Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 89-98, 2020 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-618 Citation: H. Singh, J. Singh, P. Kumar, V. Kumar Singhal, B. Singh Kholia, L. Mohan Tewari (2020) Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii Hook.f. and Elymus nutans Griseb. (Triticeae: Poaceae) from Western Himalaya, India. Caryologia 73(2): 89-98. doi: 10.13128/caryolo- gia-618 Received: September 8, 2019 Accepted: April 3, 2020 Published: July 31, 2020 Copyright: © 2020 H. Singh, J. Singh, P. Kumar, V. Kumar Singhal, B. Singh Kholia, L. Mohan Tewari. This is an open access, peer-reviewed article published 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. Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii Hook.f. and Elymus nutans Griseb. (Triticeae: Poaceae) from Western Himalaya, India Harminder Singh2, Jaswant Singh1,*, Puneet Kumar2, Vijay Kumar Sin- ghal1, Bhupendra Singh Kholia2, Lalit Mohan Tewari3 1 Department of Botany, Punjabi University Patiala, India, 147002 2 Botanical Survey of India, Northern Regional Centre, Dehradun, India, 248195 3 Department of Botany, D.S.B. Campus, Kumaun University, Nainital, India, 263001 *Corresponding author. E-mail: jaswant_rs@pbi.ac.in Abstract. Present cytological study records existing chromosome number diversity, their male meiotic course and pollen fertility analysis in the two wheatgrass species of tribe Triticeae Dumort. (Poaceae) from Western Himalaya, India. Agropyron thomsonii Hook. f. is an endemic grass of alpine zones of Western Himalaya and Elymus nutans Griseb., a widely distributed grass in sub-alpine to glacial regions of Himalaya. The gametophytic chromosome number count of n=21 (Jadh Ganga Valley, Uttarkashi) is a pioneer count for A. thomsonii. During the male meiotic course of A. thomsonii, 14.04- 16.29% and 2.97-4.17% pollen mother cells, respectively at prophase-I and metaphase- I, observed to be involved in phenomenon of cytomixis. Seven accessions of E. nutans collected from Bhagirathi Valley and Jadh Ganga Valley of Uttarkashi district, and Pan- gi Valley of Chamba district, recorded with gametophytic chromosome number count of n=21 and record of 1B-chromosome in PUN61958 is a new record for the species. In three accessions 5.56-9.41% and 2.5% pollen mother cells at prophase-I and met- aphase-I, respectively were also noted with phenomenon of cytomixis. In addition to phenomenon of cytomixis, during meiotic course of both species pollen mother cells also depicted associated meiotic course irregularities viz. non-synchronous disjunction of bivalents, chromatin bridges, laggards, micronuclei in sporads and shrivelled micro- spores. These species are growing in cold climatic condition habitats. So, cold stress seems to be a preferential inductor for cytomixis and associated meiotic abnormalities in the gametic cells of stamens of A. thomsonii and E. nutans that ultimately leads to reduction in pollen fertility. Keywords: Himalayan grasses, cold stress, polyploidy, male meiosis, cytomixis and meiotic abnormalities, pollen sterility. INTRODUCTION Tribe Triticeae Dumort. (Poaceae) includes annual and perennial grass taxa, having large-sized chromosomes, comprising a polyploid complex of 90 Harminder Singh et al. 2x, 4x, 6x, 8x, 10x and 12x ploidy levels with a uniform base number of x=7 (Dewey 1984). The Members of tribe Triticeae have distribution to almost all floristic regions of the globe. In Himalaya, Agropyron Gaertn. and Ely- mus L. are prominent grass genera of temperate to alpine zone habitats, and an inflorescence is a spike that has one to many spikelets at each rachis node. Earlier, the genus Agropyron was represented by more than 33 species in Himalaya along with adjoining regions of similar ter- rain (Bor 1960), and the modern concept of Agropyron restricts it to only those species having keeled glumes and/or pectinately arranged spikelets (Cope 1982). So, the most of the species of Agropyron are now assigned to genus Elymus (Melderis 1978; Singh 1983; Karthikeyan et al. 1989). At present in Himalayan regions of India genus Agropyron is represented by a single species, i.e. A. thom- sonii Hook. f. and genus Elymus by 31 species (Singhal et al. 2018b). A. thomsonii Hook.f. [= E. thomsonii (Hook. F.) Melederis; = E. nayarii Karthik.] and Elymus nutans Griseb. are perennial and caespitose wild grasses growing in the high altitudinal regions of Himalaya (above 3000 m). Former is an endemic grass to Western Himalaya (Pusalkar and Singh 2012) and later species have wid- er in distribution that is growing in different ecological habitats of Bhutan, China, India (Himalaya), Iran, Japan, Mongolia, Nepal, Pakistan and Russia (Bor 1960; Lu 1993; Murti 2001; Chen and Zhu 2006; Pusalkar and Sin- gh 2012; Dvorský et al. 2018). Scrutiny of published cyto- logical data reveals that A. thomsonii is still unrecorded for its chromosome number. As germplasm of grasses remained in a central position towards the breeding pro- grams and germplasm enhancement to ensure the food and fodder demands. So to make their programs suc- cessful, there is a need to have complete knowledge and understanding about the genetic diversity of available germplasm (Kawano 2018). The present study is in a line of endeavour to explore the morphological diversity in the grasses from phytogeographically distinct and unex- plored regions of Western Himalaya. So, this study aimed to record the exact chromosome number in A. thomsonii and E. nutans along with to comment on the behaviour of pollen mother cells during the male meiotic course, and pollen fertility. We also try to correlate prevalence of cold conditions in natural habitat and the possible cause of cytomixis, associated meiotic abnormalities and reduc- tion in pollen fertility in the currently studied species. MATERIALS AND METHODS Wild plant accessions of A. thomsonii and E. nutans were collected for detailed male meiotic and pollen fer- tility studies from Bhagirathi and Jadh Ganga valley, Uttarkashi district, Uttarakhand and Pangi valley, Cham- ba, Himachal Pradesh. Young and unopened spikes, fixed in Carnoy’s fixative (Ethanol: Chloroform: Glacial acetic acid= 6: 3: 1). After 48 h, the materials were transferred to 70% ethanol and stored in a refrigerator. Meiocyte prepa- rations were made by squashing the developing anthers from the unopened florets in 1% acetocarmine. Chromo- some counts and meiotic course was studied from fresh- ly prepared slides having pollen mother cells (PMCs)/ meiocytes at diakinesis, metaphase-I (M-I), anaphase-I (A-I) and telophases (T-I, T-II). Apparent pollen fertil- ity was estimated through stainability tests by squash- ing the mature anthers in glycerol and 1% acetocarmine (1:1) mixture. Well-filled pollen grains with completely stained nuclei and cytoplasm were scored as fertile/via- ble, while partially stained and shrivelled ones as sterile/ non-viable. Good preparations of PMCs with well-spread bivalents/chromosomes, meiotic irregularities, and pol- len grains selected for photomicrographs using Nikon 80i Eclipse and Leica Qwin Digital Imaging System. Pollen size was measured through microscopy. Meiotically ana- lyzed accessions were identified by studying, in detail the floral characters and by consulting the floras viz. Grasses of Burma, Ceylon, India and Pakistan (Bor 1960), Flora of Pakistan-Poaceae (Cope 1982), Flora of Cold Deserts of Western Himalaya-Monocotyledons (Murti 2001) and Flora of Gangotri National Park, Western Himalaya (Pusalkar and Singh 2012). Identifications revalidated by comparing the specimens with the vouchers already sub- mitted by taxonomists in the Herbaria, Botanical Survey of India, Northern Circle, Dehra Dun (BSD) and avail- able specimens, facilitated by other herbaria as online resource (Global Plants1). Voucher specimens of cytologi- cally examined accessions deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN) and Herbarium Botanical Survey of India, Northern Cir- cle, Dehra Dun (BSD). RESULTS In the present exploration study, cytological investi- gations made on two accessions of A. thomsonii gathered from the alpine meadows of Jadh Ganga Valley and seven accessions of E. nutans from Bhagirathi Valley, Jadh Gan- ga Valley and Pangi Valley. Data regarding the name of taxon, sites of the collection with altitude, accession num- ber (BSD, PUN), gametic chromosome number, ploidy level, pollen fertility percentage tabulated in Table 1. 1 Global plants (https://plants.jstor.org/). 91Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii and Elymus nutans Two wild accessions of A. thomsonii collected from the glacial floristic area of Nelong, Jadh Ganga Valley, an eastern part of cold deserts of India. Current meiotically analyzed accessions of A. thomsonii are of dwarf habit (plant height 28 cm; spike length 9.5 cm; spikelet length 1.5 cm), which are hexaploid (6x; x=7) in nature with gametophytic chromosome number count of n=21, that confirmed from the presence of 21 bivalents in PMCs at M-I (Fig. 1A) and 21:21 chromosomes at A-I poles (Fig. 1B). During the meiotic course, 14.04-16.29% prophase- I PMCs (Fig. 1D, E) and 2.97-4.17% metaphase-I PMCs (Fig. 1F) observed with the phenomenon of cytomixis. In the majority of PMCs, chromatin migration occurred as deformed mass or partially deformed bivalents through cytoplasmic channels. Partial/complete migra- tion of chromatin material among neighbouring PMCs leads to formation of hypoploid, hyperploid and enucle- ated PMCs (Fig. 1D, E). Some PMCs observed in a state of pycnosis (Fig. 1H), and in few instances nucleolus pioneered migration of chromatin material occurs that noted as a presence of additional nucleolus in diakine- sis PMCs (Fig. 1C). Further during the meiotic course considerable number of PMCs at M-I, A-I/II and T-I/ II observed with associated meiotic abnormalities like, Table 1. Information on taxon, locality of collection with altitude, accession number/s, meiotic chromosome number, ploidy level, pollen fertility percentage of the cytologically investigated taxa. Sr. No Taxon Locality with altitude (m) Accession number/s Meiotic chromosome number (n) Ploidy level Pollen fertility (%) 1. Agropyron thomsonii Hook. f. (= Elymus nayarii Karthik.) Nelong I, Uttarkashi, Uttrakhand, 3450 PUN 61593 21 6x 60 Nelong CP, Uttarkashi, Uttrakhand, 3500 PUN 61594 21 6x 65 2. Elymus nutans Griseb. Sural, Chamba, HInmachal Pradesh, 3008 BSD 1181196 21 6x 97 Nelong I, Uttarkashi, Uttrakhand, 3450 PUN 61955 21 6x 95 Nelong CP, Uttarkashi, Uttrakhand, 3500 PUN 61956 21 6x 90 Bhojwasa, Uttarkashi, Uttrakhand, 3700 PUN 61022 21 6x 98 Bhojwasa, Uttarkashi, Uttrakhand, 3750 PUN 61015 21 6x 98 Bhojwasa, Uttarkashi, Uttrakhand, 3800 PUN 61957 21 6x 95 Gaumukh, Uttarkashi, Uttrakhand, 3900 PUN 61958 21+0-1B 6x 95 Table 2. Data on the percentage of PMCs involved in chromatin transfer, abnormal sporads and pollen size of Agropyron thomsonii and Elymus nutans. Taxon Accession number (PUN) PMCs involved in cytomixis (%) Out of the plate bivalents/ chromosomes (%) Laggards/ Chromatin bridges (%) Sporads (%) Pollen Size (µm) Dyads with Tetrads with Prophase-I M-I M-I A-I A-I/T-I A-II/T-II Micronu- clei Micronu- clei 1-2 Shrivelled microspores Agropyron thomsonii Hook. f. 61593 14.04 (40/285) 2.97 (4/135) _ _ 12.30 (8/65) 15.00 (6/40) 10.00 (2/20) 10.29 (7/68) 4.41 (3/68) Small: 29.31 x 29.31 Medium: 36.80 x 36.80 Large: 42.68 x 42.68 61594 16.29 (57/350) 4.17 (10/240) 6.25 (15/240) 8.57 (6/70) 17.14 (12/70) 14.29 (8/56) 13.64 (3/22) 8.89 (8/90) 6.67 (6/90) Elymus nutans Griseb. 61955 9.41 (8/85) - - - 6.67 (4/60) - - - - Small: 27.86 x 27.86 Medium: 37.47 x 37.47 Large: 41.32 x 41.32 61956 8.00 (6/75) 2.50 (2/80) 12.50 (10/80) - 16.00 (8/50) - - 4.00 (2/50) - 61958 5.56 (5/90) - - - 11.43 (4/35) - - - - 92 Harminder Singh et al. Figure 1. Male meiosis in Agropyron thomsonii: (A) M-I PMCs with 21 bivalents (B) A-I PMC with 21:21 chromosomes at each pole; (B) PMCs involved in chromatin migration at P I (arrowed); (C) a diakinesis PMC possessing relatively small sized additional nucleolus PMCs; (D) prophase-I PMCs depicting phenomenon of cytomixis (arrowed); (E) diakinesis PMCs with chromatin migration and depicting forma- tion of hyperploid and enucleated PMC (arrowed); (F) M-I PMC involved in chromatin migration (arrowed); (G) A-I PMC with non-syn- chronous dysjunction of bivalents depicted by univalents and bivalent bridges (arrowed); (H) a PMC depicting abnormal spindle and state of pycnosis with fragments (arrowed); (I) A-I PMC with several laggards (arrowed); (J) early T-I PMC with dicentric bridge, fragments and laggards (arrowed); (K) T-II PMC as dyad with on subunit with laggard (arrowed); (L) T-I PMCs with miocronuclei at one pole (arrowed); (M) dyad subunits with micronuclei (arrowed); (N) tetrad microspore subunits with micronuclei (arrowed); (O) A tetrad with two shriv- elled microspore units (arrowed); (P, Q) fully stained fertile, shrivelled and unstained as sterile pollen grains. Scale bar=10μm. 93Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii and Elymus nutans chromatin bridges (Fig. 1G) and laggards/ fragments (Fig. 1I, J, K, L), and micronuclei in sporads (Figs. 1M, N), collectively depicting a syndrome of errors occurred during the meiotic course (Table 2). During, the micro- sporogenesis two types of sporads were noted, first hav- ing all normal microspores and another type with shriv- elled microspores (Fig. 1O). These meiotic abnormalities during meiotic course have resulted in low pollen fertil- ity (60-65%) and formation of heterogeneous sized pol- len grains in these accessions (Fig. 1P, Q). Seven accessions of E. nutans collected from sub- alpine and glacial vegetation zones of three valleys not- ed to have variable plant height in their natural habitat, viz. dwarf (PUN 60482, 61956: plant height, 9-15 cm; spike length, 3-5.5 cm; spikelet length, 10-12.5 mm; awn length, 7-10 mm), intermediate (PUN 61955: plant height, 25 cm; spike length, 8-8.5 cm; spikelet length, 9-10 mm; awn length, 15-17.5 mm) and tall (PUN 61022, 61957-58, BSD 1181196: plant height, 50-65 cm; spike length, 15-18 cm; spikelet length 30-45 mm; awn length, 25-45 mm). These accessions unequivocally have a gametic chromosome number count of n=21, confirmed from the presence of 21 bivalents in PMCs at M-I (Fig. 2A). 1B-chromosome was also observed in few pollen Figure 2. Male meiosis in Elymus nutans: (A) M-I PMC with equal-sized 21 bivalents; (B) M-I PMC with 1B-chromosome associated with A-bivalent (arrowed); (C) M-I PMC with independent 1B-chromosome (arrowed); (D) M-I PMC with chromatin stickiness; (E) M-I PMC depicting the phenomenon of cytomixis (arrowed); (F) M-I PMC with migrated chromatin material as pycnotic mass (arrowed); (G) A-I PMC with non-synchronous dysjunction of bivalents (arrowed); (H) heterogeneous sized pollen grains (arrowed); (I, J) fully stained fertile, partially stained and unstained as sterile pollen grains. Scale bar=10μm. 94 Harminder Singh et al. mother cells at metaphase-I (Fig. 2B, C) of an accession scored from Gaumukh. Majority of the pollen mother cells during meiotic course observed with normal mei- otic behaviour except some percentage of pollen moth- er cells of three accessions (PUN 61955, 61956, 61958) noted with the phenomenon of cytomixis at diakinesis and M-I (Fig. 2E), chromatin stickiness (Fig. 2D), with migrated chromatin material masses (Fig. 2F), late dys- junction of bivalents, chromatin bridges (Fig. 2G), and formation of variable-sized pollen grains (Fig. 2H) in these accessions (Table 2). Due to a low ratio of mei- otically abnormal pollen mother cells to normal pollen mother cells in the studied accessions, recorded high percentage of pollen fertility (90-98%) (Fig. 2I, J). DISCUSSION Chromosome number and ploidy A. thomsonii is an endemic grass to Western Hima- laya, and the analyzed gametophytic chromosome num- ber count of 2n=42 is a first record for the species. In the case of E. nutans record of 1B-chromosome is also a first record in the species. Earlier, the first chromosome num- ber count of 2n=42 for E. nutans has recorded by Gohil and Koul (1985) from Fotula, a cold desert region of Ladakh, Western Himalaya, India and from Sichuan and Qinghai regions of China by Liu (1985). Reports of chro- mosome number, 2n=42 are also known from Pakistan Himalaya (Salomon et al. 1988) and other distant regions of China (Lu et al. 1990; Lu 1993, 1994; Lu and Bothmer 1993; Chen et al. 2009, 2013; Dou et al. 2009, 2017; Yan et al. 2009, 2010). As tribe Triteceae has basic chromosome number, x=7 and species of genus Agropyron have three ploidy levels (2n=2x, 4x, 6x) with only P genome, but the species of genus Elymus have four ploidy levels (2n=2x, 4x, 6x, 8x) with genome constitution of ‘S/St’, ‘H’ and ‘Y’ in different combinations (Dewey 1984). So, A. thomsonii chromosomally exists at hexaploid ploidy level, and the genome is autopolyploid in nature. Similarly, E. nutans is also a hexaploid species, but its genome constitution is of strict allopolyploid in nature (Lu 1993, 1994). Both the species are hexaploid and during the meiotic course in pollen mother cells, noted with formation of regular bivalents without any indication of multivalents, display- ing the diploid like meiosis. The diploid like meiosis is probably the result of the selection of mutations in loci involved in chromosome pairing and chiasma formation facilitated by parental genome chromosomes (McGuire and Dvořák 1982). Analysis of synaptonemal-complex in allopolyploid grasses reveals the existence of diplo- dizing genetic system as in Festuca spp. (Thomas and Humphreys 1991, Thomas and Thomas 1993), Triticum and Aegilops spp. (Holm 1986, Holm and Wang 1988, Cuñado et al. 1996 a, b, c, Cuñado and Santos 1999) that works through the restriction of synapsis to homologous chromosomes and suppression of crossing over among non-homologous chromosomes. Cytomixis, meiotic abnormalities, and its consequent effects The abnormal meiotic course often leads to distur- bances in microsporogenesis henceforth resulting in pol- len malformation or sterility and furthermore negatively influence the reproductive success of the species in the wild (Lattoo et al. 2006; Kumar and Singhal 2008; Sin- ghal and Kumar 2008; Kumar 2010). Meiotic abnormali- ties in natural conditions act as agents of polyploidy in plants (Mason and Pires 2015). The deviation from the normal meiotic course may result in unreduced gamete formation. Such male meiotic studies in wheat grasses (Agropyron and Elymus) can be of great importance in discovering wild relatives of cultivated crops. In the cur- rent study the phenomenon of cytomixis observed pre- dominantly during the first meiotic division in both species. Cytomixis is a natural phenomenon, involving transfer of chromatin material mainly in proximate mei- ocytes/cells of plants through cytomictic channels (Mur- salimov and Deineko 2017). Körnicke (1901) was the first one to observe cytomixis in meiocytes of Crocus sativus. However it was Gates (1911) who coined the term ‘Cyto- myxis’ (nowadays ‘Cytomixis’) and was defined it as the chromatin extrusion process which is a natural part of meiosis. Nowadays, cytologists based on the observa- tions with modern tools employed in plant sciences consider it, as cell to cell communication (Kravets et al. 2017); a biological process (Sidorchuk et al. 2016) that takes place without any damage to migrated chromatin material (Mursalimov et al. 2018) and as an additional putative genetic recombination process (Mursalimov and Deineko 2017). So, cytomixis is a natural meiotic aberra- tion of potential evolutionary significance (Singhal et al. 2018a). In present study, during male meiotic course of A. thomsonii, 14.04-16.29% and 2.97-4.17% PMCs at pro- phase-I and M-I, respectively depicted the phenomenon of chromatin transfer. Similarly, 5.56-9.41% and 2.5% PMCs of E. nutans at prophase-I and M-I, respectively showed the phe- nomenon of chromatin transfer among proximate pol- len mother cells. Cytomixis was observed in the early stages of the first meiotic division only. High frequency of cytomixis during the first meiotic division results in high sterility and formation of heterogeneous sized pol- len grains (De and Sharma 1983; Consolaro and Pagli- 95Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii and Elymus nutans arini 1995; de Souza and Pagliarini 1997; Pierozzi and Benatti 1998; Singhal and Kumar 2008). The chromatin transfer involves partial or complete migration of nuclear contents, which results into origin of hyper, hypoploid and enucleated meiocytes. Also, few PMCs during chro- matin transfer along with/without migrated chromatin material also acquire additional nucleoli (Kumar 2010). Kumar and Singhal (2016) while enlisting 31 species possessing additional nucleoli suggested that in major- ity of the species with an additional nucleoli are result- ed due to phenomenon of cytomixis. PMC’s at M-I, A-I/ II and T-I/II observed with other meiotic abnormalities, depict a syndrome of errors occurred during the meiotic course. Differential extent and intensity of pairing/cross- ing over among non-sister chromatids of homologous chromosomes results to non-synchronous dysjunction of bivalents during meiosis-I, and formation of chroma- tin bridges occurs (Kumar 2010). Crossing over within paracentric inversion pairing loops or U-type exchange between non-sister chromatids during paring creates dicentric bivalent bridges and acentric fragments con- figurations observed at early T-I. Formation of dicen- tric bridge and laggards/fragment as meiotic configu- rations during meiotic course appears as a meiotic syn- drome that depicts reduced control over meiotic course (Jones and Brumpton 1971). Presence of large number of laggards, possibly due to abnormal spindle, disturbed cytoskeleton and other cellular changes. These chromatin fragments/laggards lead to formation of micronuclei in sporads. As, abnormal chromosome segregation, presence of micronuclei and reduced pollen fertility results due to formation of multiple spindles at meiosis-I (Vasek 1962). In Jadh Ganga Valley, cold conditions are prevalent and both the accessions of A. thomsonii growing in this region are prone to extreme temperature fluctuations i.e. short warm days and long cold nights. Male reproductive organs and their development are extremely sensitive to cold stress (Liu et al. 2019). So, during the microsporo- genesis, formation of shrivelled microspores noted in tetrads that may be due to cold stress-induced abnormal development mediated through malnutrition. Cold stress disrupts stamen development and prominently interferes with tapetum programmed cell death, which is crucial for progression of normal meiotic course and develop- ment of microspores to pollens (Oliver et al. 2005, 2007; Sharma and Nayyar 2014; Liu et al. 2019). The phenomenon of cytomixis and associated mei- otic abnormalities in A. thomsonii and E. nutans seems to be due to low-temperature stress conditions that are prevalent in the region and have potential to alter the expression of certain alleles controlling the vital steps of meiosis. Previous cytological studies of Bedi (1990), Bel- lucci et al. (2003), Malallah and Attia (2003), Kumar et al. (2010, 2011, 2014, 2017), Singhal and Kumar (2008, 2010) and Mandal et al. (2013) are also in the view that cytomixis is under direct control of genetic factors. Pollen development is a complex process regulated at different genetic levels. Mutants showing abnormal pollen development can be of beneficial help in under- standing the process of pollen development (Sheila 1993). Cy tomixis, coupled with associated meiotic abnormalities, leads to the formation of genetically vari- able pollen grains, affecting pollen size and fertility. Pol- len size variation depends upon the extent of chromatin material, or amount of DNA (Stebbins 1971) possessed/ lost by meiocytes during chromatin transfer, and pres- ently somewhat pollen size variation was observed. Effects of cytomixis on meiotic course, pollen size, and pollen fertility have previously been reported in grasses viz. Agropyron cristatum (Bauchan et al. 1987), Alope- curus arundinaceus (Koul 1990), Brachiaria humidicola (Boldrini et al. 2006), Elymus semicostatus (Singhal et al. 2018c), Urochloa panicoides (Basavaiah and Murthy 1987), and many other flowering plants viz. Vicia faba (Bhat et al. 2006), Nicotiana tabacum (Mursalimov and Deineko 2011), Chlorophytum borivilianum (Mandal and Nandi 2017), Anchusa spp. (Keshavarzi et al. 2017), Thalictrum cultratum (Kumar et al. 2017) and Clematis ladakhiana (Khan et al. 2018). In the end, it may be summarized that individu- als of A. thomsonii and E. nutans of cold desert habitat are of dwarf habit and whereas of other alpine regions of western Himalaya are taller. Respectively, both the species unequivocally noted with chromosome number count of 2n=42 and 2n=42+0-1B with 6x ploidy level are pioneer counts for the species. In natural habitats of these species cold climatic conditions are prevalent and seem these species are differentially affected by cold stress, which is a potential inducer for abnormal mei- otic course and sporads. The phenomenon of cytomixis and associated meiotic abnormalities observed in pollen mother cells of A. thomsonii and E. nutans affects the pollen size and pollen fertility in the species. ACKNOWLEDGMENTS The authors wish to thank the University Grants Commission (UGC), New Delhi for financial sup- port under DRS, SAP-I, II, III, ASIST program and DSA-I schemes; Department of Biotechnology (DBT), New Delhi under DBT-IPLS project [BT/PR4548/ INF/22/146/2012]; for award of UGC-BSR-Fellowship [Award letter no. 15610/Research/03/06/2015] and Sci- 96 Harminder Singh et al. ence and Engineering Research Board-Department of Science and Technology (SERB-DST) Start-Up Research Grant (Young Scientists) [vide SERB sanction No. SB/ YS/LS-182/2014 dated 8/9/2015]. Thanks are also due to the Head, Department of Botany, Punjabi University Patiala, and Director, Botanical Survey of India, Kol- kata and Head of Office, NRC, Dehradun, for providing necessary laboratory, Herbarium (PUN, BSD), Inter- net facilities and Co-ordinator, University Sophisticat- ed Instrumentation Centre for kind permission to use imaging system facility. REFERENCES Basavaiah, Murthy TCS. 1987. Cytomixis in pollen moth- er cells of Urochloa panicoides P. Beauv. (Poaceae). Cytologia. 52:69–74. Bauchan GR, Linkous LCW, Tai W. 1987. Cytomixis in Agropyron cristatum. Genome. 29:765–769. Bedi YS. 1990. Cytomixis in woody species. In: Proc Indi- an Nat Sci Acad Plant Sci. 100:23–238. Bellucci M, Roscini C, Mariani A. 2003. Cytomixis in pollen mother cells of Medicago sativa L.  J Heredit. 94:512–516. Bhat TA, Parveen S, Khan AH. 2006. MMS-induced cyto- mixis in pollen mother cells of broad bean (Vicia faba L.). Turk J Bot. 30:273–279. Boldrini KR, Pagliarini MS, do Valle CB. 2006. Cell fusion and cytomixis during microsporogenesis in Brachiaria humidicola (Poaceae).  S Af J Bot. 72:478– 481. Bor NL. 1960. Grasses of Burma, Ceylon, India and Paki- stan (excluding Bambuseae). London: Pergamon Press. Chen SL, Zhu GH. 2006. Elymus L.  Flora of China (Poaceae). Beijing and St. Louis, MO: Science Press and Missouri Botanical Garden. 22:400–429. Chen SY, Ma X, Zhang XQ, Chen ZH. 2009. Genetic vari- ation and geographical divergence in Elymus nutans Griseb. (Poaceae: Triticeae) from West China.  Bioch Syst Ecol. 37:716–722. Chen S, Zhang X, Ma X, Huang L. 2013. Assessment of genetic diversity and differentiation of Elymus nutans indigenous to Qinghai-Tibet Plateau using simple sequence repeats markers.  Can J Pl Sci. 93:1089– 1096. Consolaro MEL, Pagliarini MS. 1995. Cytomixis in pollen mother cells of Centella asiatica L. Nucleus. 38:80–85. Cope TA. 1982. Poaceae. In: Nasir E, Ali SI. (Eds.). Flora of Pakistan. No. 143. Pakistan: Department of Bota- ny, University of Karachi. Cuñado N, Santos JL. 1999. On the diploidization mech- anism of the genus Aegilops: Meiotic behaviour of interspecific hybrids. Theor. Appl. Genet. 99:1080– 1086. Cuñado N, Callejas S, García MJ, Fernández A, Santos JL. 1996a. Chromosome pairing in the allotetraploid Aegilops biuncialis and a triploid intergeneric hybrid. Genome 39:664–670. Cuñado N, Callejas S, García MJ, Fernández A, Santos JL. 1996b. The pattern of zygotene and pachytene pair- ing in allotetraploid Aegilops species sharing the U genome. Theor. Appl. Genet. 93:1152–1155. Cuñado N, García MJ, Callejas S, Fernández A, Santos JL. 1996c. The pattern of zygotene and pachytene pair- ing in allotetraploid Aegilops species sharing the D genome. Theor. Appl. Genet. 93:1175–1179. Dewey DR. 1984. The genomic system of classification as a guide to intergeneric hybridization with the peren- nial Triticeae. In: Gustafson JP. (Ed.). Gene manipu- lation in plant improvement, 16th Stadler Genetics Symposium. Boston: Springer. pp. 209–279. De M, Sharma AK. 1983. Cytomixis in pollen mother cells of an apomictic ornamental Ervatamia divari- cata (Linn.) Alston. Cytologia. 48:201–207. Dou QW, Chen ZG, Liu YA, Tsujimoto H. 2009. High frequency of karyotype variation revealed by sequen- tial FISH and GISH in plateau perennial grass forage Elymus nutans. Breed Sci. 59:651–656. Dou Q, Yu F, Li Y, Zhao Y, Liu R. 2017. High molecular karyotype variation revealed in indigenous Elymus nutans in the Qinghai Plateau. Pl Divers. 39:117–122. Dvorský M, Klimes L, Dolezal J, Wild J, Dickoré BW. 2018. A Field Guide to the Flora of Ladakh. Praha: Academia. Gates RR. 1911. Pollen formation in Oenothera gigas. Ann Bot. 25:909–940. Gohil RN, Koul KK. 1985. In: Bir SS. (ed.). SOCGI plant chromosome number reports. J Cytol Genet. 21:155. Holm PB. 1986. Chromosome pairing and chiasma for- mation in allohexaploid wheat, Triticum aestivum, analysed by spreading of meiotic nuclei. Carlsberg Res. Commun. 51:239–294. Holm PB, Wang X. 1988. The effect of chromosome SB on synapsis and chiasma formation in wheat, Triti- cum aestivum, cv. Chinese Spring. Carlsberg Res. Commun. 53:191–208. Jones GH, Brumpton RJ. 1971. Sister and non-sister chromatid U-type exchange in rye meiosis.  Chromo- soma. 33:115–128. Karthikeyan S, Jain SK, Nayar MP, Sanjappa M. 1989. Florae Indicae Enumeratio Monocotyledonae. Cal- cutta: Botanical Survey of India. 97Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii and Elymus nutans Kawano S. 2018. Karyotype and chromosome behavior analyses in three regions of the Indomalayan Realm. Cytologia 83:223–228. Keshavarzi M, Nasrollahi F, Sheidai M. 2017. Cytogenet- ic study of the genus Anchusa L. (Boraginaceae) in Iran. Caryologia. 70:57–365. Khan NA, Singhal VK, Gupta RC. 2018. First record of chromosome count and cytomixis in an endemic species of Clematis ladakhiana Grey-Wilson (Ranun- culaceae) from cold deserts of Jammu and Kashmir. Caryologia 71:233–237. Körnicke M. 1901. About change of location of cell nuclei. Meeting Reports of Society of Lower Rhine for Nature and Medicine (Bonn). pp. 14–25. (in Ger- men) Koul KK. 1990. Cytomixis in pollen mother cells of Alo- pecurus arundinaceus Poir. Cytologia. 55:169–173. Kravets EA, Yemets AI, Blume YB. 2017. Cytoskeleton and nucleoskeleton involvement in processes of cyto- mixis in plants.  Cell Biol. Int. [doi:10.1002/cbin. 10842] Kumar P. 2010. Exploration of cytomorphological diver- sity in the members of Polypetalae from Lahaul-Spiti and adjoining areas. PhD Thesis. India: Punjabi Uni- versity Patiala, Punjab. Kumar P, Singhal VK. 2008. Cytology of Caltha palustris L. (Ranunculaceae) from cold regions of Western Himalayas. Cytologia. 73:137–147. Kumar P, Singhal VK. 2016. Nucleoli migration coupled with cytomixis. Biologia. 71:651–659. Kumar P, Singhal VK, Kaur D, Kaur S. 2010. Cytomixis and associated meiotic abnormalities affecting pollen fertility in Clematis orientalis. Biol Plant. 54:181–184. Kumar P, Singhal VK, Rana PK, Kaur S, Kaur D. 2011. Cytology of Ranunculus laetus Wall. ex Royle from cold desert regions and adjoining hills of North-west Himalayas. Caryologia. 64:25–32. Kumar P, Rana PK, Singhal VK, Gupta RC. 2014. Cyto- geography and phenomenon of cytomixis in Silene vulgaris from cold regions of Northwest Himalayas (India). Plant Syst Evol. 300:831–842. Kumar P, Singhal VK, Srivastava SK. 2017. First detection of cytomixis and its consequences in  Thalictrum cul- tratum  Wall. (Ranunculaceae).  Cytol Genet. 51:384– 390. Lattoo SK, Khan S, Bamotra S, Dhar AK. 2006. Cytomixis impairs meiosis and influences reproductive success in Chlorophytum comosum (Thunb.) Jacq. – an addi- tional strategy and possible implications. J Biosci. 31:629–637. Liu B, Mo WJ, Zhang D, De Storme N, Geelen D. 2019. Cold influences male reproductive development in plants: A hazard to fertility, but a window for evolu- tion. Plant Cell Physiol. 60:7–18. Liu Y. 1985. Studies on the karyotypes of 11 species of Elymus from China. J Wuh Bot Res. 3:325–330. Lu BR. 1993. Meiotic studies of Elymus nutans and E. jac- quemontii (Poaceae, Triticeae) and their hybrids with Pseudoroegneria spicata and seventeen Elymus spe- cies. Plant Syst Evol. 186:193–211. Lu BR. 1994. Morphological identification of Elymus sibiricus, E. nutans, and E. burchan-buddae, and their genomic relationships.  Acta Phytotax Sinic. (Chi- na). 32:504–513. Lu B, Bothmer, RV. 1993. Meiotic analysis of Elymus caucasicus, E. longearistatus, and their interspecific hybrids with twenty-three Elymus species (Triticeae, Poaceae). Plant Syst Evol. 185:35–53. Lu BR, Yan J, Yang JI. 1990. Cytological observation on Triticeae materials from Xinjiang, Qinghai and Sichuan. Acta Bot. Yunn. (China). 12:57–66. Malallah GA, Attia TA. 2003. Cytomixis and its possible evolutionary role in a Kuwait population of Diplotax- is harra (Boraginaceae). Bot J Linn Soc. 143:169–175. Mandal GD, Nandi AK, Das AB. 2013. Cytomixis and associated meiotic abnormalities in pollen mother cells of Chlorophytum tuberosum (Roxb.) Baker. Cyt- ologia. 78:157–162. Mandal GD, Nandi AK. 2017. Cytomixis with associated chromosomal anomalies and the reproduction of Chlorophytum borivilianum Santapau & RR Fern. Tai- wania. 62:211–215. Mason AS, Pires JC. 2015. Unreduced gametes: meiotic mishap or evolutionary mechanism? Trends Genet. 31:5–10. McGuire PE, Dvořák J. 1982. Genetic regulation of heter- ogenetic chromosome pairing in polyploid species of the genus Triticum sensu lato. Canad. J. Genet. Cytol. 24:57–82. Melderis A. 1978. Elymus. In: Hara H, Chater AO, Wil- liams LHJ. (eds.) An enumeration of the flowering plants of Nepal. London: British Museum (Natural History). Mursalimov SR, Deineko EV. 2011. An ultrastruc- tural study of cytomixis in tobacco pollen mother cells. Protoplasma. 248:717–724. Mursalimov S, Deineko E. 2017. Cytomixis in tobacco microsporogenesis: are there any genome parts pre- disposed to migration? Protoplasma. 254:1379–1384. Mursalimov S, Zagorskaya A, Deineko E. 2018. Evalu- ation of DNA damage in tobacco male meiocytes involved in cytomixis using comet assay. Protoplas- ma.  255:413–417. [https://doi.org/10.1007/s00709- 017-1144-6] 98 Harminder Singh et al. Murti SK. 2001. Flora of cold deserts of Western Hima- laya. Vol I. Monocotyledons. Kolkata: Botanical Sur- vey of India. Oliver SN, Dennis ES, Dolferus R. 2007. ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility 90 in rice. Plant Cell Physiol. 48:1319–1330. Oliver SN, Van Dongen JT, Alfred SC, Mamun EA, Zhao X, Saini HS, Fernandes SF, Blanchard CL, Sutton BG, Geigenberger P, Dennis ES, Dolferus R. 2005. Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility. Plant Cell Environ. 28:1534–1551. Pierozzi NI, Benatti R. 1998. Cytological analysis in the microsporogenesis of ramie Boehmeria nivea Gaud. (Urticaceae) and the effect of colchicine on the chi- asma frequency. Cytologia. 63:213–221. Pusalkar PK, Singh DK. 2012.  Flora of Gangotri National Park, Western Himalaya, India. Kolkata: Botanical Survey of India. Salomon B, Bothmer RV, Yang JL, Lu BR. 1988. Notes on the perennial Triticeae species in Northern Paki- stan. Bot Jahrb Syst. 110:7–15. Sharma KD, Nayyar H. 2014. Cold stress alters transcrip- tion in meiotic anthers of cold tolerant chickpea (Cic- er arietinum L.). BMC Res. Notes. 7:717. Sheila M. 1993. Male gametophyte develpoment. Plant Cell. 5:1265–1275. Sidorchuk YV, Novikovskaya AA, Deineko EV. 2016. Cytomixis in the cereal (Gramineae) microsporogen- esis. Protoplasma. 253:291–298. Singh G. 1983. New combinations in Asiatic Elymus (Poaceae). Taxon 32:639–640. Singhal VK, Kumar P. 2008. Impact of cytomixis on mei- osis, pollen viability and pollen size in wild popu- lations of Himalayan poppy (Meconopsis aculeata Royle). J Biosci. 33:371–380. Singhal VK, Kumar P. 2010. Variable sized pollen grains due to impaired male meiosis in the cold desert plants of North West Himalayas (India). In: Benja- min JK. (Ed.). Pollen: structure, types and effects. New York: Nova Science Publishers, pp 101–126. Singhal VK, Kumar R, Kumar P. 2018a. A new percep- tion about cytomixis: Causes, mechanisms and role. Chromosome Sci. 21:61–66. Singhal VK, Singh J, Kumari V. 2018b. Chromosome counts through male meiosis in seven species of genus Elymus L.(Tribe Triticeae: Poaceae) from North West Himalayas, India. Cytologia 83: 365–368. Singhal VK, Singh J, Singh H, Kumar P, Kholia BS, Tewari, L.M. 2018c. Chromosome count, meiotic abnormalities, pollen fertility and karyotype of Ely- mus semicostatus (Nees ex Steud.) Meld. from North- west Himalaya. Caryologia. 71:322–330. de Souza A, Pagliarini M. 1997. Cytomixis in Brassica napus var. oleifera and Brassica campestris var. oleifera (Brassicaceae). Cytologia. 62:25–29. Stebbins GL. 1971. Chromosomal evolution in higher plants. London, UK: Edward Arnold. Thomas H, Humphreys MO. 1991. Progress and potential of interspecific hybrids of Lolium and Festuca. J. Agri. Sci. 117:1–8. Thomas HM, Thomas BJ. 1993. Synaptonemal complex formation in two allohexaploid Festuca species and a pentaploid hybrid. Heredity 71:305–311. Vasek FC. 1962. ‘Multiple spindle’ a meiotic irregularity in Clarkia exalis. Amer J Bot. 49:536–539. Yan XB, Guo YX, Zhao C, Liu FY, Lu BR. 2009. Intra- population genetic diversity of two wheatgrass spe- cies along altitude gradients on the Qinghai-Tibetan Plateau: its implication for conservation and utiliza- tion. Conserv Genet. 10:359–367. Yan XB, Guo YX, Liu FY, Zhao C, Liu QL, Lu BR. 2010. Population structure affected by excess gene flow in self-pollinating Elymus nutans and E. burchan-bud- dae (Triticeae: Poaceae). Popul Ecol. 52:233–241. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 2 - 2020 Firenze University Press The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) Shaimaa S. Sobieh*, Mona H. Darwish Gene flow patterns reinforce the ecological plasticity of Tropidurus hispidus (Squamata: Tropiduridae) Fernanda Ito, Danielle J. Gama-Maia, Diego M. A. Brito, Rodrigo A. Torres* The technique of Plant DNA Barcoding: potential application in floriculture Antonio Giovino1,*, Federico Martinelli2,*, Anna Perrone3 Cytogenetic of Brachyura (Decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species Alessio Iannucci1, Stefano Cannicci1,2,*, Zhongyang Lin3, Karen WY Yuen3, Claudio Ciofi1, Roscoe Stanyon1, Sara Fratini1 Comparison of the Evolution of Orchids with that of Bats Antonio Lima-de-Faria Identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection Mehdi Zahravi1,*, Panthea Vosough-Mohebbi2, Mehdi Changizi3, Shahab Khaghani1, Zahra-Sadat Shobbar4 Populations genetic study of the medicinal species Plantago afra L. (Plantaginaceae) Saeed Mohsenzadeh*, Masoud Sheidai, Fahimeh Koohdar A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum using EMA-giemsa and fluorochrome banding Timir Baran Jha1,*, Partha Sarathi Saha2, Sumita Jha2 Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii Hook.f. and Elymus nutans Griseb. (Triticeae: Poaceae) from Western Himalaya, India Harminder Singh2, Jaswant Singh1,*, Puneet Kumar2, Vijay Kumar Singhal1, Bhupendra Singh Kholia2, Lalit Mohan Tewari3 Population genetic and phylogeographic analyses of Ziziphora clinopodioides Lam., (Lamiaceae), “kakuti-e kuhi”: An attempt to delimit its subspecies Raheleh Tabaripour1,*, Masoud Sheidai1, Seyed Mehdi Talebi2, Zahra Noormohammadi3 Induced cytomictic crosstalk behaviour among micro-meiocytes of Cyamopsis tetragonoloba (L.) Taub. (cluster bean): Reasons and repercussions Girjesh Kumar, Shefali Singh* Karyotype diversity of stingless bees of the genus Frieseomelitta (Hymenoptera, Apidae, Meliponini) Renan Monteiro do Nascimento1, Antonio Freire Carvalho1, Weyder Cristiano Santana2, Adriane Barth3, Marco Antonio Costa1,* Karyotype studies on the genus Origanum L. (Lamiaceae) species and some hybrids defining homoploidy Esra Martin1, Tuncay Dirmenci2,*, Turan Arabaci3, Türker Yazici2, Taner Özcan2 Determination of phenolic compounds and evaluation of cytotoxicity in Plectranthus barbatus using the Allium cepa test Kássia Cauana Trapp1, Carmine Aparecida Lenz Hister1, H. Dail Laughinghouse IV2,*, Aline Augusti Boligon1, Solange Bosio Tedesco1