82 ACTA BOT. CROAT. 78 (1), 2019 Acta Bot. Croat. 78 (1), 82–90, 2019 CODEN: ABCRA 25 DOI 10.2478/botcro-2019-0010 ISSN 0365-0588 eISSN 1847-8476 A cytogenetic and pollen study of annual Medicago species from Soummam Valley (Northeastern of Algeria) Linda Djafri-Bouallag*, Malika Ourari, Mohamed Sahnoune Laboratory of Ecology and Environment, Department of Environment Biological Sciences, Faculty of Nature and Life Sciences, Université de Bejaia, Targa Ouzemmour, 06000 Bejaia, Algeria Abstract – This paper reports a cytogenetic study of eight Medicago L. species sampled from the Soummam Val- ley (northeastern Algeria). Chromosome numbers and meiosis irregularities during microsporogenesis were explored. Pollen viability rate and pollen size were also examined. The studied taxa are diploid and display biva- lent pairing and regular chromosome segregation during meiosis. Although meiosis appears regular, some anomalies were detected in relatively high cumulated rates (14.66%–26.14%). The most common meiotic abnor- mality examined here is related to cytomixis (from 14.66% in M. littoralis to 25.83% in M. laciniata). Other anomalies were also detected, including chromatic bridges, asynchronous divisions, micronuclei and multipolar cells. Consequently, the species exhibited varying percentages of pollen viability (from 70.11% in M. laciniata to 99.14% in M. littoralis). Pollen viability was negatively correlated with meiotic abnormalities (Pearson correla- tion coefficient R = −0.72, p = 0.043). The pollen grains were also heterogeneous in size. Medicago truncatula Gaertn. and M. laciniata (L.) Miller presented the most variable pollen size (relative standard deviation exceed- ing 19%). Medicago littoralis is distinguished from other species by possessing homogeneous and large sized pollen (relative standard deviation RSD = 6.73 %). The cytogenetic and pollen data provided by this study are discussed in the context of species systematics and in the perspective of genetic improvement. Keywords: chromosome numbers, Medicago, meiotic abnormalities, microsporogenesis, pollen grain area, pol- len viability, Soummam Valley * Corresponding author, e-mail: djafribouallaglinda@gmail.com Introduction Medicago L. is one of the largest genera in the Fabace- ae family with 87 species including shrubs and herbaceous plants (Small 2011). Medicago species are characterized by flowers exhibiting a unique explosive pollination mechanism (Small 2011). Taxonomically, this genus belongs to the Tri- folieae tribe and to the Trigonellinae sub-tribe. Basing their work on two nuclear genes (CNGC5, β-cop) and one mito- chondrial DNA sequence (rpS 14-cob), Maureira-Butler et al. (2008) estimated the divergence time between Medicago and its sister clade Trigonella as having been ca 15.9 million years ago. The ancestral Medicago forms originated from the western Asia and Mediterranean regions and are supposed to be perennial and preferentially allogamous (Lesins and Lesins 1979). The derived annual species would have differ- entiated six to seven million years ago (Prosperi et al. 1995) and now have highly variable distributions depending of hu- man interference. Annual species such as M. truncatula, M. polymorpha, M. orbicularis or M. minima are considered col- onizers (Olivieri et al. 1991, Prosperi et al. 1995) and they are widely distributed in Mediterranean-type climates. Oth- er species including M. coronata are rare or endemic and restricted to particular regions. With their ability to fix at- mospheric nitrogen, Medicago species are of real interest in improving soil fertility, in mainly arid and semiarid areas. These species are also used in crop rotation and pest man- agement plans to maintain a productive soil. In addition to their agricultural and ecological interests, Medicago species are used for animal nutrition (forage) and human consump- tion due to their high protein level. Medicago is recognized to be one of the most important genera of pasture plants in the world (Heyn 1963). Moreover, according to Small (2011), Medicago has potential for the production of biopharmaceu- ticals, bioplastics, cellulose and biofuel. Medicago truncatula is used as model plant for legume biology as a result of vari- MICROSPOROGENESIS AND POLLEN IN MEDICAGO L. ACTA BOT. CROAT. 78 (1), 2019 83 ous biological characteristics (Cannon et al. 2006) includ- ing diploidy, small genome size, self-pollination, and rapid reproductive cycle. The systematics of the genus is based on morphological and floral (Heyn 1963, Lesins and Lesins 1979, Small 2011) and biochemical (Classen et al. 1982, Jurzysta et al. 1992) traits. Pollen morphology (Small 2011) and interspecific hy- bridization (Simon 1965, Simon and Millington 1967, Les- ins and Lesins 1979) were also studied. Recent phylogenetic studies involved chloroplast (matK, trnK/matK), mitochon- drial (rpS14-cob) and nuclear (GA3ox1, ITS, ETS, CNGC5, β-cop) DNA sequences (Steele et al. 2010, Hu et al. 2014, de Sousa et al. 2016). However, homonyms and similarities still make species delineation and subspecies identification confusing. In Algeria, studies on this genus are scarce, being mainly conducted with regard to autoecology (Abdelguerfi et al. 1988b), physiology (Refoufi 1988, Amouri and Fyad- Lamèche 2012) morphology and biochemistry (Medoukali et al. 2015, Moulai and Fyad-Lamèche 2017), and Rhizobia- Medicago symbiotic interactions (Cheriet et al. 2014). The genus still remains under-investigated with regard to cytoge- netics, largely due to both small chromosome size and tech- nical difficulties encountered during staining procedures. Most of the studies conducted to date remain restricted to mitotic chromosome counts (Heyn 1963, Simon and Simon 1965, Lesins and Lesins 1979, Schlarbaum et al. 1984, Mari- ani et al. 1996). Very few karyological studies have involved Algerian Medicago populations (Abdelgeuerfi and Guitton- neau 1979, Abdelguerfi et al. 1988a, Fyad-Lamèche et al. 2016) and even fewer have considered the haploid phase of these plants, as is generally the case in the whole Medicago genus worldwide. Medicago species exhibit significant diver- sity in vegetative morphology as a result of interspecific hy- bridization (Heyn 1963). Meiotic analyses may provide valu- able information regarding the genetic or genomic causes of this diversity. Such studies could also provide significant insight into the cytological evolution of species that can be used in genetic improvement and in the conservation of ge- netic resources (Kumar and Singhal 2011). In the present study, we analyzed natural populations of eight annual Medicago species sampled from different local- ities of the Soummam Valley in northeastern Algeria. The main aim of this paper, in addition to chromosome counts, is to examine the male meiotic course and the subsequent effects of meiotic abnormalities encountered during differ- ent stages of meiosis I and II on pollen grain size and pol- len viability. Materials and methods Eight annual species, namely M. truncatula Gaertn., M. littoralis Rohde ex Lois., M. intertexta (L.) Miller, M. ciliaris (L.) Krocker, M. arabica (L.) Huds., M. polymorpha L., M. laciniata (L.) Miller and M. minima (L.) Bart. were sampled from wild populations of the Soummam Valley and neigh- borhoods (Northeastern Algeria). The geographical char- acteristics of the sampling sites are given in Tab. 1. Species identification was performed according to Lesins and Les- ins (1979) and voucher specimens were deposited at ENSA Herbarium of the botany department of the National High School of Agriculture in Algiers (dpt.botanique@ensa.dz; www.ensa.dz). Flower buds at different stages of development before an- thesis were collected, fixed in situ in Carnoy’s fixative (gla- cial acetic acid – chloroform – absolute ethanol, 1:3:6) and preserved at 4 °C in a refrigerator until used. Cytological analysis was performed on pollen mother cells (PMCs). Young flower buds were hydrolyzed in 1 N HCl at 60 °C in a water bath for three minutes. Under a binocular stereomicroscope, the removed immature anthers were immediately excised on a slide in a drop of 1% lacto- propionic orcein and squashed under a coverslip. All stages of meiosis were analyzed in order to determine chromosome numbers, to study meiotic behavior and to evaluate meiotic abnormality frequencies. About fifty individuals per popu- lation were harvested and around 1000 PMCs per species were scrutinized. Pollen viability was determined by staining mature flow- ers before anthesis using cotton blue in lactophenol (1% blue aniline in lactophenol) according to Mertens and Hammer- smith (1998). Flowers were dissected to free up pollen grains from the anther and mounted in a drop of cotton blue. The staining takes from 20 to 30 minutes. Viable pollen grains are stained uniformly dark blue, whereas defective grains are weakly stained and appear vacuolated, plasmolysed, and atypical in size and shape. Photomicrographs were taken us- ing a digital camera connected to an OPTIKA B-350 micro- scope. About 15 flowers were prepared for each population and up to 1000 pollen grains per flower were analyzed for evaluating pollen viability. An average pollen viability rate was estimated for each species. The pollen grain cell area was measured for 100 viable pollen grains per sample using Im- Tab. 1. Geographical characteristics of the sampling sites of the Algerian Medicago L. species. Taxa Sampling site Latitude, longitude Altitude (m) M. truncatula Gaertn. Vahloul 36°32'03,65"N, 4°35'46,23"E 238 M. littoralis Rohde ex Lois. Allaghane 36°23'42,76"N, 4°27'43,13"E 206 M. intertexta (L.) Miller Ideraken (Timzrit) 36°37'48,78"N, 4°46'29,97"E 75 M. ciliaris (L.) Krocker El Ghaba (Smaoune) 36°36'53,79"N, 4°50'30,86"E 305 M. arabica (L.) Huds. Taddart Tamokrante (Amizour) 36°37'6.30"N 4°59'57.80"E 511 M. polymorpha L. Allaghane 36°23'42,76"N 4°27'43,13"E 206 M. laciniata (L.) Miller Allaghane 36°23'42,76"N 4°27'43,13"E 206 M. minima (L.) Bart. Aftis 36°23'7.85"N 4°27'23.45"E 217 DJAFRI-BOUALLAG L., OURARI M., SAHNOUNE M. 84 ACTA BOT. CROAT. 78 (1), 2019 ageJ 1.48v Image Processing and Analysis in Java (Rasband 2016). Pearson correlation coefficient (R) between meiotic abnormalities frequency and pollen viability rate as well as the relative standard deviation (RSD) of pollen grain area were estimated for each species sample. The relative standard deviation formula is: RSD = 100 s / x-; where s is the sample standard deviation, x- is the sample mean. Fisher’s exact test was applied to compare frequencies of meiotic abnormali- ties and pollen viability rates; and the least significant differ- ence (LSD) test was used to check the homogeneity of pol- len viability rates and pollen grain area mean values across samples. Before running LSD test, raw data were divided by SD in order to homogenize their variances. Statistical treat- ments were performed using Statistica 8.0 (StatSoft 2008). Results Meiotic chromosome counts revealed that the M. trun- catula Gaertn., M. littoralis Rohde ex Lois., M. intertexta (L.) Miller, M. ciliaris (L.) Krocker, M. arabica (L.) Huds, M. la- ciniata (L.) Miller and M. minima (L.) Bart. populations were all diploid with a basic number x = 8 chromosomes. M. poly- morpha L. proved also diploid but the basic chromosome number was x = 7 (Figs. 1, a-h). Our samples showed no tetraploid or aneuploid cytotypes. The studied taxa displayed regular bivalent pairing and chromosome segregation at meiosis. The analysed PMCs show exclusively bivalent associations (Figs. 1, a-h). No multivalent or univalent associations were observed. Nu- merous anomalies in relatively high rates (≈ 25%) were de- tected, including cytomixis, the most common abnormality, chromatic bridges, asynchronous divisions, micronuclei and multipolar cells (Tab. 2). M. littoralis is distinguished by its lowest abnormality rate, restricted to cytomixis (14.66%). The samples show cytomixis rates varying from 14.66% in M. littoralis to 25.83% in M. laciniata (Tab. 2). M. arabica, Tab. 2. Chromosome numbers and meiotic abnormality rates in the Medicago L. species from Northeastern Algeria. Abbreviations: PMC – pollen mother cells; CM – cytomixis; CB – chromatic bridges; AD – asynchronous divisions; MN – micronuclei; MC – multipolar cells; CA – cumulative abnormality frequency; PV – pollen viability; SD – standard deviation; PGA – pollen grain area; RSD – relative standard deviation; N – number of individuals (for each species). For each column separately, values with different letters are significantly different as revealed by Fisher’s exact test (small letters) and LSD test (capital letters) at P = 0.05. Species Chromosome numbers and meiotic abnormalities Pollen data PMC 2n CM (%) CB (%) AD (%) MN (%) MC (%) CA (%) PV±SD (%) (N=15) PGA±SD (102 µm2) (N=100) PGA RSD (%) (N=100) M. truncatula 1170 16 18.29b 1.2c 0 1.88c 2.56c 23.93 0.79±0.16B 6.37±1.24C 19.56 M. littoralis 921 16 14.66a 0 0 0 0 14.66 0.99±0.01G 10.75±0.72F 6.73 M. intertexta 1137 16 15.04a 1.06c 1.32c 0.79b 0.79b 19.00 0.88±0.21B 7.01±0.99E 14.15 M. ciliaris 1035 16 14.78a 1.45c 0 0 0 16.23 0.96±0.05E 6.39±0.99D 15.58 M. arabica 1124 16 24.11cd 0 0 0 0 24.11 0.97±0.02F 6.81±0.95E 13.91 M. polymorpha 1323 14 22.45c 0.68b 1.13c 0 0 24.26 0.81±0.12C 5.01±0.53A 10.75 M. laciniata 1270 16 25.83d 0 0.31b 0 0 26.14 0.70±0.30A 7.22±1.37C 19.04 M. minima 1437 16 22.13c 0.63b 0.84c 0.42b 0 24.01 0.87±0.05D 6.34±0.73B 11.63 M. polymorpha, M. laciniata and M. minima (Sect. Leptospi- rae) exhibited cytomixis rate values exceeding 22.13%, sig- nificantly higher than those of the remaining species (Tab. 2). The occurrence of cytomixis in our samples was record- ed from early prophase I to microspore stage (Figs. 2, a-e). The chromatin transfer is performed either by one or by sev- eral strands in all the analysed species. It also occurs by di- rect contact between two or more cells (Figs. 2, a-b). An- other abnormality recorded in M. truncatula, M. intertexta, M. ciliaris, M. polymorpha and in M. minima was chromatic bridges at rates ranging from 0.63% in M. minima to 1.45% in M. ciliaris (Tab. 2, Figs. 2, f ). Asynchronous divisions were observed at anaphase II in M. intertexta, M. polymorpha, M. laciniata and in M. minima (Fig. 2, g). The rate recorded for this irregularity in the analysed samples varies from 0.31% in M. laciniata to 1.32% in M. intertexta (Tab. 2). Micronuclei were found in M. truncatula (1.88%), M. intertexta (0.79%), and in M. minima (0.42%) (Figs. 2, h-i). The last anomaly observed in the analysed taxa is the form of multipolar cells. In this case, the ultimate meiotic product corresponds to polyads instead of tetrads (Fig. 2, j-l). This abnormality was observed only in M. truncatula (2.56%) and M. intertexta (0.79%) (Tab. 2). Pollen viability rates were variable among and within populations (Fig. 3). While M. littoralis appears to be invari- able with a high percentage of viability (99.14%), M. laciniata shows significant variability ranging from 0 to 98.75% with a relatively low mean value (70.11%). Pollen viability rates seem to be inversely proportional to the cumulative rate of meiotic anomalies, since the Pearson coefficient correlation value is − 0.72 with p = 0.043. Pollen average area varies from 501.21 µm2 ± 53.86 in M. polymorpha to 1074.93 µm2 ± 72.32 in M. littoralis (Figs. 4, 5). M. truncatula and M. laciniata re- veal heterogeneous pollen grain area; with a calculated rela- tive standard deviation of pollen grain area (RSD) of around 19%. It is worth noting that M. littoralis is distinguished from MICROSPOROGENESIS AND POLLEN IN MEDICAGO L. ACTA BOT. CROAT. 78 (1), 2019 85 Fig. 1. Micromeiocyte metaphase plates in the Medicago L. species from Northeastern Algeria. a) M. truncatula (8 bivalents, 2n=2x=16); b) M. littoralis (8 bivalents, 2n = 2x = 16); c) M. intertexta (8 bivalents, 2n = 2x = 16); d) M. ciliaris (8 bivalents, 2n = 2x = 16); e) M. ara- bica (8 bivalents, 2n = 2x = 16); f ) M. laciniata (8 bivalents, 2n = 2x = 16); g) M. minima (8 bivalents, 2n = 2x = 16); h) M. polymorpha (7 bivalents, 2n = 2x = 14). Scale bars = 10 µm. Fig. 2. Abnormal meiotic behaviour in the studied Medicago L. species from Northeastern Algeria. a) Cytomixis between two micromeio- cytes at prophase I; b-d) cytomixis between several micromeiocytes at different stages; e) cytomixis between two microspores; f ) chromatic bridge (arrow); g) asynchronous division; h-i) micronucleus (arrow); j-l) multipolar cells. Scale bars = 10 µm. DJAFRI-BOUALLAG L., OURARI M., SAHNOUNE M. 86 ACTA BOT. CROAT. 78 (1), 2019 other species by the large size and the homogeneity of its pol- len (RSD = 6.73 %) (Fig. 5). Discussion In this article, chromosome numbers, meiotic behaviour and meiotic abnormalities were performed by analysis of di- viding pollen mother cells on natural populations in North- eastern Algeria. Pollen viability analyses were also carried out. The present results represent the first haploid phase cy- togenetic observations reported for Algerian populations of Medicago species. The encountered basic chromosome numbers (x = 8, x = 7) were previously reported in the genus Medicago by Heyn (1963), Simon (1965) and by Lesins and Lesins (1979). Ac- cording to Steele et al. (2010), the basic chromosome num- ber of x = 8 would be plesiomorphic in the Trifolieae tribe. The basic chromosome number of x = 7 was interpreted as an innovation deriving from x = 8 following chromosome rearrangements (Lesins and Lesins 1979). Based on the phy- logeny of plastid and nuclear sequences (trnK / matK, GA- 3ox1), the apomorphic base number x = 7 arose four times in the genus. Furthermore, this phylogeny showed that M. polymorpha and M. murex form a monophyletic group called 'Polymorpha clade' (Steele et al. 2010). As in M. murex, M. polymorpha genome would have arisen from a translocation of chromosome 8 on chromosome 3 by losing the centro- meric region and thus forming de novo a long satellite (Les- ins and Lesins 1979). The chromosome counts 2n = 2x = 14 and 2n = 2x = 16, obtained for the analysed species are in agreement with those made by several authors (Heyn 1963, Simon and Simon 1965, Lesins and Lesins 1979, Schlarbaum et al. 1984, Mariani et al. 1996). However, some authors re- ported deviating counts with different chromosome basic numbers. In fact, Humphries et al. (1978) and Vogt and Aparicio (1999) found 2n = 14 in M. truncatula Gaertn. In M. polymorpha, several authors (see Rani et al. 2012) found 2n = 16 with x = 8. In this taxon, the deviating count 2n = 16 is considered to be due to either species misidentification or presumably to the number over-estimation attributed to the presence of a pair of chromosomes with large satellites (Kamari et al. 1996). The absence of tetraploid or aneuploid cytotypes is consistent with the results obtained by the ma- jority of authors for these species (Simon and Simon 1965, Fig. 3. Pollen viability in the Medicago L. species from Northeastern Algeria. Max – maximum, M – mean; SE – standard error, Min – minimum. TRU – M. truncatula, LIT – M. littoralis, INT – M. intertexta, CIL – M. ciliaris, ARA – M. arabica, POL – M. poly- morpha, LAC – M. laciniata, MIN – M. minima. Different capital letters above bars denote significantly different results as revealed by LSD test (P = 0.05). Fig. 4. Pollen grains of the Medicago L. species from Northeastern Algeria stained by cotton blue in lactophenol. a) M. truncatula (het- erogeneous pollen); b) M. littoralis (homogeneous pollen); c) M. intertexta (homogeneous pollen); d) M. ciliaris (homogeneous pollen); e) M. arabica (homogeneous pollen); f ) M. polymorpha (homogeneous pollen); g) M. laciniata (heterogeneous pollen); h) M. minima (homogeneous pollen). Arrows indicate defective pollen grains. Scale bars = 10 µm. MICROSPOROGENESIS AND POLLEN IN MEDICAGO L. ACTA BOT. CROAT. 78 (1), 2019 87 Lesins and Lesins 1979, Schlarbaum et al. 1984, Mariani et al. 1996, Small 2011, Sadeghian and Hesamzadeh Hejazi 2014). However, polyploid levels were reported for two species (M. intertexta, M. polymorpha) by some authors. As reported by Fernandes and Santos De Fatima (Kamari et al. 1996) but not confirmed by Lesins and Lesins (1979), the individual of M. intertexta (L.) Miller from Portugal was tetraploid (2n = 32). Recently, in M. polymorpha L., a tetraploid level with 2n = 4x = 32 was reported from India by Rani et al. (2014) in a gen- eral survey on angiosperms. The few deviating chromosome counts should be verified since polyploidy is relatively rare among the annual species of Medicago. Indeed, according to Small (2011), only M. ovalis, M. rugosa and M. scutellata are certainly tetraploid. Aneuploid forms were also reported for M. ciliaris (L.) Krocker by some authors. Early on, in 1956, Heyn found in a Middle East accession of M. ciliaris a dip- loid chromosome number 2n = 18 (2n = 16 + 2B). However, Clement (1962) and Lesins and Lesins (1979) are reluctant to accept this possibility and consider the “supernumerary chromosomes” as detached satellites, not countable as com- plete chromosomes. Later, Abdelguerfi and Guittonneau (1979) in an Algerian population and Kamari et al. (1996) in a Tunisian population again found this count of 2n = 18. Analysis of meiotic chromosome behaviour in meta- phase I is essential to formulate a breeding strategy. Our sam- ples show exclusively bivalent associations. These chromo- some pairings were a prerequisite for a balanced segregation (Da Ines et al. 2014). They also generate new combinations of parental alleles that increase genetic diversity (Naranjo 2012). The natural hybridizations reported within the com- plex M. littoralis - M. truncatula - M. tornata (Heyn 1963, Lesins and Lesins 1979) appear to be absent in the analysed littoralis population since meiosis seems regular. The low rate of meiotic anomalies could explain its morphological ho- mogeneity and therefore, the studied population may corre- spond to the “pure” form of M. littoralis. On the other hand, although the studied M. truncatula population appears to be morphologically homogeneous, it showed numerous meiot- ic anomalies suggesting potential natural hybridization with some other related species, such as M. littoralis or M. tornata. The most common meiotic abnormality observed in all the studied samples is cytomixis. This is related to the migration of chromatin between meiocytes through cytoplasmic chan- nels. This chromatin transfer is under genetic control but can be influenced by environmental conditions, as pointed out by Ranjbar et al. (2011a) in Trigonella spruneriana Boiss. The analysed samples show variable cytomixis rates. In As- tragalus, thecytomixis rate was relatively low, varying from 0% to 3.82% (Ranjbar et al. 2011b). In contrast, this rate can reach 45% in Lotus (Sheidai and Jalilian 2006). In Medicago L., the few cytomictic studies reported have often dealt with polyploid taxa. The cytomixis rate recorded for the tetraploid species M. sativa reached 46% (Bellucci et al. 2003). Ranjbar et al. (2011b) reported that cytomixis in Astragalus seem to be more prevalent among polyploid taxa than in their dip- loid counterparts. In contrast, in Solanaceae, Singhal and Kumar (2008) have observed higher rates in diploids than in polyploids. In general, cytomictic activity is maximal dur- ing early prophase I due to the parallel transfer of trophic factors and signal molecules ensuring the synchronisation of meiosis (Mursalimov et al. 2013). In M. sativa, chroma- tin transfer is mostly detected in prophase I and rarely in later stages (Bellucci et al. 2003). In the analysed samples, the cytomixis was recorded from early prophase I to micro- spore stage. This report was similar to those observed in As- tragalus cyclophyllos (Ranjbar et al. 2011b), Lotus cornicula- tus (Jeelani et al. 2012) and in Nepeta govaniana (Kaur and Singhal 2014). The chromatin transfer performed either by one or by several strands and by direct contact between two or more cells in all the analysed species is also reported by several authors for other taxa such as Medicago sativa, Lo- tus corniculatus and Astragalus cyclophyllos (Bellucci et al. 2003, Sheidai and Jalilian 2006, Ranjbar et al. 2011b). The chromatic bridges recorded in M. truncatula, M. intertexta, M. ciliaris, M. polymorpha and in M. minima samples have also been reported in some species such as Onobrychis cho- rassanica (Ranjbar et al. 2010), Trigonella spruneriana (Ran- jbar et al. 2011a) and Calamagrostis emodensis (Saggoo and Kumari 2013) at variable frequencies. In the genus Passiflora (Passifloraceae), this rate can reach 26% (Souza and Pereira 2011). Chromatic bridges could be explained either by chro- mosomal rearrangement such as heterozygous paracentric inversions (Ruvalcaba-Ruiz and Rodríguez-Garay 2002, El- rod and Stansfield 2003) or by unrepaired double-strand breaks induced by Spo11 enzyme (Horlow and Doutriaux 2003). The observed asynchronous divisions in M. intertexta, M. polymorpha, M. laciniata and in M. minima samples re- sult from a mutation affecting the spindle “checkpoint” (Ris- so-Pascotto et al. 2003). In this case, only one cell of the dy- ad progresses in the cell cycle while the defective one stays blocked at metaphase II. The rate recorded for this irregu- larity was variable in the analysed samples. Variable rates are reported in many Leguminosae species. For reference, the rates vary from 0.41% to 0.79% in Trigonella and from 0 Fig. 5. Pollen grain area in the Medicago L. species from North- eastern Algeria. Max – maximum, M – mean; SE – standard error, Min – minimum. TRU – M. truncatula, LIT – M. littoralis, INT – M. intertexta, CIL – M. ciliaris, ARA – M. arabica, POL – M. pol- ymorpha, LAC – M. laciniata, MIN – M. minima. Different capital letters above bars denote significantly different results as revealed by LSD test (P = 0.05). DJAFRI-BOUALLAG L., OURARI M., SAHNOUNE M. 88 ACTA BOT. CROAT. 78 (1), 2019 to 24.98% in Astragalus (Ranjbar et al. 2011a, Ranjbar and Jahanian 2013, Jahanian and Sarpoushi 2014). Micronuclei, the other anomaly recorded in our samples, would be caused by the condensation of the chromatin originating from cyto- mixis or fragments of chromatic bridges. These micronuclei can then be removed by cytokinesis or by budding produc- ing thus sterile microcytes (Baptista-Giacomelli et al. 2000, Kiihl et al. 2011). A literature overview reveals that in angio- sperms the micronuclei rate varies according to the ploidy level. Thus, in diploid taxa, it ranges from 0% in Trigonella spruneriana to 1.66% in Astragalus ebenoides (Ranjbar et al. 2011a, Ranjbar and Jahanian 2013). However, in tetraploid taxa, this rate varies from 0% in Astragalus carmanicus to 5.52% in Vicia pallida (Singhal and Kaur 2011, Jahanian and Sarpoushi 2014). In the octoploid Alchornea triplinervia (Eu- phorbiaceae) tree species, this rate can reach 9.61% (Godoy et al. 2012). The observed polyads in M. truncatula and in M. intertexta were the result of several mutations such as ms28 and ms17, which affect the bipolar spindle formation (Albertsen and Phillips 1981, Golubovskaya and Distanova 1986). Another hypothesis, put forward by Caetano-Pereira and Pagliarini (2001), refers to the role of transposable ele- ments. Spindle orientation abnormalities have been reported in several species: in Agropyron cristatum, Carthamus tinc- torius, Fuchsia, Thunbergia mysorensis, Aloysia lycioides, Zea mays (Caetano-Pereira and Pagliarini 2001 and references therein). This abnormality is reported in many Legumino- sae species at variable rates (e.g. 0.13% in Onobrychis choras- sanica, 0.32% in Astragalus carmanicus, 10.98% in Vicia pal- lida) (Ranjbar et al. 2010, Singhal and Kaur 2011, Jahanian and Sarpoushi 2014). The pollen viability showed an important intra- and in- terspecific variation. A significant variation in viability rate was reported within populations and among species in Med- icago (Simon and Millington 1967, Lesins and Erac 1968, Singh and Lesins 1972). Sometimes, hybrids show rates similar to those of the parental taxa (Simon and Millington 1967). Our pollen samples also showed an important inter- specific variation in size. There are no data in the literature about pollen grain size in Medicago. However in other spe- cies, some authors, such as Kumar et al. (2010), explain this pollen size heterogeneity by the presence of meiotic abnor- malities. In conclusion, the eight annual Medicago populations in- vestigated in the Soummam Valley (Northeastern Algeria) showed regular meiosis with the presence of exclusively 8 bi- valents in M. truncatula Gaertn., M. littoralis Rohde ex Lois., M. intertexta (L.) Miller, M. ciliaris (L.) Krocker, M. arabi- ca (L.) Huds, M. laciniata (L.) Miller, M. minima (L.) and 7 bivalents in M. polymorpha L. at metaphase I revealing the diploid nature of these species. The observed meiotic abnor- malities related to cytomixis, multipolar cells, micronucleus, asynchronous divisions and chromatic bridges seem to be correlated with pollen size and pollen viability. 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