Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 135-143, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1375 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Egizia Falistocco, Gianpie- ro Marconi, Lorenzo Raggi, Daniele Rosellini, Marilena Ceccarelli, Emidio Albertini (2021) Variation of microsporogen- esis in sexual, apomictic and recombi- nant plants of Poa pratensis L.. Car- yologia 74(4): 135-143. doi: 10.36253/ caryologia-1375 Received: August 06, 2020 Accepted: November 30, 2021 Published: March 08, 2022 Copyright: © 2021 Egizia Falistocco, Gianpiero Marconi, Lorenzo Raggi, Daniele Rosellini, Marilena Cecca- relli, Emidio Albertini. 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. Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis L. Egizia Falistocco1,*,+, Gianpiero Marconi1,+, Lorenzo Raggi1, Daniele Rosellini1, Marilena Ceccarelli2, Emidio Albertini1 1 Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy 2 Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy *Corresponding author. E-mail: egizia.falistocco@unipg.it + Contributed equally to this work Abstract. Apomixis is a rather widespread phenomenon in plants. It is defined as the asexual formation of a seed from the maternal tissues of the ovule, avoiding the pro- cesses of meiosis and fertilization. Some species are facultative apomicts and form seeds by means of sexual and apomictic pathways to different extents. This is the case of Poa pratensis, the Kentucky bluegrass, which reproduces by aposporous pseudoga- mous facultative apomixis. This grass is one of the most studied apomictic systems, however some aspects, such as the male meiotic behavior, have not been so far investi- gated. In this study the process of microsporogenesis in genotypes of P. pratensis with a different mode of reproduction was investigated. The analysis revealed an almost regu- lar meiosis in the sexual plants whereas apomictic genotypes exhibited different levels of meiotic irregularities, mainly due to cell fusion and irregular segregation in I and II division. Our data did not reveal evident connections between the extent and types of abnormalities and the components of apomixis, apomeiosis and parthenogenesis. The meiotic behavior of the examined plants was discussed in the light of their origin. Keywords: Poa pratensis L., Kentucky bluegrass, apomixis, microsporogenesis, meiotic abnormalities. INTRODUCTION The sexual seed formation is based on two fundamental mechanisms, meiosis and fertilization. The combination of these events produces new nuclear compositions so that sexual reproduction is a means not only of gen- erating new but also variable individuals. However, in some flowering plants, seeds form asexually, from maternal tissues by a process known as apomixis (Bicknell and Koltunow 2004). Apomixis, is a complex trait resulting from the circumvention of female meiotic reduction (a process known as apomeio- sis) and fertilization (parthenogenesis). In gametophytic apomixis, a mega- 136 Egizia Falistocco et al. gametophyte (embryo sac) is originated from an unre- duced cell, and subsequently a clonal embryo develops by parthenogenesis from a 2n egg (Matzk et al. 2005). Several species need fertilization for endosperm develop- ment while others do not (Barcaccia and Albertini 2013). The three components of apomixis, namely apomeiosis, parthenogenesis and autonomous endosperm formation, have been uncoupled experimentally, as documented in numerous genera such as Taraxacum (Van Dijk et al. 1999; Van Dijk, 2003), Erigeron (Noyes and Rieseberg 2000), Poa (Albertini et al. 2001), Hypericum (Barcaccia et al. 2006; Schallau et al. 2010), Cenchrus (Conner et al. 2013), and Hieracium (Catanach et al. 2006; Henderson et al. 2017). Most plants of apomictic species are facultative apomicts and form seeds by means of sexual and apom- ictic pathways to different extents. This means that plants that reproduce by apomixis also retain the ability to reproduce sexually to varying degrees (Nogler 1994; Tucker 2003). The phenomenon of apomixis is far from rare and its pattern of distribution suggests that it evolved many times during plant evolution. Among flowering plants it occurs with high frequency in certain families such as Asteraceae, Rosaceae, Ranunculaceae and Poaceae (Bick- nell and Koltunow 2004). Most apomictic plants produce viable pollen, this implies that within apomictic populations the formation of viable pollen represents a possibility for the fertiliza- tion of unreduced eggs. However, alterations of micro- sporogenesis in apomictic individuals have not been so far extensively investigated. Poa pratensis L., Kentucky bluegrass, is an impor- tant fodder and turf grass which mainly reproduces by aposporous pseudogamous apomixis, i.e. unreduced aposporous embryo sacs develop through parthenogen- esis to viable apomictic seeds if the unreduced polar nuclei fuse with a sperm cell from the male gameto- phyte (pseudogamy). The species is highly variable when reproduction mode, chromosome number and pheno- typic traits are considered. In this species apomixis is facultative, with a frequency ranging from 0 to 100%, while chromosome numbers from 2n=18 to 150 have been reported (Matzk et al. 2005). Among the native monocot apomictic systems, P. pratensis is one of most explored. For several years, selected genotypes from wild Italian populations have been investigated with the aim of understanding the genetic control and mechanisms that regulate apomixis (Mazzucato 1995; Albertini et al. 2001; Porceddu et al. 2002; Raggi et al. 2015; Marconi et al. 2020). These studies provided a solid background for the present investigation that aimed at analyze the meiotic behavior of plants of P. pratensis exhibiting a different mode of reproduction and to find possible rela- tionships between apomixis and its components and the alterations of microsporogenesis. MATERIALS AND METHODS Plant material Genotypes of P. pratensis with different reproduc- tive systems were examined: i) a sexual genotype S1/1- 7 derived from a cross between two completely sexual genotypes selected from German cultivars (Matzk 1991); ii) an apomictic (aposporic and parthenogenetic) RS7-3 (Mazzucato 1995) and L4 (Marconi et al. 2020) plants, both from Italian natural populations and iii) several plants belonging to two F1 segregating populations pro- duced by crossing S1/1-7 x RS7-3 (Barcaccia et al. 1998) and S1/1-7 x L4 (Marconi et al. 2020). Reproductive mode and chromosome number of the above reported materials employed in this study were investigated in previous studies (Barcaccia et al. 1998; Albertini et al. 2001; Porceddu et al. 2002; Marconi et al. 2020 and ref- erences therein) and are summarized in Table 1. Plants were grown at the experimental field of the Dept. of Agricultural, Food and Environmental Sciences in Perugia (N 43°10’15.3’’, E 12°39’58.7’’). Meiotic analysis For meiotic investigations inflorescences not com- pletely emerged from the flag leaf were employed. For each plant, four-five inflorescences were collected and immediately fixed in absolute ethanol-acetic acid 3:1 (v/v) for 24 hours, then they were transferred to 70% ethanol and stored at 4°C until analysis. Cytological preparations were made by squashing the anthers of a single flower on a glass slide with some drops of 0.5% acetocarmine (Merck Life Science, Italy), intensified by ferric oxide. For each plant 150-200 pollen mother cells (PMCs) for each meiotic stage were analyzed. Slides were observed under a Microphot Nikon microscope. Images were recorded with a digital photocamera SONY ICX282AQ and then processed using Adobe Photoshop 5.0. The alterations observed in each meiotic phase were expressed as percentage of the meiocytes examined. For pollen viability analysis, pollen samples were collected from each plant, and stained with a mixture of acetocarmine and glycerol (1:1) (Ramanpreet and Gupta 2019). The pollen viability was expressed as percentage of fully stained pollen grains over a total of at least 1.000 137Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis pollen grains for each sample. The percentage of the meiotic anomalies recorded at I and II division and pol- len viability were graphically displayed. RESULTS Meiotic analysis of the parental genotypes The sexual plant S1/1-7, with 2n=36, is considered a tetraploid with four additional chromosome pairs (Matzk 1991; Barcaccia et al. 1998; Porceddu et al. 2002); it exhibited an almost regular meiotic behavior with only few exceptions consisting of cell fusion and meio- cytes linked by cytoplasmic connections at prophase I (4.3%). Pollen viability was almost complete reaching the 98.4% (Fig. 1a). In the apomictic parental plant RS7-3 (2n=64), pre- viously described as a probable octoploid having four additional chromosome pairs (Mazzuccato 1995; Por- ceddu et al. 2002), few abnormalities at different meiotic stages were observed. These included meiocytes at meta- phase I with univalents (4.0%), anaphase I with lagging chromosomes (7.0%) and irregular segregation in the second division (15.0%). A high percentage of viable pol- len was recorded (98.0%, Fig. 1b). The apomictic L4 plant, hexaploid with 2n=42 (Marconi et al. 2020), revealed numerous abnormali- ties during both first and second division. At prophase I meiocytes linked by cytoplasmic connections (4.5%) were observed (Fig. 2a), whereas lagging chromosomes (19.0%) and irregular segregation (24.0%) were detected at anaphase I and II, respectively. Dyads and triads fre- quently occurred (15.0%) at the end of meiosis. The via- ble pollen produced by this plant was reduced to 51.0% with the grains displaying a quite heterogeneous size (Fig. 1c). Meiotic analysis of F1 progenies Four plants among those obtained from the cross S1/1-7 x RS7-3 were analyzed: sexual PG-F122, aposporic and parthenogenetic PG-F115 and PG-F146, and apo- sporic PG-F15. As showed by Porceddu and colleagues (2002) all these plants have a chromosome number 2n=50. The sexual plant PG-F122 showed an almost reg- ular microsporogenesis with few exceptions consisting of anaphase I with laggards (8.6%), and few triads at telo- phase II (1.3%). The pollen viability was extremely high (99.0%, Fig. 1d). In PG-F115 events of cellular aggrega- tion at prophase I (1.9%, Fig. 2b) and numerous cells at anaphase I with lagging chromosomes (50.0%) were found. In the second division, the irregular congres- sion and segregation of chromosomes was observed in numerous meiocytes (15.0%). As a consequence, a con- siderable number of triads and dyads (13.0%) was pro- duced at the end of meiosis. The pollen displayed vari- ability in size but appeared fully stained (Fig. 1e). Gen- otype PG-F146 showed irregularities along the entire microsporogenetic process. These consisted in meiocytes at prophase I aggregated by cytoplasmic connections (1.4%), univalents at metaphase I (9.4%), lagging chro- mosomes at anaphases I (20.0%), and irregular orienta- tion of chromosomes at metaphase II and anaphases II (24.0%). A conspicuous number of triads and dyads Table 1. Name, progeny, mode of reproduction, somatic chromosome number (2n) with relative reference. Name Progeny Mode of reproduction 2n Reference for chromosome number determination S1/1-7 - Sexual 36 Porceddu et al. 2002 RS7-3 - Apomictic* 64 Porceddu et al. 2002 L4 - Apomictic* 42 Marconi et al. 2020 PG-F122 S1/1-7 ´ RS7-3 Sexual 50 Porceddu et al. 2002 PG-F115 S1/1-7 ´ RS7-3 Apomictic* 50 Porceddu et al. 2002 PG-F146 S1/1-7 ´ RS7-3 Apomictic* 50 Porceddu et al. 2002 PG-F15 S1/1-7 ´ RS7-3 Aposporic only 50 Porceddu et al. 2002 Apo143 S1/1-7 ´ L4 Aposporic only 39-42 Marconi et al. 2020 Apo40 S1/1-7 ´ L4 Parthenogenetic only 44-48 Marconi et al. 2020 Apo98 S1/1-7 ´ L4 Parthenogenetic only 39-42 Marconi et al. 2020 *Aposporic and parthenogenetic. 138 Egizia Falistocco et al. Figure 1. Percentage of meiotic abnormalities at I and II division and pollen viability recorded on parental genotypes S1/1-7 (a), RS7-3 (b) and L4 (c); on progenies from the cross S1/1-7 x RS7-3: PG-F122 (d), PG-F115 (e), PG-F146 (f), PG-F15(g) and from the cross S1/1-7 x L4: APO 143 (h), APO 40 (i) and APO 98 (j). 139Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis was recorded at the end of meiosis (20.0%). A consider- able variability of the pollen size was observed; however, pollen viability of this plant was complete (Fig. 1f ). The aposporic recombinant PG-F15 displayed a quite anoma- lous microsporogenesis. At prophase I, fusion and aggre- gation of meiocytes (4.7%) were observed, with fusions that in some cases involved meiocytes at prophasic stag- es (Fig. 2c). In addition, cells with chromosomes sepa- Figure 2. Aspects of alterations of microsporogenesis and pollen grains in some of the examined genotypes. Meiocytes at prophase I con- nected by cytoplasmic channels in L4 (a); Fusion of three cells at the beginning of prophase I in PG-F115 (b); fusion of two meiocytes at different stage of prophase I in PG-F15 (c); cell at the diakinesis stage with chromosomes separated into three groups (d); absence of chro- mosome segregation at anaphase I in PG-F15 (e); spreading of chromosomes in both cells of a meiocyte at anaphase II in PG-F15 (f); triad formed at the end of microsporogenesis in APO 40 (g); dyad formed at the end of microsporogenesis in APO 40 (h); pollen grains pro- duced by APO 40 exhibiting a large size variability (i). The bar represents 10 μm. 140 Egizia Falistocco et al. rated into two or three groups were observed (Fig. 2d). At anaphase I, numerous PMCs (33.0%) showed lagging chromosomes or absence of segregation (Fig. 2e). In the second division the spreading of chromosomes in one or both cells of meiocytes was frequently observed (15.0%, Fig. 2f), as well as dyads and triads at the end of meiosis (12.0%). Despite these abnormalities, also in this case the pollen appeared fully viable (Fig. 1g). Among the progeny of the cross S1/1-7 x L4, the aposporic APO143 and parthenogenetic APO40 and APO98 were analyzed. The chromosome numbers of these genotypes, 2n=44-48 for APO40 and 2n=39-42 for APO98 and APO143, was previously estimated by means of flow cytometry (Marconi et al. 2020). In APO143 most of the anthers resulted empty and the few mey- ocites that was possible to analyse showed anomalies consisting of linked cells (4.5%) at prophase I and lag- ging chromosomes at anaphase I (5.0%). The few pollen grains produced, were not viable (Fig. 1h). The meiotic irregularities detected in APO40 mainly affected the first division where about 6.0% of PMCs at prophase I were connected by cytoplasmic channels and 34.0 % of meiocytes at metaphase I showed univalents. Anoma- lies of the second division were due to the absence of segregation of chromosomes at anaphase II (8.0%) and formation of triads and dyads (9.0%) at the completion of meiosis (Fig. 2g, h). The pollen showed a remarkable variability in size but all grains appeared fully stained (Fig. 1i, Fig. 2i). In APO48 a high number of cells at ana- phase I with laggards were recorded (48.0%). The scarci- ty of meiocytes in the second division suggests a possible degeneration of PMCs before the dyad stage. A remark- able number of cells that entered the second division displayed irregular segregation of chromosomes (26.0%) and the pollen was not viable (Fig. 1j). DISCUSSION In this work the meiotic behavior of sexual, apom- ictic and F1 recombinant genotypes of P. pratensis was investigated. In sexual plants an almost regular micro- sporogenesis was obser ved, whereas the apomictic genotypes displayed meiotic abnormalities at different degrees, mostly consisting in events of cell fusion and irregular segregation of chromosomes during I and II division. Data did not reveal relationships between the amount and types of such abnormalities and the repro- ductive mode of the apomictic genotypes. However, the origin of the parental genotypes S1/1-7, RS7-3 and L4 may offer useful indications for interpreting their meiot- ic behavior and those of their progenies. In fact, the sex- ual S1/1-7 plant was derived from a cross between two completely sexual genotypes selected from German cul- tivars (Matzk 1991; Barcaccia et al. 1998). The achieve- ment and persistence of sexuality by means of meiosis and fertilization is guaranteed by regular processes of microsporogenesis and gametogenesis. The fact that S1/1-7 was obtained by crossing two completely sexual genotypes contributed to preserving its fertility. Con- versely, the apomictic RS7-3 and L4 were collected in the wild and did not undergo any anthropogenic pres- sure (Mazzuccato 1995; Marconi et al. 2020). These gen- otypes differ in chromosome number, meiotic behavior and pollen fertility, and also their progenies, obtained by crossing them with S1/1-7 as female parent, showed vari- ability in the same traits. Given that both crosses have the same female parent, it is possible to evaluate the con- tribution of each male parent to the characteristics of the corresponding offspring. Since all plants from the cross S1/1-7 x RS7 had the same chromosome number 2n=50 (Porceddu et al. 2002) this evidence demonstrates that a regular chromosome segregation occurred and that fer- tilization took place between normal haploid female and male gametes with n=18 and n=32, respectively. On the contrary, two different ranges of chromosome number (2n=39-42 and 2n=44-48) were detected in plants from S1/1-7 x L4 (Marconi et al., 2020). This suggests that L4 produced functional gametes with a different chromo- some number and that they accomplished fertilization. It has been suggested that the meiotic events are controlled by a large number of genes, some controlling the meiotic phases and others post-meiotic events and gametogenesis. The mutation of any of these genes can cause anomalies affecting the gamete fertility (Ma 2005). Most of the meiotic abnormalities observed in this study were common to all plants examined; for example, the irregular segregation of chromosomes, which is prob- ably due to the defective spindle formation or its total absence (Kaul and Murthy 1985). Generally, these altera- tions are not directly responsible for pollen viability but rather for pollen chromosome number; so that they are considered one of the principal sources of polyploid or aneuploid-polyploid pollen grains (Stebbins 1963; Podio et al. 2012). This may explain why, despite their meiotic disturbances, RS7-3 and the apomictic and recombinant progenies PG-F15, PG-F115 and PG-F146 showed a high level of pollen fertility. The considerable number of triads and dyads and the heterogeneous size of pollen detected in PG-F115 and PG-F146 are a further evidence that these meiotic alterations influence the chromosome constitu- tion of pollen grains (Stanley and Linskens 1974). The genotype L4 as well as genotypes APO143, APO40 and APO98 displayed a different situation. The 141Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis meiotic alterations detected in L4 could explain the pro- duction of aneuploid pollen grains, as above suggested, but do not clarify the cause of the reduction in pollen fer- tility of this plant. This, most likely, is the result of post- meiotic events, such as the alteration of the normal activ- ity of genes controlling the steps following the completion of meiosis and gametogenesis (Lalanne and Twell 2002). The scarcity of PMCs detected in APO143 could be the consequence of mutations affecting the normal development of anthers and the formation of meiocytes. A certain degree of PMCs scarcity has been already reported in Boechera (Rojek et al. 2018) while genetic and molecular analyses in Arabidopsis demonstrated that a high number of genes controls several aspects of anther development and that their mutations can seri- ously damage the anther cell differentiation, tapetum function and microspore development (Ma 2005; Sand- ers et al. 1999). Studying mutants in Arabidopsis, Yang and col- leagues (2003) demonstrated that mutations of genes controlling the meiotic progression can result in pro- grammed cell death with the consequence of the death of most meiocytes before cytokinesis. The dramatic reduc- tion in the number of meiocytes observed in APO98 could be the result of a phenomenon of cell degeneration similar to the one described in Arabidopsis. Among those obtained from the cross S1/1-7 x L4, the parthenogenetic recombinant APO40 was the only genotype producing viable pollen. Moreover, the micro- sporogenesis pathway of this plant was not affected by the scarcity of meiocytes that characterized APO143 and APO98. A possible explanation for the different meiotic behavior of the F1 plants from the cross S1/1-7 x L4 could be the different number of mutations that these plants inherited from the male parent; the differ- ent chromosome number characterizing these genotypes could support this hypothesis. Further considerations can be done taking into account the cytological data and the reproduction mode of the plants obtained from the cross S1/1-7 x RS7. It can be observed that the apomictic and recombinant geno- types have similar behavior as the male parent, whereas the sexual PG-F122 reflects the meiotic characteristics of the female parent. This suggests that the meiotic behav- ior and the mode of reproduction are together inherited from one of the parents. However, the progeny from S1/1-7 x L4 does not provide enough evidence support- ing this hypothesis because all the examined genotypes were apomictic recombinant. Cellular aggregations due to cytoplasmic channels and cell fusion represent sporadic events in the exam- ined plants. Such aggregations involved only few cells (2-4) and did not damage the pollen fertility because they did not proceed to meiosis but degenerated, as dem- onstrated by the fact that they were not observed from prophase I onwards. Cell fusion has been reported in several plant species and may result from suppression of cell wall formation during premeiotic mitoses (Nirmala and Rao 1996). Instead, the cytoplasmic connections originate from the pre-existing system of plasmodes- mata which form within anther tissues and subsequently become completely obstructed by the progressive depo- sition of callose (Heslop-Harrison 1966). In some cases, due to the scarce production of callose, the connections remain giving origin to meiocytes aggregation. The dis- covery of this phenomenon in P. pratensis is interest- ing because it has been demonstrated that the defective deposition of callose is a critical step in the anomalous development of female gametophyte in apomictic plants (Peel et al. 1997; Dusi and Willemse 1999). Further investigations based on a higher number of samples could clarify the hypotheses made in this study. Considering that the fertility of plants is a complex mechanism, it would be useful to combine the meiotic analysis with the analysis of the reproductive structures and, in particular, of the anthers. FUNDING This work was supported by Fondazione Cassa di Risparmio di Perugia (Italy), project code: 2019.0317.029. REFERENCES Albertini E, Porceddu A, Ferranti F, Reale L, Barcaccia G, Romano B, Falcinelli M. 2001. Apospory and parthe- nogenesis may be uncoupled in Poa pratensis: a cyto- logical investigation. Sex Plant Reprod. 14:213–217. doi:10.1007/s00497-001-0116-2. Barcaccia G, Mazzucato A, Albertini E, Zethof J, Gerats A, Pezzotti M, Falcinelli M. 1998. Inheritance of par- thenogenesis in Poa pratensis L.: auxin test and AFLP linkage analyses support monogenic control. Theor Appl Genet. 97:74–82. doi:10.1007/s001220050868. Barcaccia G, Arzenton F, Sharbel TF, Varotto S, Parrini P, Lucchin M. 2006. Genetic diversity and reproduc- tive biology in ecotypes of the facultative apomict Hypericum perforatum L. Heredity 96, 322–334. doi. org/10.1038/sj.hdy.6800808 Barcaccia G, Albertini E. 2013. Apomixis in plant repro- duction: a novel perspective on an old dilemma. Plant Reprod. 26, 159–179. doi:10.1007/s00497-013-0222-y. 142 Egizia Falistocco et al. Bicknell RA, Koltunow AM. 2004. Understanding apo- mixis: recent advances and remaining conundrums. Plant Cell. 16:S228-S245. doi:10.1105/tpc.017921. Catanach AS, Erasmuson SK, Podivinsky E, Jordan BR, Bicknell R. 2006. Deletion mapping of genetic regions associated with apomixis in Hieracium. Proc Nat Acad Sci USA. 103:18650–18655. doi:10.1073/ pnas.0605588103. Conner JA, Gunawan G, Ozias-Akins P. 2013. Recombi- nation within the apospory specific genomic region leads to the uncoupling of apomixis components in Cenchrus ciliaris. Planta. 238:51–63. doi:10.1007/ s00425-013-1873-5. Dusi DMA, Willemse MTM. 1999. Activity and localiza- tion of sucrose synthase and invertase in ovules of sex- ual and apomictic Brachiaria decumbens. Protoplasma. 208:173-185. https://doi.org/10.1007/BF01279088 Henderson ST, Johnson SD, Eichmann J, Koltunow AMG. 2017. Genetic analyses of the inheritance and expres- sivity of autonomous endosperm formation in Hiera- cium with different modes of embryo sac and seed formation. Ann Bot. 119:1001–1010. doi:10.1093/ aob/mcw262. Heslop-Harrison J. 1966. Cytoplasmic connections between angiosperms meiocytes. Ann Bot. 30:221- 230. http://www.jstor.org/stable/42908662. Kaul MLH, Murthy TGK. 1985. Mutant genes affecting higher plant meiosis. Theor Appl Genet . 70: 449-466. https://doi.org/10.1007/BF00305977 Lalanne F, Twell D. 2002. Genetic control of male germ unit organization in Arabidopsis. Plant Physiol. 129:865-875. doi:10.1104/pp.003301. Ma H. 2005. Molecular genetic analyses of microsporo- genesis and microgametogenesis in flowering plants. Annu Rev Plant Biol. 56:393-434. doi:10.1146/ annurev.arplant.55.031903.141717. Marconi G, Aiello D, Kindiger B, Storchi L, Marrone A, Reale L, Terzarol, N, Albertini E. 2020. The role of APOSTART in switching between sexuality and apo- mixis in Poa pratensis. Genes. 11: 941. doi:10.3390/ genes11080941. Matzk F, Prodanovic S, Baumlein H, Schubert I. 2005. The inheritance of apomixis in Poa pratensis con- firms a five locus model with differences in gene expressivity and penetrance. Plant Cell. 17:13–24. doi:10.1105/tpc.104.027359. Matzk F.1991. New efforts to overcome apomixis in Poa pratensis L. Euphytica. 55:65–72. Mazzucato A. 1995. Italian germplasm of Poa pratensis L. II. Isozyme progeny test to characterize genotypes for their mode of reproduction. J Genet Breed. 49:119– 126. Nirmala A, Rao PN. 1996. Genesis of chromosome numerical mosaicism in higher plants. The Nucleus. 39:151-175. Nogler GA. 1994. Genetics of gametophytic apomixis: a historical sketch. Pol Bot Stud. 8:5-11. Noyes RD, Rieseberg LH. 2000. Two independent loci control agamospermy (apomixis) in the triploid flow- ering plant Erigeron annuus. Genetics. 155:379–390. doi: 10.1093/genetics/155.1.379 Peel MD, Carman JG, Leblanc O. 1997. Megasporocyte callose in apomictic buffelgrass, Kentucky Blue- grass, Pennisetum squamulatum Fresen, Tripsacum L., and Weeping Lovegrass. Crop Sci. 37:724-732. doi:10.2135/cropsci1997.0011183X003700030006x. Podio M, Siena LA, Hojsgaard D. 2012. Evaluation of meiotic abnormalities and pollen viability in apo- sporous and sexual tetraploid Paspalum notatum (Poaceae). Plant Syst Evol. 298: 1625-1633. doi 10.1007/s00606-012-0664-y. Porceddu A, Albertini E, Barcaccia G, Falistocco E, Fal- cinelli M. 2002. Linkage mapping in apomictic and sexual Kentucky bluegrass (Poa pratensis L.) geno- types using a two way pseudo-testcross strategy based on AFLP and SAMPL markers. Theor Appl Genet. 104:273–280. doi:10.1007/s001220100659 Ramanpreet A, Gupta RC. 2019. Meiotic studies in genus Withania Pauquy, from Indian Thar Desert. Caryolo- gia. 72:15-21. doi:10.13128/cayologia-247. Raggi L, Bitocchi E, Russi L, Marconi G, Sharbel TF, Veronesi F, Albertini E. 2015. Understanding genet- ic diversity and population structure of a Poa prat- ensis worldwide collection through morphological, nuclear and chloroplast diversity analysis. PLoS one. 10(4):e0124709. doi:10.1371/journal.pone.0124709. Rojek J, Kapusta M, Kozieradzka-Kiszkurno M, Majcher D, Górniak M, Sliwinska E, Sharbel TF, Bohdanow- icz J. 2018. Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicace- ae). Ann Bot. 122:513-539. doi:10.1093/aob/mcy114. Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu Y-C, Lee PY, Truong MT, Beals TP, Goldberg RB. 1999. Anther developmental defects in Arabidop- sis thaliana male-sterile mutants. Sex Plant Reprod. 11:297-322. doi:10.1007/s004970050158. Schallau A, Arzenton F, Johnston AJ, Hähnel U, Koszegi D, Blattner FR, Altschmied L, Haberer G, Barcac- cia G, Bäumlein H. 2010. Identification and genetic analysis of the APOSPORY locus in Hypericum perfo- ratum L. The Plant Journal. 62:773–784. doi:10.1111/ j.1365-313X.2010.04188.x. Stanley RG, Linskens HF. 1974. Pollen. In Biology Bio- chemistry Management. Springer Verlag. Berlin. 143Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis Stebbins GL. 1963. Variation and evolution in plants. Columbia University Press. New York and London. Tucker MR, Araujo AC, Paech NA, Hecht V, Schmidt DL, Rossell JB, de Vries SC, Koltunow AM. 2003. Sexual and apomictic reproduction in Hieracium subge- nus Pilosella are closely interrelated developmen- tal pathways. Plant Cell. 15:1524-1537. doi:10.1105/ tpc.011742. Van Dijk PJ, Tas IC, Falque M, Bakx-Schotman T. 1999. Crosses between sexual and apomictic dandeli- ons (Taraxacum). II. The breakdown of apomixis. Heredity 83, 715–721. https://doi.org/10.1046/j.1365- 2540.1999.00620.x Van Dijk PJ. 2003. Ecological and evolutionary oppor- tunities of apomixis: insights from Taraxacum and Chondrilla. Phil Trans R Soc London Ser B, Biol Sci 358, 1113–1121. doi:10.1098/rstb.2003.1302. Yang X, Makaroff CA, Ma H. 2003. 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