Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(4): 93-104, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-164 Citation: Z. Bouziane, R. Issolah, A. Tahar (2019) Analysis of the chromo- some variation within some natural populations of subterranean clover (Tri- folium subterraneum L., Fabaceae) in Algeria. Caryologia 72(4): 93-104. doi: 10.13128/caryologia-164 Published: December 23, 2019 Copyright: © 2019 Z. Bouziane, R. Issolah, A. Tahar. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Analysis of the chromosome variation within some natural populations of subterranean clover (Trifolium subterraneum L., Fabaceae) in Algeria Zahira Bouziane1,3, Rachida Issolah2,*, Ali Tahar3 1 Université Abbès Laghrour, Khenchela, Algérie 2 INRAA, CRP Mehdi Boualem, Division de Recherche sur les ressources phytogénétiques, BP 37, Baraki, Alger, Algérie 3 Université Badji Mokhtar, Laboratoire de Biologie végétale et de l’environnement, Anna- ba, Algérie *Corresponding author: rachida.issolah@yahoo.com Abstract. Nine natural populations of subterranean clover (Trifolium subterraneum L.) coming from different eco-geographical sites of the North-East Algeria, have been stud- ied for their chromosome number and karyotype features. The study is part of the evalu- ation and valorization of plant genetic resources of fodder and pastoral interest in Alge- ria. The results of mitosis detect two groups of populations, and reveal diversity in the number among and within populations. The Algerian populations of T. subterraneum are characterized by two chromosomic formulas. The first formula (2n=2x=16m) (median), more common in most of the studied populations, is in conformity with previous reports in this species. The karyotype of these populations is symmetrical for size and form. The second (2n=2x= 18m), is detected for the first time and described as a new chro- mosomal formula in T. subterraneum. The latter is relatively more frequent than the first one and characterizes the populations coming from high altitude areas. The karyotype (2n=2x=18m) is relatively symmetrical. At the level of the two established Karyotypes, satellites are highlighted at the first pair. A variation in the size and frequency of these satellites is observed. The species exhibits regular meiotic behaviour, confirming the pres- ence of two basic chromosome numbers (x=8 and 9). The study also highlights the role of ecological factors (Altitude and Rainfall) of the originating environment of Algerian populations in the variation and evolution of chromosome numbers in T. subterraneum. The new cytogenetic data can be exploited in the taxonomy of the species in Algeria in order to select and develop this plant genetic resource in the agricultural field. Keywords. Chromosomes, Intraspecific variability, Karyotype, Subterranean clover, Trifolium subterraneum L. INTRODUCTION The genus Trifolium is one of the largest genera of the Fabaceae family (sub-family, Papilionoideae). It has more than 255 annual and perennial spe- 94 Zahira Bouziane, Rachida Issolah, Ali Tahar cies (Zohary and Heller 1984; Gillet and Taylor 2001). Most of them are of great agricultural importance and widely grown as fodder and green manure (Ellison et al. 2006). The genus Trifolium is originating from Medi- terranean, because of the greatest diversity of numbers and chromosome forms have been found in this region (Taylor 1985). It has been subdivised into eight sec- tions: Lotoidea, Paramesus, Mystillus, Vesicaria, Chrono- semium, Trifolium, Trichocephalum and Involucrarium (Zohary and Heller 1984). The principal geographical centers of diversity of Trifolium are the Mediterranean basin, the West of North America, and the highlands of Eastern Africa (Ellison et al. 2006). The cytotaxonomic studies carried out on Trifolium have shown that it pre- sents a surprising variety of chromosome numbers, and the changes in the number of chromosomes have played a large part in its evolution (Falistocco et al. 2013). Brit- ten (1963) and Pritchard (1969) have shown that an ane- uploid series of basic numbers x=5, 6, 7 and 8 are found in this genus. The presence of x=8 in about 80% of the species suggests that x=8 is the ancestral number of the genus (Senn 1938; Pritchard 1969; Zohary and Heller 1984; Ellison et al. 2006), from which the numbers x = 7, 6 and 5 are derived. Polyploidy is more common in per- ennial species (Kiran et al. 2010; Falistocco et al. 2013). Subterranean clover (Trifolium subterraneum L., sect Trichocephalum), commonly known as the burrow- ing clover or sower, is a winter annual species, native to the Mediterranean Basin, West Asia and the Atlantic coast of Western Europe (Gladstones and Collins 1983  ; Zohary and Heller 1984). The plant of subterranean clover is autogamous, characterized by mechanisms of burial of reproductive structures, ensuring thus, its own self-regenerating (Masson 1997). The species constitutes an heterogenous complexesis, divided into three sub- species:  subterraneum, brachycalycinum and yanninicum (Katznelson 1984), identifiable enough by their morpho- physiology, karyotypes, isozymes and polymorphisms for molecular markers (Piluzza et al. 2005). In Algeria, the subterranean clover is very common in the Tell and the mountain meadows (Quezel and Santa 1962), adapt- ed to different ecological conditions (Issolah et al. 2015). This species is represented by three varieties belonging to the subterraneum subspecies (Subsp. subterraneum Var. subterraneum, Var. brachycladum, Var. flagelliforme) on the eight varieties described in Algeria (Zohary and Heller 1984). Despite the agronomic importance of the species in the world, as cattle feed and soil improvement, its cytological characterization remains very restricted. This is because of the small size of chromosomes like all the other species of Trifolium (Zohary and Heller 1984). The first investigations on T. subterraneum focused only on the determination of the chromosome number (2n=16), but without establishing the karyotype (Weselx- en 1928; Yates and Brittan  1952; Brock 1953; Hutton and Peak 1954; Zohary and Katznelson 1958; Kliphuis 1962; Britten 1963; Katznelson and Morley 1965a). Later, some karyotype studies were performed in Spain (Angelo et al. 1975, 1977, 1983), Iran (Hezamzadeh Hijazi and Ziaeinasab 2006) and Italy (Falistocco et al. 1987; Falis- tocco et al. 2013). The present study is interested in the evaluation and the valorization of the phytogenetic resources of fodder and ¦pastoral¦ interest in Algeria. Its aim is the analysis of the chromosomal diversity presents in the natural populations of Trifolium subter- raneum L., and the establishment of its karyotype. It follows the different studies carried out on natural fodder legumes (Issolah and Abdelguerfi 1999a; Issolah and Khalfallah 2007;  Issolah et al. 2006,  2012,  2015, 2016). MATERIAL AND METHODS Plant materials The Trifolium subterraneum specimens were col- lected by INR AA (National Institute of Agronomic Research of Algeria), in July 2010. Nine natural popu- lations sampled from North-East Algeria (Issolah et al. 2015), were the subject of a karyological study (Table 1). Chromosome counting The seeds belonging to the nine studied popula- tions, were scarified to remove in tegumentary hardness, and then germinated on wet filter paper in Petri dishes at room temperature. The root tips meristems (1 to 1.5 cm in length) were excised in the morning between 8 am- 8.30 am and pretreated with α-bromonaphthalene (1%) at room temperature for 2h45mn. The use of this pretreatment increases the number of metaphase mitot- ic cells, allows the chromosomes to be well spread in the cell, straightens the chromatids, and contracts the chromosomes, which makes primary and secondary constrictions very noticeable (Singh 2018). For chromo- somes analysis, root tips were hydrolyzed in 1N HCl and stained in lactopropionic orcein (Dyer 1963). The chro- mosomic observations were repeated several times. For each population, five plates of chromosomes were select- ed from at least 30 individuals (seeds). Then, they were observed and photographed using a Primo Star Zeiss 95Analysis of the chromosome variation of subterranean clover (Trifolium subterraneum) in Algeria microscope. Chromosome counts were performed on metaphase plates with well individualized chromosomes. Karyotype analysis The karyomorphological analysis was carried out according to the following parameters: the lenght of long arm (L), short arm (S), the total length of the chro- mosome (LT = L + S), the difference between arms (d = L-S), and the relative length (LR (‰) = 1000 x TL/ ΣTL). Centromere position and chromosome types were determined from the two parameters: arm ratio (r= L/S), and centromeric index (CI % = S/LT x100) according to the nomenclature of Levan et al. (1964). For determin- ing the asymmetry of the karyotype, three parameters were estimed: [(Ias. K% = (Σ L/Σ LT) x100 (Aran and Saito 1980)], the ratio between the longest and the short- est chromosome pairs (R), and the inter-chromosomal asymmetry coefficient (A2) (standard deviation of chro- mosome length / mean chromosome length) (Romeo Zarco 1986). Chromosome measurements, based on five plates per population, were performed using the Axio- vision software (1999-2009). The different karyotype cal- culations were made thanks to Excel (2007). Meiosis To confirm the results corresponding to the num- bers found by mitosis (presence of supernumerary chromosome pair for certain populations), the meiotic behaviour of the nine populations was also analysed. For this purpose, a trial has been conducted at the experi- mental station of INRAA (November 2014). Each popu- lation was represented by twenty individuals (seeds) and sowed in total randomization (field) for identifying the different phases of meiosis (laboratory). The flower buds collect period was spread over a month before flowering (recovering flower buds of variable size). For each plant, at least five flower buds were collected (April 2015) in the early morning (from 8h), then fixed in Carnoy solu- tion (Ethanol-acetic acid 3  : 1, v/v) for at least 48 h at 4C°. After dissection of the anthers, the pollen mother cells (PMC) were crushed in an acetic carmine drop 1% (Jahier et al. 1992). Observations and photographs at dif- ferent phases were performed using a Primo Star Zeiss microscope. RESULTS Chromosome counting All mitotic metaphase plates of investigated popu- lations of the species Trifolium subterraneum L. showed a diploid number of chromosomes (2n = 16) (Figure 1). This number is frequently observed in individuals of the populations 12/10; 13/10; 19/10; 20/10 and 33/10. Howev- er, the somatic metaphases of the four populations 22/10; 23/10; 25/10; 26/10, have presented along with the char- acteristic number of the species (2n = 16), a second and new number of chromosomes (2n = 18), often encoun- tered during this study in these later populations (Fig- ure 1). The two chromosome numbers (2n = 16 and 18) are observed within the cells of the same individual, and also in different individuals of the same population. This indicates a chromosomical variation within and between the populations of Trifolium subterraneum. The analysis of 15 individuals per population, indi- cated that the variation of the chromosome numbers (2n = 16 and 18) was not in the same frequency in these Figure 1. Mitotic metaphases of Algerian natural populations of Trifolium subterraneum L. with two chromosomes numbers 2n=16 and 2n=18 respectively: (a) population 12/10; (b) population 13/10; (c) population 19/10; (d) population 20/10; (e) population 22/10; (f ) population 23/10; (g) population 25/10; (h) population 26/10; (i) population 33/10; (j) population 22/10 (2n=18); (k) population 25/10 (2n=18); (l) population 26/10(2n=18) . Arrows: satellites. Bar: 2.5µm. 96 Zahira Bouziane, Rachida Issolah, Ali Tahar latter populations (Figure 2). Indeed, within the popu- lations (22/10 and 23/10), the frequency of the number (2n = 16) represents twice the frequency of the num- ber (2n=18) (0.67 and 0.33; 0.64 and 0.36, respectively). However, very similar frequency values are shown in the other two populations (25/10 and 26/10) (0.56 and 0.44; 0.5 and 0.46 respectively) (Figure 2). Karyotype analysis In all investigated populations, the morphology and chromosome structure are almost identical (Table 2-5). Our results showed that the chromosomes of the Alge- rian population of the species Trifolium subterraneum L. are small. The size of the chromosomes varies from 1.02 μm (Table 3) to 3.01 μm (Table 2). The total lengths of diploid chromosome set are comprised between 12.82 μm (Table 4) and 18.87 μm (Table 2). The mean value of the total length (TLG) of all studied populations is 1.92μm. The results of this study indicate also that the population 22/10 (2n =16) is characterized by the high- est values for the selected parameters, like the mean value of chromosome length, which gives an estimated size of the genome (18.87 μm) and the largest first pair and eighth pair (3.01μm-1.72μm) (Table 2). Thus, we note that the two additional chromosomes present in the populations (2n = 18), have the same form, with a mean size of 1.09 μm (Figure 1, Table 3 and 5). The results of this study indicated also that satellites are located at the first chromosome pair within all investigated popula- tions. A variation of the size and an abundance of these satellites are noticed. Thus, the metaphase plates of the populations characterized by (2n= 16), present a consid- erable size of these satellites compared to that noted on the plates of the populations characterized by (2n=18) with 0.25 μm ± 0.022; 0.18 μm ± 0.025, respectively. These satellites are more abundant in the meta- phases of populations with 2n = 18 compared to those with 2n = 16. Their frequencies are 0.70 and 0.44, respectively (Figure 1). Otherwise, the results of the centromeric index (Ic) and the ratio between the long arm and the short arm (r) allowed us to determine the homologous chromosomes and to classify the different chromosomal types. Therefore, all the studied popula- tions are characterized by the karyograms, presenting median chromosomes (Figure 3b). Figure 2. Frequencies of the two chromosomes numbers (2n=16 and 18) in the four populations of Trifolium subterraneum L. (15 individuals / population). Table 1. Geographical origin and ecological characteristics of the sampling sites of nine populations of Trifolium subterraneum L. in Algeria Nº of populations Origin Altitude (m) Rainfall (mm) 12/10 Guelma 170 600 13/10 Guelma 200 558 19/10 Tarf 665 661 20/10 Tarf 555 661 22/10 Souk Ahras 950 800 23/10 Souk Ahras 1040 700 25/10 Souk Ahras 800 900 26/10 Souk Ahras 1110 700 33/10 Skikda 110 562 Source (Issolah et al. 2015) Table 2. Morphometric data within the population 22/10 (2n=16) of Trifolium subterraneum L. in Algeria. Ch p L (µm) (±SD) S (µm) (±SD) TL (µm) RL ‰ d r Ci % Ct 1 1.69 (0.41) 1.32 (0.26) 3.01 159.36 0.37 1.28 43.90 m-sat 2 1.42 (0.31) 1.26 (0.43) 2.68 141.96 0.17 1.13 46.86 M 3 1.53 (0.38) 1.08 (0.35) 2.61 138.52 0.45 1.42 41.33 M 4 1.26 (0.50) 1.09 (0.40) 2.35 124.38 0.17 1.15 46.45 M 5 1.23 (0.50) 1.05 (0.43) 2.28 120.85 0.18 1.17 45.98 M 6 1.18 (0.40) 1.05 (0.31) 2.23 118.11 0.13 1.12 47.12 M 7 1.05 (0.29) 0.94 (0.32) 1.99 105.65 0.11 1.12 47.16 M 8 0.92 (0.31) 0.80 (0.19) 1.72 91. 17 0.12 1.15 46.61 M I1as% =54.48 ∑TL=18.87 TLG=2.36 R1=1.75 A2(1)=0.14 97Analysis of the chromosome variation of subterranean clover (Trifolium subterraneum) in Algeria The values of the asymmetry index Ias% (Arano and Saito, 1980), the ratio between the largest and the smallest chromosome pairs (R), and the interchromo- somal index A2 (Romero Zarko 2006) gives indications on the evolution of chromosomes in plants. The results of the three parameters [(R1: 1.75, R3 =1.78), (I1as% = 54.48, I3as% = 55.81), and (A2 (1) = 0.14, A2(3) = 0.19)] (Table 2 and 4) are weak and indicate that the karyotype (2n=16m) is very symmetrical for the size and the form. It is therefore primitive. Nevertheless, although the asymmetry indices are low (I2as%: 56.69, I4as% 55.07) in the populations (2n = 18), they showed a karyotype with more or less uniform sizes except for the ninth pair. This is reflected by relatively high values of the ratio (R) and interchromosomal asymmetry A2, compared to those found for the karyotype (2n = 16) (Table 3 and 5). Meiosis analysis The study of meiotic behaviour showed that the nine natural populations of the species Trifolium subterra- neum exhibit normal and regular meiosis, with domi- nance of bivalents at the diakinesis, metaphases I and Figure 3. Karyotype of Trifolium subterraneum L. in Algeria. (a) Somatic metaphasis (2n=18, population 23/10); (b) Karyogram; (c) Idiogram; arrow (satellites). Bar: 2µm. Table 3. Morphometric data within the population 23/10 (2n=18) of Trifolium subterraneum L. in Algeria. Ch p L (µm) (±SD) S (µm) (±SD) TL (µm) RL ‰ d r Ci % Ct 1 1.45 (0.17) 1.05 (0.14) 2.50 146.23 0.40 1,38 42.06 m-sat 2 1.39 (0.13) 1.02 (0.16) 2.41 140.86 0.37 1,37 42.27 M 3 1.33 (0.11) 0.93 (0.08) 2.26 132.46 0.39 1,42 41.31 M 4 1.09 (0.32) 0.89 (0.08) 1.98 116.14 0.20 1,23 44.84 M 5 1.07 (0.25) 0.80 (0.22) 1.87 109.50 0.27 1,33 42.97 M 6 1,01 (0.24) 0.81 (0.19) 1.82 106.51 0.21 1,26 44.30 M 7 0.92 (0.25) 0.81 (0.19) 1.73 101.41 0.11 1,13 46.86 M 8 0.85 (0.30) 0.63 (0.04) 1.48 86.93 0,21 1,34 42.77 M 9 0.57 (0.24) 0.45 (0.15) 1.02 59.96 0.11 1,25 44.42 M I2as%=56.69 ∑TL=17.08 TLG=1.90 R2=2.45 A2(2)=0.25 Table 4. Morphometric data within the population 25/10 (2n=16) of Trifolium subterraneum L. in Algeria. Ch p L (µm) (±SD) S (µm) (±SD) TL (µm) RL ‰ d r Ci % Ct 1 1.14 (0.23) 0.91 (0.02) 2.05 159.96 0.22 1.24 44.57 m-sat 2 1.12 (0.10) 0.82 (0.01) 1.94 151.56 0.31 1.37 42.14 M 3 0.96 (0.09) 0.80 (0.01) 1.76 137.50 0.16 1.20 45.60 M 4 0.99 (0.01) 0.73 (0.02) 1.72 134.38 0.26 1.35 42.30 M 5 0.83 (0.06) 0.67 (0.02) 1.50 117.00 0.16 1.24 44.50 M 6 0.80 (0.02) 0.61 (0.15) 1.41 110.16 0.19 1.31 43.09 M 7 0.73 (0.06) 0.56 (0.20) 1.29 100.78 0.17 1.30 43.41 M 8 0.60 (0.17) 0.56 (0.18) 1.15 90.04 0.04 1.08 48.16 M I3as%=55.81 ∑TL=12.82 TLG=1.6 R3=1.78 A2(3)=0.19 98 Zahira Bouziane, Rachida Issolah, Ali Tahar Table 5. Morphometric data within the population 26/10 (2n=18) of Trifolium subterraneum L. in Algeria. Ch p L (µm) (±SD) S (µm) (±SD) TL (µm) RL‰ d r Ci % Ct 1 1.36 (0.49) 1.25 (0.36) 2.61 160.10 0.11 1.09 47.94 m-sat 2 1.22 (0.43) 1.04 (0.41) 2.26 138.20 0.18 1.17 46.01 M 3 1.25 (0.58) 0.90 (0.33) 2.15 131.45 0.35 1.38 41.96 M 4 1.14 (0.50) 0.85 (0.39) 1.99 121.50 0.29 1.34 42.75 M 5 1.00 (0.52) 0.75 (0.43) 1.75 107.40 0.25 1.33 42.94 M 6 0.91 (0.38) 0.70 (0.28) 1.61 98.97 0.21 1.30 43.50 M 7 0.82 (0.16) 0.68 (0.23) 1.50 92.08 0.14 1.20 45.42 M 8 0.77 (0.30) 0.71 (0.25) 1.48 90.55 0.06 1.09 47.88 M 9 0.56 (0.05) 0.47 (0.02) 1.03 62.48 0.09 1.20 45.15 M I4as%=55.07 ∑TL=16.38 TLG=1.82 R4=2.54 A2 (4)=0.26 Ch p: chromosome pair, L: long arm, S: short arm, LT: total length of chromosome, LR (‰)  : relative length, d  : long arm - short arm  ; r  : long arm / short arm, Ic : centromeric index, Ct : chromosome type, Ias% : asymmetry index, R  : longest / shortest pair, ΣTL  : total lenght of diploid set, TLG : average of total length, A2: interchromosomal asymmetry index, (SD) : standard deviation, sat: satellites. Figure 4. Pollen meiosis in some natural populations of Trifolium subterraneum L. in Algeria L. (a) pollen cell  ; (b) Diakinesis (population 22/10, n=x=8)  ;  (c) Diakinesis (22/10, n=x=9)  ;  (d) Diakinesis (population 23/10, n=x=8)  ; (e) Metaphase I (population 23/10, n=x=8)  ; (f ) Metaphase I (population 23/10 n=x=9) ; (g) Anaphase I ; (h) Telophase I ; (i) Tetrade. Bar: 2 µm. 99Analysis of the chromosome variation of subterranean clover (Trifolium subterraneum) in Algeria anaphases I (Figure 4). This allowed us to authenticate the basic haploid number (x = 8) for the populations (12/10; 13/10; 19/10; 20/10; 33/10). Likewise, it confirms the presence of the two chromosome numbers (2n = 16 and 18) detected in mitosis, within the four populations (22/10; 23/10; 25/10; 26/10), through the appearance of two basic haploid numbers (x = 8) and (x = 9). DISCUSSION In this study, the chromosome numbers, karyogram, idiogram and karyotype asymmetry of naturel popu- lations of Trifolium subterraneum, were determined. Mitotic metaphases showed both the same chromosome number (2n=16) in all studied populations. This number was previously reported by several authors within dif- ferent ecotypes and varieties from several areas (Senn 1938; Angelo 1975, 1977, 1983; Zohary and Heller 1984; Hezamzadeh Hijazi and Ziaeinasab 2006; Vizintin et al. 2006; Falistocco et al. 1987; Falistocco et al. 2013), con- sidering x=8, as being the ancestral basic chromosome number of the species. Meanwhile, four populations presented two numbers of chromosome (2n=16 and 18) within the cells of the same individual, and also in dif- ferent individuals of the same population. The number of chromosomes, as one of the genetic variations, is extremely variable ranging from low num- bers to relatively high numbers (Eroğlu and Per 2016). A change in the basic chromosome number of a species represents dysploidy (Yakovlev 1996). According to the same author, this change can occur either in the direc- tion of an increase (ascending dysploidy) or a decrease (downward dysploidy). In plants, this last case seems to be the most frequent, it results from the simultaneous or successive action of several cytogenetic mechanisms (rob- ertsonian translocation, deletion …) (Yakovlev 1996). Contandriopoulos (1978) reports 2n = 30, 32 and 34 for Sideritis libanotica Labill. This author notes that dysploidy still seems anarchic and has not succeeded to form populations with stable karyotypes having their own geographical distribution and a particular mor- phological differentiation. In such case, according to the same author, it would seem more judicious to speak about hyper and hypoaneuploidy. Aneuploidy may pre- sent the beginning of the mechanism leading to dys- ploidy, provided that the individuals carrying the ane- uploid number are able to multiply then impose itself in the population (Contandriopoulos 1978). Yakovlev (1996) considers that a variable chromo- some number within the same population is both an aneuploidy and dysploidy phenomenon, witch is dif- ficult to draw the line between these two phenomena, especially when it is polyploid taxa. An Intra-specific dysploidy represents a transitiona l step towards a def initive change in t he basic chromosome num- ber (Yakovlev 1996). The populations in which such change has occurred and fixed represent, well prob- ably, the direct ancestors of future dysploïde species (Yakovlev 1996). In the genus Trifolium, many variations of the nom- bre de chromosomes (2n = 16, 14, 12, and 10) character- ize different diploid species, and in some instances cyto- logical variants occur within the same species (Falistoc- co et al. 2013). Brock (1953) counted two different chromosome numbers (2n = 12 and 16) in the species Trifolium sub- terraneum growing in various regions. This author sug- gested that the difference could be the result of a chro- mosomal rearrangement without loss of genetic material. In the same genus, two basic numbers (X = 8 and 9) were highlighted within the populations of two species of Trifolium: T. ornithopodiodes from the British Isles (Rutland 1941; Muñoz-Rodríguez 1995), and also in T. montanum var. montanum. of Iberian Peninsula (Bleier 1925a; Muñoz-Rodríguez 1995). Issolah and Abdelguerfi (1999b), evenly showed the presence of two basic chro- mosomes numbers (x = 5 and 6) in the Algerian popula- tions of Trifolium scabrum. According to Pritchard (1969) and Zohary and Hel- ler (1984), the dysploidy is consistently linked to the annual species, and are most common within sections that are at a more advanced stage of evolution, such as Trifolium and Tricocephalum, in which all the four basic numbers (x =8,7,6 and 5) may be found. Uslu (2012) has shown that taxa in the Trifolium section, growing in Turkey, have three numbers (x = 6, 7 and 8). Within the tribe Trifolieae, Darlington and Jamaki (1945) and Darlington and Wylie (1945) reported three basic numbers (x = 7, 8, and 9). The last basic number (x = 9) was detected in Europe in Trigonella ornithopodi- odes L. (DC) (Darlington and Wylie 1945). This species was reclassified later, for taxonomic reasons, in the Trifo- lium genus (Allen and Allen 1981). Within the Fabaceae family, several cases, observing more than one basic chromosome number, have been reported in different genera including Onobrychis, with x = 7 and x= 8 (Hejazi et al. 2010, Arslan et al. 2012) and Genista where the most common number of chro- mosomes is 2n = 48, with the exception of the aneuploid number (2n = 44) revealed in Genista ovina (Bacchetta et al. 2012). The same process was detected in species of the genus Hedysarum, among which, H. pallidum (2n = 16 and 18) (Benhizia et al. 2003); H. coronarium (2n = 100 Zahira Bouziane, Rachida Issolah, Ali Tahar 16 and 2n = 18) (Issolah et al. 2006)  and H. perrauderi- anum (2n = 32 and 18) (Benhizia et al. 2013). In the Poaceae family, dysploidy was observed in Lygeum spartum L., whose cytogenetic study revealed two basic chromosome numbers, in two Algerian popu- lations of different origins (2n = 16 and 40) (Abddaim- Boughanmi et al. 2009). According to the same authors, the population (2n = 40), also presented a variability of the chromosome number within the same individual. Yakovlev et al. (2017) have shown that constitutive heterochromatin, DNA GC rich and rRNA are involved in chromosomal rearrangements during the change in basic chromosome numbers in Mediterranean species of the genus Reichardia Roth. (Asteraceae). These species are characterized by three basic chromosome numbers (x = 9, 8 and 7), which have contributed to the evolution of the genus in the Mediterranean region (Yakovlev et al. 2017). Concerning chromosome size, our results (1.02- 3.1μm) seem to be relatively inferior to those found by Falistocco et al. (2013) on Italian accessions of Trifolium subterraneum (2.5-3.5μm). But then, this size appears to be very similar to that recorded in T. lappaceum species of Iran (3.03 μm), but smaller than the sizes reported in other Trifolium species of Iran (T. angustifolium: 14.56 μm, T. leucanthum: 12.32 μm, T. tumens: 11.09 μm) (Ali- mardani et al. 2014). Our data are also close to those found within some Trifolium species in Turkey, such as T. echinatum (1.41-2.74 μm)  and T. phleoides (1.73-2.78 μm) (Uslu 2012), and appear to be superior to those recorded by kiran et al. (2010) in T. speciosum Willd. (0.99-1.64 μm) and T. campestris Scherb (1.13-1.73 μm). Within the same family (Fabacaea), the size of T. subterraneum chromosomes, found during our study, is relatively close to those reported for some species of the genera Hedysarum, Astragalus and Asparagus studied in Algeria (Benhizia et al. 2003 ; Issolah et al. 2006, Benhizia et al. 2013; Baaziz et al. 2014 and Boubetra et al. 2017). Our observations highlighted satellites at the first pair of chromosomes. The presence of satellites and their location on the first chromosome pair joins the result found by Falistocco et al. (2013) on Italian accessions. According to Falistocco et al. (1987)  and Falistocco et al. (2013), these satellites are present in the three sub- species of T. subterraneum (subterraneum, brachycalyci- num, yanniniccum), and their size can be used for dis- criminating the three subspecies. The satellites are more important in yanninicum and medium in the other two subspecies (Falistocco et al. 1987). In all populations, the chromosomes are median. This confirm the results of Falistocco et al. (2013) on Italian accessions, characterized also by median chro- mosomes, whereas, Angelo et al. (1983) have described two chromosomes types (median and submedian) for Spanish ecotypes. Moreover, two types of karyotypes were identified for the Iranian accessions: the first con- sists on eight median pairs; the second karyotype is composed by six median pairs and two submedian pairs (Hezamzadeh Hijazi and Ziaeinasab 2006). Karyotype asymmetry is an important parameter in karyological studies (Eroğlu 2015). In our case, the kar- yotype (2n=16) of Algerian populations of Trifolium sub- terraneum is very symmetrical. This seems to be a com- mon trait with Italian populations of T. subterraneum karyotype (Falistocco et al. 2013), but differs from the Iranian ones. The latter populations of T. subterraneum (2n = 16) are characterized by low intrachromosomal symmetry (Hezamzadeh Hijazi and Ziaeinasab 2006). On the other hand, the karyotype of the population 2n = 18 is considered relatively symmetrical because of the high value of interchomosomal asymmetry. Thus, Muñoz-Rodríguez (1995) does not consider the karyo- type of the species Trifolium ornithopodioides (2n=18) as asymmetrical, despite the high value of the asymme- try index A2 (0.20). The author noticed this, because of the more or less uniform sizes of the chromosome pairs, except for the first pair, which was larger than the others (Muñoz-Rodríguez 1995). In the species Reichardia picroides (Asteraceae), Yak- ovlev (1986) has suggested that this is a case of second- ary symmetry due to chromosomal rearrangements. The analysis of pollen meiosis confirmed the results obtained in mitosis. At the end of these results we have found that the Algerian populations of T. subterraneum are characterized by two chromosomal formulas. The first, (2n = 2x = 16m) (median) usually reported by pre- vious authors, and the second (2n = 2x = 18m) revealed for the first time in this species throughout our present work. It is important to note that the new formla (2n = 2x = 18m) is observed particularly in populations sam- pled from high altitude sites (800-1110 m), belonging to the same biogeographic area and characterized by a high rainfall (700-900 mm). Consequently, the variation in the chromosome number observed in the populations of this species and the appearance of a new chromosome pair seems to be influenced by these two ecological fac- tors (altitude and rainfall). Meanwhile, t he same popu lations considered through our study have been the subject of previous work on the ecological characterization of the natu- ral habitat of T. subterraneum in Algeria (Issolah et al. 2015). Thus, the results of this latest study have shown that the variation of the edaphic, climatic, and topo- graphic characteristics of the origin sites of these popu- 101Analysis of the chromosome variation of subterranean clover (Trifolium subterraneum) in Algeria lations influences the distribution of this species in the North-Est Algeria (Issolah et al. 2015). Significant rela- tionships were found between altitude and rainfall and the physico-chemical parameters of the soils of these populations, and the effect of altitude was relatively more pronounced notably on the nitrogen, clay, pH and C / N ratio (Issolah et al. 2015). Abdelguerfi et al. (2006) indicate that T. subterraneum is more prevalent in heavily watered and moist regions. Rossiter and Collins (1988a, 1988b) and Cocks (1992) also observed greater variability of subterranean clover populations in high rainfall areas in Australia. Various studies have shown that differences in the origin’s areas of populations and the variation of the environmental factors of the natural habitat may explain the intra-specific differences. Thus, they can affect the variation of chromosome numbers, ploidy level, chromo- some structure, and asymmetry of karyotype in certain species belonging to the genera: Trifolium (Issolah and Abdelguerfi, 1999b, Issolah 2006); Hedysarum (Issolah et al. 2006, Benhezia et al. 2013); Bellevalia and Muscari (Azizi et al. 2016); Asparagus (Boubetra et al. 2017). Envi- ronmental factors also, influenced karyotype parameters in Aegilops (Poaceae) species (Baik et al. 2017). Significant relationships were found between Altitude, total lengths chromosome set and interchromosomal asymmetry on the one hand and, on the other hand, between rainfall and intrachromosomal asymmetry (Baik et al. 2017). According to Hayward and Breese (1993), natural habitats are rarely, if ever, uniform in space and time and can encompass several distinct micro-niches or go through large seasonal fluctuations. Although Trifolium subterraneum is a self-pollinating species, Allard and Adams (1969) and Hayward and Breese (1993), report that fluctuations and variation in edaphic conditions at the site of origin trigger in self-pollinated species, a dis- ruptive selection that produces and maintains high levels of variability in wild populations. In Italy, a relationship between many morphological characteristics and the ecological factors of the environ- ment of origin (altitude and rainfall) has been deter- mined in several populations of T. subterraneum from Sicily (Piano et al.1993, Pecetti and Piano 1998). In a large collection of subsp. subterraneum germ- plasm of Sardinia, Piano et al. (1996, 2002) found that the level of complexity for various traits varied greatly among populations and was influenced by the climatic characteristics of the collection sites. Within the genus Trifolium, interesting relation- ships have been found between many morphological characteristics and some ecological factors (altitude and rainfull) of the environment of origin of several spon- taneous Algerian populations belonging to various spe- cies (T. campestre, T. glomeratum, T. tomontosum, T. resupinatum, T. scrabrum, T. lampaceum, T. spumo- sum) (Issolah and Abdelguerfi 1993, 1995, 2003 ; Issolah 2006). In addition, Medoukali et al. (2015), do not report any significant relationship between the morphological characteristics and the environment of origin of popu- lations belonging to several Trifolium species (T. angus- tifolium, T. lappaceum, T. resupinatum, T. tomentosum, T. scabrum, T. campestre, T. fragiferum, T. pallidum, T. pallescens, T. squarrosum, T. glomeratum, T. cherleri, T. stellatum, T. repens and T. spumosum). Nevertheless, a large genetic variation of isoenzymes has been observed (Medoukali et al. 2015). Although the species is self-pollinated with cleis- togamous flowers (Katznelson and Morley 1965), there is a possibility of occasional cross breeding, and this exceptional rarefaction could be of great importance for the evolution of T. subterraneum. Marshall and Broué (1973) estimated the cross-pollination rate of the Aus- tralian clover populations at 0.15%. Variation released by occasional hybridization can then be fixed by selfing and made available to natural selective pressures (Cocks 1992b). According to Piano (1984), natural populations of subterranean clover were formed by clusters of several genetically distinct strains. This would probably explain the chromosomal variation observed in this study within and between populations. As a result, the dif- ferent populations of T.subterrarenum would have been crossed. Meanwhile, four populations from the same region exhibited the same somatic behaviour (2n = 16 and 18) (within the same individual and between different indi- viduals) and meiotic (n = x = 8 and n = x = 9). These populations would probably be evolved in time, since they belong to a species of the “Trichocephaleum” section considered, according to Zohary and Heller (1984), as the most evolved section compared to other sections of the genus Trifolium. This section is therefore composed of species, whose interaction, with the various ecologi- cal characteristics of the natural habitat, would affect the chromosomal rearrangements and evolutionary trends of the populations within T. subterraneum species. CONCLUSION This study permitted to identify and analyse the intraspecific diversity of the chromosome numbers and karyotypes within nine natural populations of Trifo- lium subterraneum, originating from the different areas of the north eastern Algeria. Two chromosome numbers 102 Zahira Bouziane, Rachida Issolah, Ali Tahar are distinguished in this species: 2n=16 (x=8) and 2n=18 (x=9). The first number (2n=16), is widely detected by previous authors, while the second one (2n=18) is new- ly observed in algerian populations of this species. The latter number (2n=18) is frequently met in populations coming from the high altitude areas. The ecological con- ditions of the origin’s environment of the populations would have an effect on the changes in the genetic and karyological structure, particularly the altitude factor. This karyological approach provides new information that will help researchers to elucidate and complete the systematics and the nature of diversity within Trifolium subterraneum species. However, thorough investigations of the morphological and molecular aspects of these nat- ural populations would be necessary, to determine the limits of dysploidy. Furthermore, comparative analysis with other populations from different origins would help to understand more about the genome evolution process of T. subterraneum populations in their environment of origin. This would permit to valorize and develop this plant genetic resource in the Mediterranean area, espe- cially in Algeria. REFERENCES Allen O N, Allen E.K.1981. The leguminoseae, Macmillon.C.O, London. Abdeddaim-Boughanmi k and Kaid-Harche M. 2009. Structure, ultrastructure of the Anther pollen micro- sporogenesis and morphology of pollen grains of two populations of Lygeum spartum L. in Algeria. Americ J. Agric. and Biol Sci. 4 (3) 201-205. Abdelguerfi A, AbdelguerfI–Laouar M, M’hammedi Bouzina M, Guittonneau GG, Huguet T, Abbas K, Mebarkia A, Aouani M E and Madani T. 2006. Dis- tribution et écologie de quelques Fabaceae spon- tanées d’intérêt pastoral et / ou fourrager en Algérie. Workshop international sur la Diversité des fabacées fourragères et de leurs symbiotes : Applications bio- technologiques, agronomiques et environnementales. Alger, 19-22 Février 2006: 27-36. Alimardani F, Torabi S, Naghavi R, Ebrahimi A. 2014. Study of cytological among some Trifolium species of Iran. Interciencia. 39 (4) 147– 151. Allard RW. 1965. Genetic systems associated with colo- nizing ability in predominantly self-pollinated spe- cies. In: Baker H.G. and Stebbins G.L. (Eds), Proc. First International Union of Biological Sciences Symp. Academic Press, New York, pp. 49– 75. Allard RW, Adams J. 1969. Population studies in predom- inantly self-pollinating species. XII. Intergenotypic competition and population structure in barley and wheat. Am. Nat. 103: 621– 645. Angulo MD, Sanchez de Rivera A.M. 1975. Studies on Tri- folium subterraneum ecotypes. Cytologia, 40: 415–423. Angulo MD, Sanchez de Rivera.1977. Comparative chro- mosomal study of Spanich ecotyes and Autralien cultivars of Trifolium Subterraneum L. Cytologia 42: 473-482. Angulo MD, Sanchez de Rivera.1983. Karyological stud- ies on spanich taxa of Trifolium subterraneum L. Cyt- ologia 48: 305-312. Arano H, Saito H. 1980. Cytological studies in family Umbelliferae.V. Karyotypes of seven species in sub- tribe Seselinae. Chromosoma. 2 (17): 471–480. Arslan E, Ertuğrul K, Tugay O, Dural H. 2012. Karyo- logical studies of the genus Onobrychis Mill. and the related genera Hedysarum L. and Sartoria Boiss. & Heldr. (Fabaceae, Hedysareae) from Turkey. Caryolo- gia. 65 (1): 11–17. AxioVision 1999- 2009. By Carl Zeiss. Release 4.8.1. Azizi N, Amirouche R, Amirouche N. 2016. Karyological investigations and new chromosome number reports in Bellevalia Lapeyrouse, 1808 and Muscari Miller 1758 (Asparagaceae) from Algeria. Comp Cytogen 10: 171-187. doi : 10.3897/CompCytogen.v10i1.6445. Bacchetta G, Brullo S, Velari TC, Chiapella LF, Kosovel V. 2012. Analysis of the Genista ephedroides group (Fabaceae) based on karyological, molecular and morphological data. Caryologia. 65(1):47–61. Baaziz K, Benamara-Bellagha M, Pustahija F, Brown CS, Siljak-Yakovlev S, Khalfallah N. 2014. First karyo- type analysis, physical rDNA mapping and genome size assessment in four North African Astragalus taxa (Fabaceae). Turkish J. Bot., 38: 1248–1258. doi: 10.3906/bot-1405-40. Baik N, Maamri F, Bandou H. 2017. Karylogical sty- dy and meiotic analysis of four species of Aegilops (Poaceae) in Algeria. Carylologia.70 (4)  : 324-337. Doi.org/10.1080/00087114.2017.1387340. Benhizia H, Benhizia Y, Ghernoub L, Siljak-Yakovlev S, Khalfallah N. 2013. Meiotic behaviour and karyo- type features of endangered endemic fodder spe- cies Hedysarum perrauderianum (Fabaceae) in some populations from Algeria.Caryologia _Firenze. doi: 10.1080/00087114.2013.821838. Benhizia H, Rached-Mosbah O, Benhizia Y, Kouachi A, KhalfallahN. 2003. Etude cytogenetique d’Hedysarum pallidum Desf. Espece endemique Nord-africaine, tolerante a l’antimoine. Universite de Constantine. Sciences et Technologie C. 20: 7–13. Boubetra K, Amirouche N, Amirouche R. 2017. Com- parative morphological and cytogenetic study of 103Analysis of the chromosome variation of subterranean clover (Trifolium subterraneum) in Algeria five Asparagus (Asparagaceae) species from Algeria including the endemic A. altissimus Munby. Turk J Bot. 41: 588-599. doi: 10.3906/bot-1612-63 Bleier, H. 1925 b. Chromosomenzahlen und Kern volu- mina in der Gattung Trifolium. Ber. Deutsch Bot. Ges. 43(5): 236-238. Britten E. 1963. Chromosome number in the genus Trifo- lium. Cytologia, 28: 428-449. Brock RD.1953. Species formation in Trifolium subterra- neum. Nature, 171:939. doi 10.1038/171939a0. PMID: 13054785. Cocks, P.S. 1992b. Evolution in sown populations of sub- terranean clover (Trifolium subterraneum L.) in South Australia. Aust. J. Agric. Res. 43:1583–1595. Contandriopoulos J. 1978. Contribution à l’étude cytotax- inomique des Sideritis section Empedoclea (Labiatae). Plant Syst. Evol. 129(4): 277-289. Darlington CD, Janaki Ammal, E.K. 1945. Chromosome Atlas of cultivated plants. Allen and Unwin, London. Darlington CD, Wylie AP. 1945. Chromosome Atlas for flowring plants, George Allen and Unwin Ltd., Lon- don. Dyer AF. 1963. The use of lactopropionic orcein in rapid squash methods for chromosome preparations. Stain. Technol. 38: 85–90. Ellison NW, Liston A, Steiner JJ, Williams WM, Tay- lor NL. 2006. Molecular phylogenetics of the clover genus (Trifolium–Leguminosae), Mol. Phylogenet. Evol. 39 (2): 688-705. Eroğlu HE. 2015. Which chromosomes are subtelocentric or acrocentric? A new karyotype symmetry/asymme- try index. Caryologia. 68: 239- 245. Eroğlu HE, Per S. 2016. Karyotype analysis of Zygoribat- ula cognata (Oudemans) (Acari: Oribatida: Oribatuli- dae). Turk Entomol. Derg. 40: 33-38. Excel 2007. Windows 8. Microsoft office. Falistocco E, Piccirilli M, Falcinelli M. 1987. Cytotax- onomy of Trifolium subterraneum L. Caryologia. 40: 123–130. Falistocco E, Marconi G, Falcinelli M. 2013. Comparative cytogenetic study on Trifolium subterraneum (2n = 16) and Trifolium israeliticum (2n = 12). Genome 56: 307–313. Gillett J M, Taylor NL. 2001. The World of Clovers. Iowa State University Press, Ames, Iowa, USA. Gladstones JS, Collins WJ.1983. Subterranean clover as a naturalized plant in Australia. J. Aust. Inst. Agric. Sci. 49: 191–202. Hayward MD, Breese EL. 1993. Population structure and variability. In: Hayward M.D., Bosemark N.O. and Romagosa I. (Eds), Plant breeding. Principles and Prospects. Chapman & Hall, London, pp. 16–29 Hezamzadeh Hijazi S M, Ziaeinasab M. 2006. Karyologi- cal study on some of species of Trifolium Genus in Iran. Iran. J. of Biol. 19 (3): 299 - 313. Hejazi H, Mohsen S, Nasab MZ. 2010. Cytotaxonomy of some Onobrychis (Fabaceae) species and populations in Iran. Caryologia. 63(1):18–31. Hutton EM, Peak JW. 1954. The Effect of autotetraploidy in five varieties of subterranean clover (Trifolium sub- terraneum L.) J.agrc .Res. 5: 356-364. Issolah R, Abdelguerfi A. 1999 a. Variability within 31 spontaneous populations of Trifolium scabrum L., nature of relations with factors of the site of origin. Cahiers options méditerranéennes, 39: 123-127. Issolah R, Abdelguerfi A. 1999b. Chromosome numbers within some spontaneous populations of Trifolium species in Algeria. Caryologia, 52: 151-154. Issolah R. 2006. Synthese de travaux réalises sur des popu- lations algeriennes de plusieurs espèces du genre Trifo- lium L. Workshop international « Diversité des Faba- cees fourrageres et de leurs symbiotes  : Applications Biotechnologiques, Agronomiques et Environnemen- tales ». Alger, Algérie. 19-22 Février 2006: 81-83. Issolah R, Benhizia H, Khalfallah N. 2006. Karyotype variation within some natural populations of Sulla (Hedysarum coronarium L., Fabaceae) in Algeria. Genet Resourc Crop. Evol. 53(8):1653– 1664. Issolah R, Khalfallah N. 2007. Analysis of the morpho- physiological variation within some Algerian popula- tions of Sulla (Hedysarum coronarium L.; Fabaceae). J. Biol. Sci., 7: 1082-1091. Issolah R, Tahar A, Derbal N, Zidoun F, Ait Meziane MZ, Oussadi A, Dehiles I, Bradai R, Ailane M, Terki N, Aziez F, Zouahra, A, Djellal L. 2012. Caracterisation ecologique de l’habitat naturel du Sulla (Fabaceae) dans le Nord-Est de l’Algérie. Rev. Ecol. (Terre et Vie), 67: 295-304. Issolah R, Bouazza L, Tahar A, Terki N, Dehiles I, Man- sour B, Nagoudi T. 2015. Caracterisation ecologique de l’habitat naturel du trefle souterrain (Trifolium subterraneum L., Fabaceae) dans le Nord- Est de l’Algerie. Rev. Ecol., 70: 182-193. Issolah R, Tahar A, Sadi S, Adjebi M, Alane F, Chelling- Siziani, Lebied M. 2016. Analysis of the behaviour and the chemical composition within populations of Trifolium subterraneum L. J.Biol.Sci., 16: 148-154. doi:10.3923/jb.2016.148.154. Jahier J, Chevre AM, Delourme R, Eber F, Tangay AM. 1992. Techniques de cytogénétique végétale. INRA. Paris, pp. 1-184. Kiran Y, Sahin A, Turkoglu I, Kursat M, Emre I. 2010. Kar- yology of seven Trifolium L. taxa growing in Turkey. Acta Biologica Cracoviensia Series Botanica, 52: 81-85. 104 Zahira Bouziane, Rachida Issolah, Ali Tahar Kliphuis E. 1962. Chromosome numbers of some annual Trifolium species, occuring in the Netherlands. Acta Bot. Neerland 11: 90-92. Katznelson J. 1974. Biological flora of Israel. 5. The sub- terranean clovers of Trifolium subsect. Calycomor- phum Katzn. Trifolium subterraneum L. (sensu lato). Isr J Bot 23: 69-108. Katznelson J, Morley F. 1965 a. Speciation processus in Trifolium suberraneum L. Israel J. Bot. 14:15-35. Levan A, Freda K, Sandberg AA 1964. Nomenclature for centromeric position on chromosomes. Hereditas. 52: 201-220. Marshall DR, Broue´ P. 1973. Outcrossing rates in Aus- tralian populations of subterranean clover. Aust. J. Agric. Res. 24: 863–867. Masson P. 1997. Des prairies de très longue durée avec des espèces annuelles à ressemis spontané : les pâtures à trèfle souterrain. Fourrages, 153: 139-146. Medoukali I, Bellil I, Khelifi D. 2015. Evaluation of Genetic Variability in Algerian Clover (Trifolium L.) Based on Morphological and Isozyme markers. Czech J. Genet. Plant Breed. 51(2): 50–61. Muñoz-Rodríguez AF. 1995. Trifolium sect. Paramesus and Sect. Trifoliastrum in the Iberian Peninsula II. Karyological study. Stud Bot. 14:103–128. Pecetti L, Piano E. 1998. Leaf size variation in subterra- nean clover (Trifolium subterraneum L. sensu lato). Gene.Res.crop. Evol. 45: 161–165 Pecetti L, Piano E. 2002. Variation of morphological and adaptive traits in subterranean clover populations from Sardinia (Italy). Gene.Res.crop. Evol. 00:0-1. Piano E. 1984. Preliminary observations on the structure and variability of Sardinia populations of subterrane- an clover. Genet. Agr. 38: 75–90. Piano E, Spanu F, Pecetti L. 1993. Structure and variation of subterranean clover populations from Sicily, Italy. Euphytica 68: 43–51. Piano E, Pecetti L. 1996. Selecting subterranean clover varieties for Mediterranean environments in Italy. In: Parente G., Frame J. and Orsi S. (Eds), Grassland and land use system. Proc 16th EGF Meet. ERSA, Gori- zia, pp. 283–286. Piluzza G, Pecetti L, Bullitta S, Piano E. 2005. Discrimi- nation among subterranean clover (Trifolium subter- raneum L. complex) genotypes using RAPD mark- ers. Genetic Resources and Crop Evolution 52, 193– 199. Pritchard A J. 1969. Chromosome numbers in some spe- cies of Trifolium. Austral. J. Agric. Res. 20: 883- 887. Quezel P, Santa S. 1962. Nouvelle flore de l’Algerie et des régions desertiques meridionales. Tome I. Ed. CNRS, France. Rossiter RC, Collins WJ. 1988 a. Genetic diversity in old subterranean clover (Trifolium subterraneum L.) pop- ulations in Western Australia. 1. Pastures sown ini- tially to the Dwalganup strain. Aust. J. Agric. Res. 39: 1051–1062. Rossiter RC, Collins WJ. 1988 b. Genetic diversity in old subterranean clover (Trifolium subterraneum L.) pop- ulations in Western Australia. 2. Pastures sown ini- tially to the Mount Barker strain. Aust. J. Agric. Res. 39: 1063–1074. Romero Zarco C. 1986. A new method for estimating karyotype asymmetry. Taxon 35:526-530. Rutland J.P. 1941. The menton catalogue. A list of chro- mosome numbers of British plants. Suppl. 1. New Phytol. 40: 210. Senn HA. 1938. Chromosome number relationship in the Leguminosae. Biblioth. Genet. 7: 175- 336. Siljak-Yak- ovlev S 1996. La dysploïdie et l’évolution du caryo- type. Bocconea. 5: 211–220. Siljak-Yakovlev S, Godelle B, Zoldos V, Vallès J, Garnatje T , Hidalgo O.2017. Evolutionary implications of het- erochromatin and rDNA in chromosome number and genome size changes during dysploidy: A case stydy in Reichardia genus. Plosone 12(8): e0182318. doi.org/ 10.1371/ journal. Pone .0182318. Singh R J. 2018. Practical manual on plant cytogenetics. CRC Press, Boca Raton, Boca Raton, FL 33487-2742 © 2018 by Taylor & Francis Group, LLC, pp 1-347. Taylor NL. (Editor). 1985. Clovers around the world. In Clover science and technology. American Society of Agronomy, Madison, Wisc. pp. 1–6. Uslu E. 2012. Karyology of nine Trifolium L. taxa from Turkey. Caryologia. 65(4): 304–310. Vizintin L, Javornik B, and Bohanec B. 2006. Genetic characterization of selected 15 Trifolium species as revealed by nuclear DNA content and ITS rDNA region analysis, 16 Plant. Sci. 170: 859-866. Yates JJ, Brittan NH. 1952. Cytological studies of subter- ranean clover (Trifolium subterraneum L.). Aust. J. Agric. Res. 3: 300–304. doi: 10.1071/ AR9520300. Weselxen H. 1928. Chromosome number and morphol- ogy in Trifolium. Univ .of Calif. Publ. Agric. Sci.2: 255-376. Zohary D, Katznelson J. 1958. Two species of subter- ranean clover in Israel. Aust. J. Bot. 6: 177–182. doi: 10.1071/BT9580177. Zohary M, Heller D. 1984. The genus Trifolium. Israel Academy of Sciences and Humanities. Jerusalem. pp. 1-606. 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