Karyotype Analysis and Chromosome Evolution in Species of Lathyrus (Fabaceae)
Salwa Fahmy Badr
The karyotypes of 21 accessions of Lathyrus L. belonging to four sections were investigated. Although all the species have a chromosome number of 2n = 14, they could be differentiated by their karyotype formula and quantitative parameters of the karyotypes. Phenetic distance showed that in spite of the differences observed among entitied, they can be grouped in clusters that coincide with the taxonomic sections established by Kupicha and with the life cycle of the species. The section Clymenum can be distinguished by the presence of a subtelocentric pair. From an evolutionary point of view, variation in genome size, however, is congruent with morphological variation as well as with the life cycle.
Lathyrus is distributed throughout the temperate regions of the Northern
Hemisphere with 52 species in Europe, 30 species in North America, 78 species
in Asia and 24 species extending into tropical East Africa and 24 species into
temperate South America (Kupicha, 1983; Allkin et al., 1985; Goyder,
1986). The genus Lathyrus (Fabaceae, Papilionoidae, Vicieae) consists
of 13 sections and comprises about 150-160 species of annual or perennial herbs
with erect or, more usually, climbing and sprawling habit, which are predominantly
self-pollinating (Kupicha, 1983). The main center of diversity is the eastern
Mediterranean region, with two smaller centers in North and South America (Kupicha,
1983; Allkin et al., 1985; Simola, 1986). Lathyrus species occur
in a diversity of habitats, including open woods, forest margins, meadows, pastures,
fields, slopes, marshes, seashores, sand dunes and roadsides. Lathyrus
exhibits a typical bee-pollinated papilionoid flower, which may be yellow, orange,
red, purple, violet, bluish, or white (Conny et al., 1998). There are
many species cultivated for forage and human food: L. sativus (grass
pea), L. hirsutus (rough pea), L. cicera (flatpodded vetchling),
L. odoratus (sweet pea), L. ochrus (ochrus), L. sylvestris
Cytological investigations have shown that all species were diploid with 2n = 14 chromosomes, the basic chromosome number of x = 7 is constant throughout the genus and that most of the species are diploid, with polyploids as rare exceptions (Senn, 1938; Yamamoto et al., 1984; Broich, 1989; Battistin and Fernández, 1994; Klamt and Schifino-Wittmann, 2000; Seijo and Fernández, 2001). Despite this stability in chromosome number, large variations in chromosome size have played an important role in the evolution of Lathyrus species, which are associated with a fourfold variation in 2C nuclear DNA amount (Narayan and Durrant, 1983). The species show marked similarity in their chromosome shapes and karyotype arrangements within complements. The amounts of nuclear DNA within this genus are discontinuously distributed. Many karyotypic studies have been performed on Old World members of Lathyrus (Lavania and Sharma, 1980; Yamamoto et al., 1984; Sahin et al., 2000), but there is a paucity of data for American species, with the karyotypes of only five South American entities described so far (Battistin and Fernández, 1994; Klamt and Schifino-Wittmann, 2000). From the available information, a number of conflicting observations have arisen. Some authors claim that, in addition to the numerical constancy, Lathyrus species display morphological uniformity of chromosomes and homogeneous karyotype arrangement (Lavania and Sharma, 1980; Narayan and Durrant, 1983; Klamt and Schifino-Wittmann, 2000). However, others have found enough interspecific karyotype differences to allow species characterization (Yamamoto et al., 1984; Murray et al., 1992a; Battistin and Fernández, 1994). Such discrepancy was also observed at the infraspecific level, mainly in the widely studied L. odoratus L. and L. sativus (Bhattacharjee, 1954; Sharma and Datta, 1959; Verma and Ohri, 1979; Murray et al., 1992b). From a karyosystematic point of view, Yamamoto et al. (1984) have noted that Old World species could be grouped according to their karyotype morphology and that some of them were coincident with the taxonomic sections proposed by Davis (1970). However, these authors did not propose any relationship, either among groups or considering the world infrageneric classification proposed by Kupicha (1983), Przyblska et al. (1999), Seijo and Fernández (2003) and Wong et al. (2006).
Genetic relationships within species of the genus Lathyrus were reported
by Datta (1955) and Chaudhuri (1966). In addition to this numerical chromosome
constancy species display uniformity in chromosome morphology (all chromosomes
are metacentric or submetacentric). Evolution, nevertheless, has resulted in
a large increase in chromosome size (Narayan and McIntyre, 1989). In general,
the total length of somatic chromosome set was longer in perennial species than
in annual ones as reported by Rees and Hazarika (1969). Species of the section
Clymenus, Nissolia and Aphaca have chromosome complements
larger than those in the section Cicercula. The main karyotypic difference
between species of the genus Lathyrus involves the shape of satellite
chromosome (Yamamoto et al., 1984). The divergence and evolution within
this genus is accompanied by a 3-fold increase in chromosome size which is directly
correlated with 4-fold increase in their nuclear DNA amounts. Comparisons of
total DNA amounts showed discontinuity in the variation among species of this
genus (Narayan, 1982).
It has been reported that inferageneric Lathyrus classification has varied markedly during its history and classifications based on morphology are problematic due to homoplasy and lack of diagnostic characters (Conny et al., 1998). Thus, the present investigation is concerned with karyotype analysis and idiogram of the genus Lathyrus to clarify the taxonomic relationships among species of this genus and to examine the pattern of chromosome variation in relation to the taxonomic position and the life cycle of the taxa.
MATERIALS AND METHODS
In the present work, cytological study was carried out on 21 accessions samples obtained as a donation from Western regional plant introduction station 59 Johnson Hall Pullman (PI), Washington 99164 and ICARDA Genetic Resource (IG). The accessions represent 13 species belonging to four sections. For cytological preparations, seeds were germinated on moist filter paper in petri dishes at room temperature (18-22 C).1-2 cm long roots were detached and pretreated with a saturated solution of 1,4-dichlorobenzene for 3-4 h. Roots were then washed briefly in water and fixed in a mixture of 3:1 (v/v) ethyl alcohol: glacial acetic acid for 24 h and kept in a refrigerator until use.
Cytological preparation were carried out using the Feulgen squash technique for Feulgen staining root tips were hydrolyzed in 1N HCl at 60°C for 8-10 min, washed in distilled water and stained in Leuco-basic fuchsin for at least 1 h. The terminal 1-2 mm of the root tips were squashed in a drop of 45% acetic acid on a clean slide. Cover slips were separated by the freeze-drying method. samples were then dehydrated in absolute ethanol for 2-3 min and made as permanent preparation by mounting in Depex slides were allowed to dry at room temperature for few days. Cells with good spreading of chromosomes were photographed using a zeiss Ultraphoto microscope equipped with automatic camera. The nomenclature used for the description of the chromosome morphology is that proposed by Levan et al. (1965). The abbreviations m, sm and st designate metacentric, submetacentric and subtelocentric chromosomes, respectively. Idiograms were drawn based on mean centromeric index and arranged in order of decreasing size.
For the numerical characterization of the karyotypes the following parameters
were calculated: (1) total chromosome length of the haploid complement (TCL);
(2) mean Chromosome Length (CL); (3) mean Centromeric Index (CI); Comparisons
of chromosome morphological features were made by arranging the chromosomes
of each karyotype in pairs in order of their arm ratio and length as determined
from the photographic prints. An idiogram for each sample was constructed using
the total length of each pair of homologous chromosomes to represent the haploid
chromosome number. The relative position of the centromere and their variation
within the karyotype has been expressed. A cluster analysis of the karyotype
data was coned out to examine karyotype similarity among species and sections.
A data matrix 21 OTUs (operational taxonomic units) x 7 variables was constructed.
The TCL, CI, number of m, sm and st chromosomes as well as the life cycle were
considered. The SYSTAT ver. 7 program was used to standardize the data matrix,
to calculate the average taxonomic distance and to generate a phenogram. Clustering
was performed using the unweighted pair-group method (UPGMA).
All the studied samples have 2n = 14 chromosomes. Karyotype formulae and parameters for the studied species are summarized in Table 1. Figure 1 shows the mitotic metapbases and Fig. 2 the respective idiograms.
As a whole, karyotypes of the analyzed species, in the four sections, have
a predominance of m chromosomes. The most common formula among section Cicercula
species is 12 m+2 sm (five species), followed by 14 m (two species). In
the section Lathyrus the dominance formula is 8 m+6 sm (four species)
and the second formula is 10 m+4 sm (three species).
Somatic chromosomes of Lathyrus 1: L. sylvestris1
2: L. sylvestris2 3: L. hirsutus1 4:
L. hirsutus2 5: L. hirsutus3 6: L.
hirsutus4 7: L. annus 8: L. sphaerious 9: L.
latifolius 10: L. ochrus 11: L. clymenum 12: L. articulatus
13: L. aphaca 14: L. inconspicus 15: L. blepharicarpus
16: L. cicera1 17: L. cicera2 18: L.
cicera3 19: L. cicera4 20: L. cicera5
21: L. marmoratus
||Locality, Accession number (A No.), Karyotype Formula (KF),
total length of the haploid complement (TCL), mean Centromeric Index (CI),
life cycle (CY) and of the studied Lathyrus species (m) metacentric
(sm) sub metacentric (st) subteleocentric (P) perennial (A) annual
Idiograms of Lathyrus Somatic chromosomes of Lathyrus,
1: L. sylvestris1, 2: L. sylvestris2,
3: L. hirsutus1, 4: L. hirsutus2, 5:
L. hirsutus3, 6: L. hirsutus4, 7: L.
annus, 8: L. sphaerious, 9: L. latifolius, 10: L. ochrus,
11: L. clymenum, 12: L. articulatus, 13: L. aphaca,
14: L. inconspicus, 15: L. blepharicarpus, 16: L. cicera1,
17: L. cicera2, 18: L. cicera3, 19:
L. cicera4, 20: L. cicera5 and 21: L.
Relationship between the total lengths of the haploid complement
(TCL) and the mean Centromeric Index (CI). Values of TCL and CI are summarized
in Table 1
. These variables grouped species mainly by
sections. Each symbol in the plot represents once species
||Dendrogram showing the phenetic relationships among the studied
species of Lathyrus, constructed using the matrix of karyotype similarities
The section Clymenum characterized by the presence of two subtelcentric
chromosomes and different number of m and sm chromosomes. Section Aphaca
has a formula 10 m+4 sm. Considering the life cycle, perennial species have
a greater TCL than the annual. Considering the mean value of TCL and CI among
the studied section, the section Lathyrus has the longest Total Chromosome
Length (TCL) and but the centromeric index is almost the lower than those in
section Cicercula. The idiograms as well as the parameters presented
in this paper represent the means of the populations analyzed for each species.
However, at the interspecific level, TCL and CI significantly differentiate
some species (Table 1).
The relationship between TCL and CI of each species is plotted in Fig. 3. ANOVA of TCL discriminated among species of the four sections analyzed (F = 20.140, p<0.01) and the CI (F = 4.201, p<0.0021).
The UPGMA phenogram constracted on the basis of karyotype similarities shows two major clusters (Fig. 4). The first cluster is comprised the two samples of L. sylvestris in section Lathyrus, which are characterized by the highest CI, TCL and perennial species and the second cluster include the rest of studied samples. This cluster comprised two major groups, the first one includes the samples of section Clymenum, which characterized by the presence of two subtelcentric and the second groups separated into three subgroups. The samples of L. cicera, L. inconspicus and L. blepharicarpus, which are belonging to section Cicercula are delimited in one subgroup. The second subgroup includes the samples of L hirsutus. The species L. annus, L. sphaercus, L. latifolius, L. aphaca and L. marmoratus are delimited in the last subgroup. L. marmoratus is delimited with L. latifolius due to the similarities in the CI and life cycle.
The results of this study revealed a detailed picture of the chromosome features of some Lathyrus species belonging to four sections and of their pattern of variation in relation to their systematic position and life cycle. Like most species of Lathyrus, all the species analyzed here were diploids with 2n = 14. The chromosome numbers of the species agree with those published previously (Senn, 1938; Lavania and Sharma, 1980; Yamamoto et al., 1984; Battistin and Feranández, 1994; Klamt and Schifino-Wittmann, 2000). In addition to this numerical chromosome constancy species displayed uniformity in chromosome morphology (all chromosomes were either metacentric or submetacentric) and in karotype arrangements. Evolution, nevertheless, has resulted in a large increase in chromosome size which is associated with a five-fold increase in 2C nuclear DNA amounts (Rees and Hazarika, 1969). Cytological and molecular investigations into the organization of chromosomes and the composition and distribution of nuclear DNA within and between chromosome complements have given evidence that there are strong constraints controlling evolutionary changes in genome organization (Narayan, 1982; Narayan and Durrant, 1983; Narayan and Rees, 1976).
L. clymenum and L. ochrus belonging to the section Clymenum, these two species are separate in cluster tree from L. articulatus. The lower degree of similarity among them may be attributed to a considerable difference in karyotype, including differences in chromosome length between individual chromosomes of both species and also difference in some morphological variations such as pod shape and structure, these agree with protein bands reported by El-Shanshoury (1997).
Karyotype formulae and quantitative analysis have a great uniformity among populations of any species, except those that correspond to different taxonomic varieties. These results support the hypothesis that claims infraspecific stability of karyotypes in Lathyrus species (Murray et al., 1992b). At the interspecific level, quantitative and qualitative data allowed the differentiation of several of the taxa studied. Among species of section Lathyrus and Cicercula, the most variable characters were the number of m chromosomes, as well as the karyotype formulae were more similar, but species still could be differentiated mainly by the number and type of chromosomes. These facts show that the karyotypes of Lathyrus species are not as fully constant as has been postulated (Narayan and Durrant, 1983; Klamt and Schifino-Wittmann, 2000) and that entities may be characterized by their chromosome features as was suggested by other authors (Yamamoto et al., 1984; Murray et al., 1992a; Battistin and Fernández, 1994).
In relation to the genome size variation, there are differences in the chromosome length among the studied taxa. These differences among complement length of diploid species are in accordance with that cited for nuclear DNA amounts of Old World species of Lathyrus (Rees and Hazarika, 1969; Narayan, 1982) and support the statement that variation in genome size is, perhaps, one of the more striking changes that have occurred during the divergence and evolution of the chromosome complements of this genus (Narayen and Rees, 1976). As a whole, Lathyrus is characterized by symmetrical karyotypes, with a predominance of sm chromosomes (Yamamoto et al., 1984), but the sections under this study have a predominance of m chromosomes and the presence of a st pair is characterized to section Clymenum. In spite of the observed interspecific variation, the bulk of karyotype data-formula, TCL and CI showed a conservative tendency toward the maintenance of the general structure of the karyotypes among different clusters of species, in accordance with observations by Yamamoto et al. (1984). Analysis of karyotype formulae showed that, in general, species of section Clymenum have a pair of st. chromosome; they differ from the other sections, Lathyrus, Aphaca and Cicercula. Therefore, karyotype features may become good taxonomic characters to define members of Clymenum.
The bulk of available karyotype data showed that most of karyotype groups found in Lathyrus could be related either to different sections sensu Kupicha (1983) or to the life cycle of the entities. The phenctic analyses of karyotype characters support this postulate. At the interspecific level, within section Lathyrus and Cicercula, some species can be distinguished clearly by their karyotype formulae and when quantitative karyotype data are added, the majority of entities can be differentiated. The constancy in chromosome number observed in the species studied here and in those cited in the literature (Senn, 1938; Battistin and Fernández, 1994; Klamt and Schifino-Wittmann, 2000; Seijo and Fernández, 2003) indicates that numerical changes have not been important in the evolution of South American species, as noted for most of the entities of Lathyrus (Hitchcock, 1952; Yamamoto et al., 1984; Sahin et al., 2000). However, this constancy differs from the situation described for North America, where several endemic polyploid species were found, so that North America was considered as a center of polyploid origin for Lathyrus (Broich, 1989).
Present findings that TCL varies without significant changes in karyotype formula, as seen among annual and perennial species of sections Cicercula and Lathyrus, suggest that changes in genome size may have been nonrandom and that the variation in DNA amounts is equally distributed among all chromosomes of the complements. These observations agree with those cited for Old World species of Lathyrus, in which variation of genome size was attributed to proportional distribution of mainly moderately repetitive DNA throughout the complement (Narayan and Durrant, 1983). Data obtained from banding patterns also support the nonrandomness of genomic change in Lathyrus because bands with similar base composition tend to have equilocal disposition in the karyotypes (Unal et al., 1995; Seijo, 2002). This pattern of evolution at molecular and subchromosomal levels suggests that species within each group evolved in a concerted fashion, maintaining the karyotype morphology.
The reduction of genome size that accompanied the evolutionary change from
perennial to annual in sections Lathyrus and Cicercula species
coincides with different reports on angiosperm groups (Price and Baehmanm, 1976;
Greilhuber and Ehrendorfr 1988; Seijo and Fernández, 2003). Moreover,
annual species of this section, in addition to having lower TCL, present smaller
pollen grains and lighter seed than perennials (Seijo, 2002). These observations
are in agreement with a considerable amount of data that show that the size
of reproductive organs may be related to the genome size (Choi, 1971; Chung
et al., 1998); as was postulated in the nucleotype hypothesis (Bennett,
The author is most grateful to Prof. R.H. Sammour a Prof. of Molecular Systematic, Tanta University, Egypt, for kind supply of seed materials and his helping in computer analyses and revising the manuscript.
1: Waling, I., W.V.J.C. Houba and J.J. Vander Lee, 1989. Soil and plant analysis, a series of syllabi. Part 7. Plant Analysis Procedures Wageningen, Agricultural University.
2: Bhattacharjee, S.K., 1954. Cytogenetics of Lathyrus sativus Linn. Caryologia, 6: 333-337.
3: Battistin, A. and A. Fernandez, 1994. Karyotypes of four South America NATIVE species and one Cultivated species of Lathyrus L. Carynlagia, 47: 325-330.
4: Bennett, M.D., 1972. Nuclear DNA content and minunum generation time in herbaceoas plants. Proc. Royal Soc. Lond. Ser. B, Biol. Sci., 181: 109-135.
5: Broich, S.L., 1989. Chromosome ntmsbers of North American Lathyrus (Fabaceae). Madrono, 36: 41-48.
6: Chaudhuri, P.R., 1966. Karyotypic analysis of different strains of Lathyrus odorantus L. Trans. Base Res. Inst., 29: 97-104.
7: Choi, W.Y., 1971. Variation nuclear DNA content in the genus Vicia. Genetics, 68: 195-211.
8: Chung, J., J. Lee, K. Arumuganatiian, G.L. Graef and J.E. Specitt, 1998. Relationships between nuclear DNA content and seed and leaf size in soybean. Theor. Applied Genet., 96: I464-1468.
Direct Link |
9: Conny, B., A. Smussen and A., Aron, 1998. Chloroplast DNA characters, phylogeny and classification of Lathyrus (Fabaceae). Am. J. Bot., 85: 387-401.
Direct Link |
10: Datta, P.C., 1955. Studied on the structure and behaviour of chromosomes of a few species of the genus Lathyrus as a means of detecting inter relations. Genet. Iber., 7: 85-115.
11: Davis, P.H., 1970. Lathyrus. In: Flora of Turkey and the East Aegean Islands, Davis, P.H. (Ed.). Edinburgh University Press, Edinburgh, UK., pp: 328-369.
12: El-Shanshoury, A., 1997. The use of seed proteins revealed by SDSPAGE in taxonomy and phylogeny of some Lathyrus species. Biol. Plant, 39: 553-559.
Direct Link |
13: Greilhuber, J. and E. Ehrendorfr, 1988. Karyological approach to plant taxonomy. ISI Atlas of science. Plant Anim. Sci., l: 289-297.
14: Hitchcock, C.L., 1952. A revision of North American species of Lathyrus. Univ. Washington Publ. Biol., 15: 1-104.
15: Klamt, A. and M.T. Scittfino-Wittmann, 2000. Karyotype morphology and evolution in some Lathyrus (Fabaceae) species of southern Brazil. Gene. Mol. Biol., 23: 463-467.
Direct Link |
16: Kupicha, E.K., 1983. The infrageneric structure of Lathyrus. Notes R. Botanic Garden Edinburgh, 41: 209-244.
17: Lavania, U.C and A.K. Sharma, 1980. Giemsa C banding in Lathyrus L. Botanical Gazette, 142: 199-203.
18: Levan, A., K. Fredga and A.A. Sandberg, 1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201-220.
CrossRef | Direct Link |
19: Murray, B.G., M.D. Bennett and K.R.W. Hammett, 1992. Secondary constrictions and NORs of Lathyrus investigated by silver staining and in situ hybridization. Heredity, 68: 473-478.
Direct Link |
20: Murray, B.G., K.R. Hammett and L.S. Standring, 1992. Genomic constancy during the development of Lathyrus odoratus cultivars. Heredity, 68: 321-327.
Direct Link |
21: Narayan, R.K.J., 1982. Chromosome Changes in the Evolution of Lathyrus. In: Kew Chromosome Conference Ii, Brandham, P.E. and M.D. Bennett (Eds.). George Allen and Unwin, London, UK., pp: 243-250.
22: Narayan, R.K.J. and H. Rees, 1976. Nuclear DNA variation in Lathyrus. Chromosoma, 54: 141-154.
CrossRef | Direct Link |
23: Narayan, R.K.J. and A. Durrant, 1983. DNA distribution in chromosomes of Lathyrus species. Genetica, 61: 47-53.
24: Narayan, R.K.J., 1988. The role of genomic constrains upon evolutionary changes in genome size and chromosome organization. Ann. Bot., 82: 57-66.
25: Narayan, R.K.J. and F.K. McIntyre, 1989. Chromosomal DNA variation genomic constraints and recombination in Lathyrus. Genetica, 79: 45-52.
26: Price, H.J. and K. Bachmann, 1976. DNA content in higher plants. Bot. Rev., 46: 27-52.
27: Przyblska, J., Z. Zimniak-Przybylska and P. Krajewski, 1999. Diversity of seed albumins in some Lathyrus species related to L. sativus L.: An electrophoretic study. Gen. Resour. Crop Evol., 46: 261-266.
Direct Link |
28: Rees, H. and M.H. Hazarika, 1969. Chromosome evolution in Lathyrus. Chromosomes Today, 2: 157-165.
29: Sahin, A., H. Genc and E. Bagci, 2000. Cytotaxonomic investigations on some Lathyrus L. species growing in eastern mediterrenean and southern aegean regions-II. Acta Bot. Gallica, 147: 243-256.
CrossRef | Direct Link |
30: Seijo, G. and A. Fernandez, 2001. Cytogenetic analysis of Lathyrus japonicus Willd. (Leguminosae). Caryalogia: Int. J. Cytol. Cytosyst. Cytogenet., 54: 173-179.
CrossRef | Direct Link |
31: Seijo, J.G., 2002. Estudios citogeneticas en especies sudarnericanas del genero. Lathyrus, seccion Notolathyrus (Leguminosae). Ph.D Thesis. Universidad Nacional de Cordoba, Cordoba, Argentina.
32: Senn, H.A., 1938. Experimental data for a revision of the genus Lathyrus. Am. J. Bot., 25: 67-78.
33: Sharma, A.K. and P.C. Datta, 1959. Application of improved technique in tracing Karyotype difference between strains of Lathyrus odoratus L. Cytolagia, 24: 389-402.
34: Simola, L.K., 1986. Structural and chemical aspects of evolution of evolution of Lathyrus species. Proceedings of the International Symposium of IBEAS, September 9-13, 1985, Paris, pp: 225-239.
35: Unal, E.A., J. Wallace and R.S. Callow, 1995. Diverse heterochromatin in Lathyrus. Caryologia, 48: 47-63.
36: Wong, T.S., D. Zhurina and U. Schwaneberg, 2006. The diversity challenge in directed protein evolution. Comb. Chem. High Throughput Screen, 4: 271-288.
Direct Link |
37: Verma, S.C. and D. Ohri, 1979. Chromosome and nuclear phenotype in the legume Lathyrus sativus L. Cytologia, 44: 77-90.
38: Yamamoto, K., T. Fujiware and L.D. Blumenreictt, 1984. Karyotypes and morphological characteristics of some species in the genus Lathyrus L. Jap. J. Breed., 34: 273-284.
39: Allkin, R., T.D. Macfarlane, R.J. Witte, F.A. Bisby and M.E. Adey, 1985. The geographical distribution of Lathyrus. Vicieae Database Project, 7: 1-75.
40: Goyder, D.J., 1986. The Genus Lathyrus. In: Lathyrus and Lathyrism, Kaul, A. and D. Combes (Eds.). Third Worled Medical Research Foundation, New York, pp: 334.