following parameters must be calculated:
(1) Total length of all chromosome complement (TCL).
(2) Mean chromosome length ± standard error (SE).
(3) Total value of arm ratio (TAR) of all chromosomes.
(4) Mean arm ratio ±SE. (MAR).
(5) Relative length (RL) of each chromosome pair.
(6) Relative arm ratio (RAR) of each pair.
(7) Ratio between the longest and the shortest chromosome pairs (L/S).
Based on these measurements a karyotype for any species is constructed as follows: Chromosome images from well-spread cells were cut from the photomicrograph, matched in pairs according to their length and arm ratio and arranged in order of decreasing length. In karyotype preparation the terminology of Levan et al. (1965) may also followed. They indicated six types of chromosomes depending upon the position of the centromere.
Fig. 7: An idiogram of a metaphase chromosome.
Chromosome type as defined by Levan et al. (1965).
Location of centromere
1.0 - 1.7
1.7 - 3.0
3.0 - 7.0
7.0 - <!--[if supportFields]>SYMBOL 165 \f "Symbol"
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Perfect symmetry is obtained when TF% is 50 (Sarbhoy, 1980).
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Where, A1 is the intrachromosomal asymmetry index that ranges from zero to one. The equation is formulated in order to obtain lower values when chromosomes tend to be metacentric. n is the number of homologous chromosome pairs or groups. bi is the average length for short arms in every homologous chromosome pair or group. Bi is the average length for long arms in every homologous chromosome pair or group.
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The interchromosomal asymmetry index (A2) provides an easy way of estimating variation in chromosome length which does not depend on chromosome number. Because of the use of the standard deviation instead of the variance, A2 index is independent from the chromosome size and has no units.
3- Image Analysis of Karyotype:
Imaging technologies now play a vital role in the areas of biological research where visual information has special importance, e.g., cytology. The application of image technology to human chromosome research began in the 1960s as diagnostic demands increased (Carlson et al., 1963; Granum & Lundsteen, 1979 and Mendelsohn et al., 1980). Automatic detection of good chromosomal spreads and subsequent automatic karyotyping to detect chromosomal aberration were objectives in clinical cytology (Castleman and Melnyk, 1976).
For plant chromosomes, the first comprehensive system was developed in 1985 and named Chromosome Image Analyzing System (CHIAS) (Fukui, 1985 & 1986). CHIAS automatically detected barley and rice metaphase spreads with an efficiency of 80% or better and this was followed by semiautomated karyotyping of up to 120 chromosomes in single metaphase spread within a few minutes (Fukui et al., 1987; Fukui, 1988; Kamisugi & Fukui, 1990 and Fukui & Kakeda, 1990).
It has been shown that the imaging methods are indispensable not only in routine chromosome analysis, like diagnosis, karyotyping, etc., but also in chromosome research because it offers several new approaches such as generation of new image parameters for identifying small plant chromosomes, quantitative idiograms, simulated optical and staining images of chromosomes and quantitative mapping of in situ hybridization signals on chromosomes (Fukui and Nakayama, 1996).
Aid of the karyotype in the taxonomy of some families
Chromosomes have been considered as sources of valid taxonomic criteria. In fact the classification of a number of families has been aided or substantiated by information on chromosome criteria (Moore, 1978 and Jackson, 1984).
Chromosome number by itself may be a useful systematic character. Similar chromosome numbers may indicate close relationship; different chromosome numbers often create some reproductive isolation through reduced fertility of hybrids. Chromosome size, the position of the centromere, special banding patterns and other features may also be systematically informative (Judd et al., 1999).
Chromosome number is generally constant within a species, although exceptions to this generality are fairly frequent. Chromosome number may be constant within large groups. Andropogoneae, the large grass tribe that includes Zea, Sorghum and many important range grasses, consistently has x = 10; the great majority of the approximately 1000 species of the subfamily Maloideae are x = 17 and almost all members of Pinaceae are diploids (2n = 24). In some species, chromosome number varies without correlated morphological variation. Moreover, differences in chromosome number, when associated with morphological differences may be recognized taxonomically, as in subspecies of Asplenium trichomanes. As an example of the taxonomic utility of chromosome number, consider Lantana. These tropical shrubs are taxonomically confused due to hybridization, polyploidy and poorly resolved generic limits. There are two base numbers (11 and 12) and perhaps a third (9) in the genus (Judd et al., 1999).
The classification of many families has been aided or substantiated by information on chromosome number and morphology. The Gramineae provide many classical examples of the value of the chromosome data in tribal delimitation. Spartina, for example, was for long placed in tribe Chlorideae (x = 10), although its chromosomes (x = 7) were at variance. Marchant (1968) showed the genus to have in fact x = 10 small chromosomes and dilemma was solved (Moore, 1978).
The removal of Paeonia with a basic chromosome number of x = 5 and large chromosomes from Ranunculaceae to the distantly related Paeoniaceae, has clarified the boundaries of the former family, which Gregory (1941) showed to be dominated by groups of genera with either large chromosomes (x = 8) or small chromosomes (x = 7) is another classical example of the role of chromosomes in plant taxonomy at the family level.
Another example is the segregation of Yucca and Agava from their previous families; Liliaceae and Amaryllidaceae respectively and placing them together in the Agavaceae (Moore, 1978).
In 2000, cytological studies were carried out by Benko-Iseppon and Morawetz in 98 species (22 genera out of eight families: Caprifoliaceae, Sambucaceae, Viburnaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Morinaceae and Calyceraceae of the Dipsacales sensu lato and the eventually related families Cornaceae and Hydrangeaceae point to a very close relationship between the Viburnaceae, Sambucaceae and Adoxaceae, corroborating the inclusion of these families into an order different from the Dipsacales, namely the Viburnales. Many cytological features shared by these families differ strongly from the Dipsacales sensu stricto, especially (1) chromosome size and morphology, (2) the presence of cold induced chromosome regions (CIRs), (3) interphase nuclear structure and (4) chromosome condensing behaviour at prophase. They found that, Cornaceae and Hydrangeaceae present similar interphase nuclei, but differ from the Viburnales by other karyomorphological characters. They discussed the results with respect to previous morphological, embryological and molecular findings.
Other examples demonstrating the role of chromosomal criteria in the taxonomy at the family level include the work of Behera and Patnaik (1974) on Amaranthaceae, Badr et al. (1997) on the Solanaceae, Kamel (1996) on the Asteraceae and Aboel Atta et al. (1999) on the Ranunculaceae.
The karyotype data also appear to be of taxonomic value in providing a logical basis for the redistribution of genera in tribes. Karyotype studies were principally based on the idea that symmetrical karyotypes are more primitive than asymmetrical ones; longer chromosomes than shorter ones; median centromeres with chromosome arms of equal length were more primitive than chromosomes with arms of unequal length; low basic numbers had given rise to higher ones. These features are based on the comparison between karyotypes of known relative antiquity, as determined through classical taxonomy (Sharma, 1990).
The Karyotype data also appear to be of taxonomic value in providing a logical basis for the redistribution of genera in tribes. Harrison (1969) gave some examples of the Ranunculaceae. The genera Aquilegia and Isopyrum were placed in the Helleboreae and Thalictrum and Anemonella in Anemoneae.
At the generic level and below, the chromosomes provided a range of cytological possibilities for understanding the limitations, affinities and evolution of taxa. Examples illustrating the importance of chromosomal data in the treatment of plant genera are found in Crepis (Babcock, 1947), Clarkia (Lewis & Lewis, 1955), Lentodon (Rousi, 1973), Allium (Badr, 1977), Mentha (Harley & Brighton, 1977), Launaea (Amin, 1978), Artemisia (Vallés, 1987), Trifolium (El-Kholy, 1990), Panicum (Haroun, 1991), Plantago (Badr, 1992), Hypochoreis (Ruas et al., 1995), Astragalus (Badr et al., 1996), Phaseolus (Mercado-Ruaro & Delgado-Salinas, 1998), Sesbania (Abou El-Enain et al., 1998), Lathyrus (Abou El-Enain, 1999), Vicia (Kamel, 1999b), Artemisia (Vallés et al., 2001) and several others.
Other chromosomal studies which dealt with karyotype morphology and its aid in taxonomic classifications and generic delimitation, are works of; Yamamoto (1973) on karyotaxonomical studies on some annual species of Vicia, Badr & El-Kholy (1987) on karyotype studies in the genus Plantago L., Badr et al. (1987) on cytology and taxonomic relationships of some taxa in the genus Silene L., Maxted et al. (1991) on cytotaxonomic studies of Eastern Mediterranean Vicia species (Leguminosae), Tita (1991) on contributions to the study of karyotypes of Vicia pannonica Cr., Li & Wei (1992) on the karyotype analysis of some new taxa of Glycyrrhiza, Wang & Zhang (1992) on the studies on chromosome numbers of some species of Vicia L., Galasso et al. (1993) on cytotaxonomic studies in Vigna, Heering & Hanson (1993) on karyotype analysis and interspecific hybridization in three perennial Sesbania species (Leguminosae), Pundir et al. (1993) on morphology and cytology of Cicer canariense, a wild relative of chickpea, Roti Michelozzi (1993) on karyotype variation by whole arm translocation in Italian specimens of the Vicia cracca group (Fabaceae), Salimuddin (1993) on karyological studies in the genus Sesbania, Ugborogho & Obute (1993) on studies on the vegetative morphology, floral biology, and karyomorphology of Vigna unguiculata (L.) Walpers species complex (Papilionaceae) in southern Nigeria, Ward et al. (1993) on the chromosome numbers for Dalea species (Fabaceae) from southwestern New Mexico and southeastern Arizona, Li & Wang (1994) on karyotype analysis of Vicia (Willed. ex. Link) Maxim and V. ramuliflora (Moxim), Prathepha (1994) on chromosome number of the genus Afgekia Craib (Leguminosae), Salimuddin (1994) on karyotype, nuclear, and chromosomal DNA variation in Lens culinaris Med., Wang et al. (1994) on preliminary karyomorphological study on the plants in genera Oxytropis and Astragalus from Qinghai-Xizang Plateau, Zhang et al. (1994) on cytotaxonomical studies on 14 species of Oxytropis DC. from Nei Menggu, Baranyi & Greilhuber (1995) on flow cytometric analysis of genome size variation in cultivated and wild Pisum sativum (Fabaceae), Bidak & Brandham (1995) on intraspecific uniformity of chromosome number and nuclear DNA quantity in two Egyptian weedy species, Malva parviflora (Malvaceae) and Trigonella stellata (Leguminosae), Coulaud et al. (1995) on first cytogenetic investigation in populations of Acacia heterophylla, endemic from La Reunion Island, with reference to A. melanoxylon, Fuchs et al. (1995) on telomere sequence localization and karyotype evolution in higher plants, Jun et al. (1995) on karyotypes of Vicia sect. Vicilla from Northeast China, Palomino et al. (1995) on chromosome numbers and DNA content in some taxa of Leucaena (Fabaceae: Mimosoideae), Sahin & Babac (1995) on cytotaxonomic studies on some Vicia L. species in east and south-east Anatolia II, Vijayakumar & Kuriachan (1995) on karyomorphology of five taxa of Sesbania from South India, Badr et al. (1996) on the chromosomal relationships in the genus Astragalus L. (Fabaceae) and their taxonomic inferences, Ruas & Aguiar-Perecin (1997) on chromosome evolution in the genus Mikania (Compositae), Dimitrova & Greilhuber (1999) on karyotype and DNA-content evolution in ten species of Crepis (Asteraceae) distributed in Bulgaria, El-Nahas (1999) on karyotype and heterochromatin pattern of some Allium taxa using C-banding technique as revealed by Giemsa, El-Shazly & Abou El-Enain (1999) on chromosomal criteria of some Sesbania species and their taxonomic inferences, Jianquan et al. (2000) on karyological studies on the Sino-Himalayan genus, Cremanthodium (Asteraceae: Senecioneae), Richardson et al. (2000) on heterochromatin banding patterns in Rutaceae-Aurantioideae and chromosomal evolution.
1- Family Asteraceae:
The cytological criteria in the Asteraceae show considerable variation. Compilations of the recorded chromosome numbers in the family made by Fedorov (1969) and Goldblatt & Johnson (1981-1998) show that the chromosome numbers vary from a low of n = 2 in Haplopappus gracilis (Nutt.) A. Gray and Brachycome lineariloba (DC.) Druce to as high as n = 103 in Wernaria apiculata Sch. Bip., n = 106 in Wernaria nubigena kunth. and n = 110-120 in Montanoa guatemalensis Robins. and Greenm.
Two contrasting views have been proposed with regard to the basic chromosome number in Asteraceae. Solbrig (1978) noted that x = 9 is the most common basic number and proposed it as the model number of the family. He further observed that high frequency of this number (x = 9) is particularly common among trees and shrubs. The correlation between this basic number and habit of members of Asteraceae support evidence that ancestors of the family are woody with a basic number of x = 9. However, Mehra (1977), on the other hand proposed x = 5 as the basic number in the family. He further argued for a correlation of basic number variations and the tribal delimition in the family. He assumed that original basic number of the ancestors was x = 5 which became stabilized in many of the tribes at the tetraploid level with x = 10, but there are others where the stabilization has been achieved at the aneuploid level (e.g. x = 9) as in Astereae, Anthemideae and Cichorieae, coupled undoubtedly with major gene mutations and perhaps structural chromosomal alternations.
Stebbins (1971) reported contrasting sizes of somatic chromosomes in species belonging to the Asteraceae. In Cichorieae, Agoseris heterophylla (Nutt.) Greene has n = 9 and a mean chromosome length of 2.4 ?m, while in Chaetadelpha wheeleri A. Gray, also with n = 9, the mean chromosome length is 6.4 ?m.
Various ploidy levels have been observed within numerous genera and species of the Asteraceae. Mehra et al. (1965) listed somatic chromosome number of 2n = 18, 54 and 64 for Sonchus arvensis L. Two more counts (2n = 32 and 2n = 36) were added by Gupta et al. (1972). On the other hand in the index of plant chromosome numbers compiled by Goldblatt and Johnson (1981-1991), 2n = 18, 36, 37 and 54 for the same species were also recorded.
Ashri (1957) and Ashri & Knowles (1960) recognized four sections in genus Carthamus L. having 10,12,22, and 32 pairs of chromosomes. Hanelt (1961 & 1963) assigned taxa with n = 22 and n = 32 chromosome pairs to C. lanatus L. A somatic chromosome number of 2n = 44 was recorded for this species by Goldblatt and Johnson (1981, 1984 & 1990). In section Centaureinae of the genus Centaurea L. Garcia-Jacas and Susanna (1992) recorded 2n = 20 in C. protongi Boiss.; 2n = 40 in C. crocata Franco. and 2n = 60 in C. acaulis Desf.
Maffei et al. (1993) recorded the presence of polyploidy in species of Achillea L. A tetraploid chromosome number of 2n = 36 was found in A. setacea Waldt. & Kit. and A. collina Becker ex. Reichenb. and a hexapolid 2n = 54 in A. distans Waldst. et Kit., while 2n = 36 and 54 were recorded in A. millefolium L. (Lavrenko and Serditov, 1991).
Polyploidy has also taken place in Artemisia L. In this genus most species are diploid with 2n = 18. Tetraploid occur with 2n = 36, although some have lost a pair of chromosomes and have 2n = 34 (new base-number x = 17), while the hexaploid A. verlotiorum can have the full complement of 2n = 54 but also exists as hypotetraploids with 2n = 52, 50 or 48 (James et al., 2000), with new base-numbers x = 13, 25 or 12.
In the genus Chrysanthemum L., Banerji & Datta (1993) recorded the presence of ploidy series of 2n = 36, 54, 55 and 63. On the other hand, Endo and Inada (1990) reported that in C. morifolium Ramat, somatic chromosome number ranged from 2n = 53 to 2n = 66. Also, in the wild species of Chrysanthemum L. (Dendranthema vestitum) 2n = 6x = 54 was recorded (Nakata et al., 1991).
In Aster L. polyploid records of 2n = 54 in A. schreberi Nees. and A. jonesiae and 2n = 73 in A. macrophyllus L. were recorded by Lamboy et al. (1991).
In Calendula officinalis L. n = 8 and n = 16 were recorded by Mehra et al. (1965) and Gupta (1969), while 2n = 28, 32, and 36 in this species are reported in Fedorov (1969) and Goldblatt and Johnson (1981; 1988 & 1991).
Ballard (1986) recorded different gametic chromosome numbers in Bidens L., he scored n = 12 in B. odorata Ballard; n = 24 in B. alba (L.) DC. and n = 36 in B. pilosa L. Three chromosome numbers were also reported in Helianthus L., 2n = 2x = 34 (H. strumosus L.), 2n = 4x = 68 (H. rigidus (Cass) Desf. and 2n = 6x = 102 H. laevigatus Torr. et A. Gray (Atlagic et al., 1992).
In Senecio L. chromosome counts of 2n = 10, 20 and 40 were recorded (Fedorov, 1969; Lavrenko & Serditov, 1991 and Ashton & Abbott, 1992). Other numbers (2n = 10, 20, 32, 38, 40, 44, 46, 48, 50, 60, 78, 80, 88, 90, 92, 96, 100, 120, 126, 130, 132, 140, 160 and 180) were scored in the genus (Goldblatt & Johnson, 1981-1991 and Knox & Kowal, 1993). In Artemisia L. The increase in ploidy level is correlated with increased karyotype asymmetry (Oliva and Vallés, 1994). On the other hand, in Hypochoeris L. aneuploidy is shown clearly from x = 6 to 5, 4, and 3, but the reduction in the number of chromosomes was not followed by changes in chromosome asymmetry (Ruas et al., 1995). However, in Centaurea L. there is a tendency towards reduction in basic chromosome number and increase in karyotype asymmetry (Garcia-Jacas et al., 1996). A similar trend is known in other genera of the Asteraceae e.g. Crepis L. (Stebbins, 1971).
Several studies have dealt with chromosomal features of members of Asteraceae in different regions of the world. Examples of these include the chromosome numbers of Artemisia L. in Iran (Tavassoli and Derakhshandeh-Peikar, 1993) and in the east African giant senecios and lobelias (Knox and Kowal, 1993). Cytotaxonomic relationships of Lactuca L. in Iberian Peninsula (Mejias, 1993), chromosome numbers and karyotypes of Italian Achillea L. (Maffei et al., 1993), chromosome studies in Latin American Compositae (Strother and Panero, 1994) and in Mexico (Keil et al., 1988). The chromosomes numbers of North American species of Artemisia L. (Stahevitch and Wojtas, 1988), the cytology of some weedy species of the family in Nigeria (Husaini and Iwo, 1990), chromosome numbers in Compositae from Pakistan (Razaq et al., 1994) and karyological studies on the Egyptian Asteraceae (Badr et al., 1997; Kamel, 1999a & 2001) (Fig.8 & 9).
The chromosomal studies of Kamel (1996) on 106 species of the Asteraceae revealed that most of the karyotypes studied are found to be symmetric, thus supporting previous observations that the karyotype in the Asteraceae is symmetric. Longer chromosomes are associated with karyotype asymmetry in the tribes; Lactuceae, Cardueae, Astereae, Heliantheae and Senecioneae, but this association is not evident in tribe Anthemideae.
The cytological aspects confirm the isolation of the genus Cichorium from tribe Lactueae based on the analysis of the morphological characters. Also, the delimitation of Staehelina dubia from the tribe Cardueae is supported by the cytological criteria. Within the Heliantheae, the isolation of Coreopsis from other species of Coreopsidinae based on the morphological characters is also supported by the cytological criteria, this may support the treatment of Coreopsidinae as tribe Coreopsideae.
2- family ranunculaceae:
Several studies have dealt with chromosome count and chromosomal features (karyotype) of members of the family. Examples of these include; Pastor et al. (1984) on Nigella, Delphinium, Anemone, Clematis, Ranunculus & Thalictrum; Dzmitryeva & Parfenau (1985) on Caltha; Lavrenko & Serditov (1986) on Ranunculus; Bhattarai (1989); Chen et al. (1989); Heyn & Pazy (1989); Serov (1989) on Anemone, Clematis, Adonis & Atragene; Baltisberger (1991) & Kadota (1991) on Ranunculus & Aconitum; Diosdado et al. (1993); Pandit & Babu (1993); Yang & Wu (1993); Yang et al. (1993) on Ranunculus, Coptis, Adonis, Delphinium, Anemone & Aconitum; Beyazoglu et al. (1994) on Aconitum; De-Caneloda & Martinez (1995) ; Yang & Gong (1995) on Ranunculus & Aconitum; Liao et al. (1996) ; Starmuhler (1996) ; Tak & Wafai (1996) ; Yang
Fig. 8: Karyotypes of different species belonging to the family Asteraceae. (From Kamel, 1999a)
Fig. 9: Karyotypes of different species belonging to the family Asteraceae. (From Kamel, 2001)
(1996) on Ranunculus & Aconitum, Anemone & 15 species of tribe Delphinieae and Ozyurt et al. (1997) on Clematis.
Within the Ranunculaceae, chromosome numbers vary from 2n = 12 in Nigella L. and 2n = 14 in Anemone L., Aquilegia L., Isopyrum L., & Ranunculus L. to 2n = 84 in Thalictrum flavum L. (Fedorov, 1969 and Goldblatt & Johnson, 1996).
Within the Ranunculaceae, in the study of Kamel (2000), long chromosomes and symmetric karyotypes characterized most of the studied species (Fig. 10). The separation of Nigella, Ranunculus and Thalictrum in three different tribes (Mabberley, 1997) is supported by the presence of different basic numbers. On the other hand, chromosome measurements of the species of Clematis were found to be similar. These may confirm the isolation of Clematis in a separate tribe (Clematideae) as proposed by Heywood (1993). The same result was found between the two genera, Delphinium and Cosolida of tribe Delphinieae.
3- Family solanaceae:
Little attention has been focused on the role of cytology in the classification of the Solanaceae. However, Badr et al. (1997), discussed the taxonomic relationships in the Solanaceae based on chromosomal criteria of 45 species, belonging to 15 genera representing eight of the 14 currently recognized tribes. Cytological data indicate that subfamily Cestroideae is less evolved than subfamily Solanoideae. The data further support the isolation of Cestrum in separate tribe. The isolation of tribe Salpiglossideae in a separate family as proposed by Hutchinson (1973) and the delimitation of both Datura and Hyoscayamus as two separate tribes as done by D’ Arcy (1991) is strongly supported by data.
Fig. 10: Karyotypes of different species belonging to the family Ranunculaceae. (From Kamel, 2000)
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