Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(4): 41-49, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-298 Citation: A.R. Andrada, V. de los Á. Páez, M.S. Caro, P. Kumar (2019) Mei- otic irregularities associated to cyto- mixis in Buddleja iresinoides (Griseb.) Hosseus. (Buddlejaceae) and Castilleja arvensis Schltdl. & Cham. (Oroban- chaceae). Caryologia 72(4): 41-49. doi: 10.13128/caryologia-298 Published: December 23, 2019 Copyright: © 2019 A.R. Andrada, V. de los Á. Páez, M.S. Caro, P. Kumar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. Meiotic irregularities associated to cytomixis in Buddleja iresinoides (Griseb.) Hosseus. (Buddlejaceae) and Castilleja arvensis Schltdl. & Cham. (Orobanchaceae) Aldo Ruben Andrada1,*, Valeria de los Ángeles Páez1, M.S. Caro1,2, P. Kumar3 1 Fundación Miguel Lillo. Miguel Lillo 251, San Miguel de Tucumán, Tucumán, Argen- tina 2 Facultad de Ciencias Naturales e Instituto Miguel Lillo. Miguel Lillo 205, San Miguel de Tucumán, Tucumán, Argentina 3 Botanical Survey of India, Northern Regional Centre, Dehradun - 248 195, Uttara- khand, India *Corresponding author: arandrada@lillo.org.ar Abstract. The current paper analyzes the male meiotic behavior in wild populations of Buddleja iresinoides and Castilleja arvensis from Piedmont areas of the Northwest Region of Argentina. Castilleja arvensis showed tetraploid number of chromosome of 2n = 24. Our results are not in agreement with the previously reported base number x = 19 for Buddleja and the chromosome number n = 28 found for B. iresinoides is atypi- cal in the genus. Around 7 % pollen mother cells were aneuploid as they showed mei- otic chromosome count of n = 20-21 bivalents. Possible origin for such atypical chro- mosome number has been discussed in this paper. During the cytological studies we also came across pollen mother cells showing meiotic abnormalities such as cytomixis, chromatin stickiness and anaphase bridges with lagging chromatin. Consequently microsporogenesis was also irregular showing dyads and triads. However, the percent- age of these irregularities during meiosis and microsporogenesis was not higher, and pollen fertility was not affected to a great extent. Cytomixis and other meiotic abnor- malities in these species are reported here for the first time. Keywords. Buddleja iresinoides, Castilleja arvensis, Chromatin, pollen mother cells, cytomixis, chromosome numbers. INTRODUCTION The migration of chromatin from the nucleus of one pollen mother cell (PMC) through specialized channels (named cytomictic channels) into an adjacent PMC was observed by Gates (1911) who called it cytomixis. Subse- quently, Risueno et al. (1969) during their investigations noticed that these intercellular channels were sufficiently large to permit the migration of chro- 42 A.R. Andrada et al. matin/chromsomes and other cytoplasmic organelles. In addition, the studies of Mursalimov et al. (2018) have established that plastids can pass into another cell through cytomictic channels. Cytomictic connections were observed for the first time Körnicke (1901) in PMCs of Crocus sativus, but as the cytogenetic studies in plants advanced this phenom- enon was also reported in meristematic, tapetal, integu- mental, nucellar and ovary cells in both Angiosperms and Gymnosperms (Cooper, 1952; Koul, 1990; Guzicka & Wozny, 2005; Wang et al., 2004; Oliveira-Pierre & Sousa, 2011; Kumar et al., 2015; Kumar & Chaudhary, 2016; Kumar & Singhal, 2016; Reis et al., 2016; Mursali- mov & Deineko, 2017; Mursalimov & Deineko, 2018). As the cytogenetic analysis in higher plants expanded, cases of cytomixis were observed more frequently in acces- sions of cultured or natural plants populations. During our previous investigations, we observed the presence of cytomixis in different families of Angiosperms such as Pipperaceae, Cuscutaceae, Ranunculaceae and Cacta- ceae from the Northwest of Argentina (NOA) (Andrada et al., 2009; Lozzia et al., 2009; Páez et al., 2013 a, b). We also investigated Buddleja iresinoides (Griseb.) Hosseus. (Buddlejaceae) and Castilleja arvensis Schltdl. & Cham. (Orobanchaceae) for male meiosis and pollen fertility and we observed cytomixis and meiotic irregularities. The genus Buddleja consist of ca. 100 species and cultivars that occur in warm, tropical, and subtropical climates from the Americas, Africa and Asia (Tallent- Halsell & Watt, 2009). Buddleja iresinoides is a shrubby plant, native to South America, distributed from Bolivia to the Northwest of Argentina were it is found in Cata- marca, Jujuy, Salta and Tucumán provinces. It is a dioec- ious plant with quadrangular stems and ovate-lanceolate leaves, tomentose flowers with a bell-shaped calyx and corolla, the latter of white or yellow color (Fig.1A and B) (Carrizo & Isasmendi, 1994). Castilleja Mutis ex L. f. comprises approximately 200 species native from western North to South America (González, 2013). Castilleja arvensis is an annual hemi- parasitic herb, growing on humid soils from Mexico to the central region of Argentina. This species is charac- terized by its erect, simple, hispid, leafy stems (Fig. 1C). The leaves at the top of the stem are bract-like, gradually become smaller than the lower ones, and generally are red or purple colored (Botta & Cabrera, 1993). Figure 1. Morphological overview of the studied plants, A) General appearance of Buddleja iresinoides, and B) inflorescence detail; C) Gen- eral appearance of Castilleja arvensis. 43Meiotic irregularities associated to cytomixis in Buddleja iresinoides and Castilleja arvensis For Buddleja chromosome numbers of 18 species are listed in IPCN (Index to plant chromosome numbers) (Goldblatt & Johnson, 1979+). The basic chromosome number x = 19 is accepted and the majority of species pre- sent this number or higher ploidy levels as gametophytic number (Norman, 2000; Tallent-Halsell & Watt, 2009). Chromosome numbers of 54 species are listed in IPCN for the genus Castilleja (Goldblatt & Johnson, op. cit.). Based on the published literature the basic chro- mosome number of x = 12 and one or more polyploid levels have been suggested (Heckard, 1968; Heckard & Chuang, 1977; Chuang & Heckard, 1982; Tank & Olm- stead, 2008; Tank et al., 2009). The aim of the current paper is to analyze the male meiotic behavior in wild populations of B. iresinoides and C. arvensis in order to establish that meiotic irregu- larities are related to the phenomenon of cytomixis and, furthmore, to evaluate if they influence pollen fertility. MATERIALS AND METHODS Analyzed materials All the material studied in this investigation was collected from natural populations of Buddleja iresin- oides (Figure 1A-B) and Castilleja arvensis (Figure 1C) in Tucumán province. Voucher samples were deposited at the phanerogamic herbarium of Miguel Lillo Founda- tion (LIL). Buddleja iresinoides: ARGENTINA, Prov. Tucumán, Dpto. Concepción, Loc. Cochuna, 27°10’20” S, 65°55’39” W, alt. 1160 m, Andrada R. S/N (LIL 610862). Castilleja arvensis: ARGENTINA, Prov. Tucumán, Dpto. Tafí Viejo, Loc. Camino a la Toma, 26°43’05,122” S, 65°17’45.53” W, alt. 878 m, 29-lX-2007, Andrada R. S/N (LIL610759). Analysis of meiosis The material used consisted of flower buds from 5 randomly selected plants which were fixed in Farmer solution (3 ethanol : 1 glacial acetic acid) for one day, immediately transferred to 70% ethanol and stored at 4 °C. Anthers were first hydrolyzed in 1 N HCl at 60 °C for 20 minutes and then washed in distilled water. Pollen mother cells were prepared by the squash technique and stained with a drop of hematoxylin propionic with fer- ric citrate (Sáez, 1960; Núnez, 1968). 100 PMCs at each stage of the meiosis were observed. Size and fertility of pollen grains Fixed flowers immediately after anthesis were select- ed in order to estimate pollen fertility rates. At least 100 pollen grains of each species were measured to deter- mine the typical pollen size range. Pollen grains were stained using Müntzing solution (glycerin-acetic carmin 1:1) (Sharma & Sharma, 1965). Well-filled pollen grains with uniformly stained cytoplasm were scored as appar- ently fertile/viable while the shrivelled/flaccid ones with unstained or poorly stained cytoplasm were counted as apparently sterile/unviable. At least 1000 pollen grains were analyzed for each taxon. Photomicrographs were taken using a Nikon Eclipse E-200 microscope equipped with a Moticam 1000 digital camera (1.3 MP). The graphics were designed with the software CorelDRAW X3. RESULTS Analysis of meiosis: Buddleja iresinoides: Generally the meiosis at pro- metaphase I started totally normal (97%) with the pres- ence of 28 bivalents at diakinesis (Fig. 2A and B). Cyto- mixis was a common phenomenon in different stages of meiosis. About 2% of the PMCs of telophase I (TI) showed simple cytomictic channels indicating transfer of chromatin and cytoplasmic material among proximate PMCs (Fig. 2C), simple cytomictic channels connecting two or more cells were observed in 25% of MII (Fig. 2D). Furthermore, at TII cytomixis consisting of 1-2 channels between two cells were found in 45% of PMCs (Fig. 2E). Different kinds of irregularities were observed (Table 1). At diakinesis, 7% of PMCs were aneuploid, and showed 20-21 bivalents. At metaphase I (MI), irregularities such as out of plate bivalents were observed in 9% of the PMCs (Fig. 2F). In addition, 8% of the PMCs at TI stage were found to show anaphase bridges with lagging chromatin between two nuclei (Fig. 2G). At MII and AII, respectively 6% and 4% of PMCs exhibited chromatin stickiness between contiguous nuclei. (Figs. 2H-I). At the end of meiosis, abnormal sporads such as dyads and triads were present. (Fig. 2J). The mean diameter for B. iresinoides pollen, as determined by light microscopy, was 13.3 μm (range of 12.9 to 13.6 μm) (Table 1). Pollen viability rates in B. iresinoides was 89 % (Fig. 4A). Castilleja arvensis: Chromosome numbers in PMCs were not constant. The diakinesis showed 78% regular PMCs with a gametophytic number of n = 12 (Fig. 3A). Cytomixis was revealed to be a very frequent phenom- enon during pachytene, and 92% of the PMCs were con- 44 A.R. Andrada et al. nected by 1-5 cytomictic channels linking two or more adjacent meiocytes (Fig. 3B). To a great extent, these channels were filled by chromatin strands indicaticating material transfer from one PMC to another. The donor cell sometimes transferred almost the whole of its chro- mosome complement to a recipient meiocyte leaving only a chromosome-like heteropycnotic body beside the nucleolus; the recipient meiocytes had bigger agglomera- tions of chromatin material (Fig. 3C). At diakinesis, 22% meiocytes showed 1-5 cytomictic channels (Fig. 3D). In 8.5% of PMCs, 1 or 2 cytomictic channels were found between the neighbour tetrads at the end of second divi- sion (Fig. 3E). Irregularities observed in this species occurred in different stages (Table 1). During diakine- sis there were present hyperploid PMCs with up to n = 20 (Fig. 3D). During this stage, small-sized meiocytes with only a small nucleus were found. These small sized cells were covered by thick callose walls giving them an aspect of monads (Fig. 3F). Subsequent stages of first meiotic division (MI, AI and TI) were not observed in the preserved material. In MII, up to 7% of meiocytes were found to possess the hyperploid chromosome num- ber of 14 at one pole (Fig. 3G). In 5% PMCs at metaphase II, it was found that chromosomes do not align on the metaphase plate and tend to lie towards the periphery of the cell wall (Fig. 3H). Dyads were also observed in 3% Figure 2. Meiosis in Buddleja iresinoides. A) Diakinesis with n = 28, B) Graphic representation of figure A; C) cytomictic channel in TI connecting 2 cells; D) MII with cytomictic channels connecting 3 cells; E) Cytomixis between tetrads; F) MI with two chromosomes away from the equatorial plate; G) TI showing anaphase bridges with lagging chromatin; H) MII with chromatin stickiness between two equatorial plates; I) AII with chromatin stickiness connecting 2 neighbour poles; J) Dyad and triad. Scale = 10 μm. Figure 3. Meiosis in Castilleja arvensis. A) Diakinesis with 12 biva- lents; B) Cells with multiple cytomictic channels in pachytene; C) Donor PMC transferring almost all the chromatin to a neighbor PMC; D) Two cells in diakinesis connected by 3 cytomictic chan- nels; E) Cytomictic channels between tetrads; F) Small-sized meio- cytes with a small nucleus during the division I; G) MII showing a plate with 14 chromosomes; H) MII showing chromosomes discon- nected from equatorial plate; I) Dyad; J) small-sized meiocytes with a small nucleus at the end of the division II. Scale = 10 μm. 45Meiotic irregularities associated to cytomixis in Buddleja iresinoides and Castilleja arvensis cases (Fig. 3I). Interestingly, 2% of PMCs were of small sizes and small nuclei (Fig. 3J). Pollen size of Castilleja arvensis was observed to range from 20.1 μm to 20.8 μm (the mean diameter was 20.5 μm) (Table 1). Pollen viability rates in Castilleja arvensis was 95%, (Fig. 4B). DISCUSSION Our results are not in agreement with the base num- ber x = 19 previously reported for Buddleja (Norman, 2000; Tallent-Halsell & Watt, 2009) and the chromo- some number n = 28 found for B. iresinoides is atypical in the genus. However, Gadella (1980) suggested that x = 19 may have been derived from ancestral hybridization between two basic stocks with x = 12 and 7 (Norman, 2000; Oxelman et al., 2004). Our results suggested that B. iresinoides could be an octoploid with a putative basic number x = 7 or its chromosome number, n = 28 may have derived through secondary aneuploidy from a dip- loid parent having n = 36. Another hypothesis is that the unusual chromosome number n = 28 (2n = 56) in this population may have derived by fusion of an unreduced gamete n = 36 from a putative parent and another normal gamete n = 19 (total 2n = 55) followed by a chromosome gain (e.g. through cytomixis) to reach 2n = 56. Similarly, the rest of irregu- lar gametes found n = 20-21 could have increased their chromosome number by cytomixis; this is after nor- mal gametes n = 19 “acted” like recipient cells increas- ing their chromosome complement in 1 or 2 additional chromosomes. The chromosome number of x = 12 had been sug- gested for the genus Castilleja (Heckard, 1968; Heck- ard & Chuang, 1977; Chuang & Heckard, 1982; Tank & Olmstead, 2008; Tank et al., 2009) and C. arvensis showed tetraploid number of chromosome of 2n = 24. The phenomenon of cytomixis as well as dyads and chromosomes that didn’t attach to the equatorial plate were observed in both analyzed species. These latter kinds of meiotic irregularities could be caused by the cytomixis (Kumar, 2010; Kumar et al., 2010; Kumar et al., 2013). The origin of cytomixis is still not clear and dif- ferent opinions exist with respect to its causes and permanence during meiosis. Oliveira-Pierre & Sousa (2011) concluded that the cytomixis could have multi- ple origins. However, these authors have cited relatively recent investigations which show that cytomictic chan- nels always structurally occur in the same way: 1) in the beginning, plasmodesms loss their connections with smooth endoplasmic reticulum (desmotubules) and then starts the intrusion of cytoplasmic material into the plasmodesms that inncrease their size forming cytom- ictic channels (Wei-cheng et al.,1988; Oliveira-Pierre & Sousa, op. cit.); 2) During the cytomixis process both cellulase and pectinase enzymes are presented as play- ing a role in digesting the cell walls of PMCs involved in this phenomenon (Wang et al.,1998); 3) in cells of ger- minal tissues of anthers during callose depositions that should block up the plamodesms occur disturbances, the connector channels increase their size and in this way facilitate the formation of cytomictic channels (Falis- tocco et al., 1995; Sheidai & Fadaei, 2005; Sheidai et al. 2006; Sidorchuk et al., 2007). Some authors argued that cytomixis is a process that occur in the early stages of meiotic division (at pro- phase I generally) supporting the idea that after passage of chromatin from one PMC to another, cells which acquired or donated chromatinic material tend to degen- erate. This kind of result was obtained by Koul (1990) through investigations carried out in Alopecurus rundi- naceus Poir. On the other hand, there are authors that state that cytomixis could develop in all stages of meio- sis (Basavaiah & Murthy, 1987 in Urochloa panicoides P. Beauv.; Bellucci et al., 2003 in Medicago sativa L.; Malal- lah & Attia, 2003 in Diplotaxis harra Boiss.; Singhal & Kumar, 2008 in Meconopsis aculeata Royle; Singhal et al., 2009 in Anemone rivularis Buch.-Ham. ex DC.). Authors have different positions regarding the transfer of cell components through cytomixis. During cell divi- sion, Heslop-Harrison (1966) suggested that intercellu- lar connections occur to foment the synchrony between meiocytes allowing homogeneity of organelles and cyto- plasmic components among them. However, Guanq-Qin & Gou-Chang (2004) refused this hypothesis because they considered it inconsistent, being that in plants the tapetal cells are responsible for providing nutrients to the PMCs (not the passage from one PMC to another), Figure 4. A-J: Fertile/stained and Sterile/unstained pollen grains in; A) Buddleja iresinoides and, B) Castilleja arvensis. Scale = 10 μm. 46 A.R. Andrada et al. between the last cells never has hitherto been observed cytoplasmic connections together the meiocytes. These authors attributed to cytomixis a more general function such as the mechanism that allows share regulatory and structural genetic products (e.g., mRNAs, oragnelles, etc.) between connected cells, favoring thus a necessary homogenization of cytoplasmatic restructural events occurred during prophase I which could cause hetero- geneity in the meiocytes and consequently could lead to loss of their quality (generating more abnormal but less normal gametes). During the pachytene stage, we have never observed cytomixis in PMCs of B. iresinoides but these started from telophase I. Our observations are not in agreement with Koul’s hypothesis (1990) according to which the cytomixis occurs in first stages of meiotic division. How- ever, in C. arvensis cytomictic channels were present in prophase I in most of the meiocytes (92% of PMCs) sug- gesting that absence or presence of cytomixis does not essentially depend on the stage of cell division. Partici- pation of other factors that may play some role in cyto- mixis is still not clear. It is evident that the cytomixis can occur in different stages of meiosis from prophase I to tetrad formation as revealed in our results. Our find- ings agree with Guanq-Qin & Gou-Chang (2004) who reported that cytomictic channels always are formed among cells at the same division stage. Nevertheless, the above cited authors mentioned that cytomixis promote homogeneity between meio- cytes, observations that contradict our results because we observed in C. arvensis pachytene the transfer of almost all the chromatin from donor cell to recipient cell and the presence of hypoploid and hyperploid PMCs. Altogether, these irregularities (heterogeneity), probably produced by cytomixis, made up more than 30% of the observed cells. According to morphological characteristics, the small-sized meiocytes with a little nucleus observed in both the division I and the division II correspond to apoptotic cells similar to the ones cited by different authors in both plants and animals (Fuzinatto et al., 2007; Kravets, 2013; Andrada et al., 2016). The abnor- mal cells could be degraded by means of apoptosis, thus explaining the high percentage of pollen grains viability observed in C. arvensis. In this species this phenomenon occured two times: after pachytene at diakinesis and between the tetrads at the end of TII (both in division I and division II after or during the two stages with major percentage of cytomixis). Although this kind of abnor- mal cell was not observed in B. iresinoides it is possi- ble that some similar mechanism could occur and the abnormal cells would be eliminated. By removing the abnormal pollen grains these plants ensure that gametes transferred being viable. In B. iresinoides transfer of chromatin from a donor cell to a recipient cell is not limited only to neighbouring meiocytes but also occurs between PMCs at same stage of division. According to Ortíz et al. (2006) and Andra- da & Páez (2014), this kind of connections could dis- turb the normal development of the phragmoplast dur- ing cytokinesis causing irregularities which could finish as unbalanced gametes and give rise to dyads as it was observed in B. iresinoides. Although in C. arvensis con- nections among meiocytes from the same PMC was not observed, these were present in dyads once meiosis had been completed. In both species chromosomes which did not align to metaphase plate and stood near cell wall were found but in different stages in the two species examined. In B. iresinoides they occured during MI, whereas in C. arven- sis this kind of irregularite was observed at MII even at the hyperploid cells. These chromosomes could occur due to transfer from a PMC to other neighbouring PMC through cytomixis channels during the early metaphase. In Buddleja iresinoides, during the división II the 57% of PMCs showed irregularities (stickness, cyto- mixis and bad debelop phragmoplast that finished in dyads and triads). This percentage is far to the 11% of irregular and inviable pollen grains revealed by Münt- ing’s stain, however, before start the division II the reg- ulatory mechanism that controls the normal course of the meiosis or the method to eliminate irregular PMCs still remains unknown. In addition, in B. iresinoides it is evident that the cytomixis (reaching the maximum value of 45% at TII) does not have a large impact on the development of non-viable pollen grains. Gernand et al. analyzed the mechanisms underlying selective elimina- tion of the paternal chromosomes during the develop- ment of wheat × pearl millet hybrid embryos and found that chromosome elimination frequently took place dur- ing meiosis. These cytological observations showed that parental genomes were spatially separated within the hybrid nucleus, and the pearl millet chromatin destined for elimination occupied peripheral positions. A similar phenomenon was found in this study; chromosomes were spatially separated within the PMCs where the chro- matin occupied a predominantly peripheral position at metaphase I from B. iresinoides and at pachitene and MII from C. arvensis. In addition, given that the B. iresinoides and B. stachyoides Cham. & Schltdl. (chromosome num- ber unknown) grow together, it is likely that the taxon studied contain chromosomes from B. stachyoides. This would have given rise to populations of hybrids with this atypical gametophytic number (n = 28). 47Meiotic irregularities associated to cytomixis in Buddleja iresinoides and Castilleja arvensis In Castilleja arvensis, among the frequent abnor- malities (as stickness, cytomixis and bad debelop phrag- moplast that finished in dyads and triads) the cytomic- tic channels at pachitene (Table 1 and Figure 3B) were salient. However, the cytomixis does not seem to be the main cause of pollen inviability. In this species, the apoptosis which was observed in both prophase and TII where occur the most of irregularities could regulate the number of abnormal PMCs during the meiosis and the non viable pollen grains would be obtained when sim- ply some PMCs with different type of irregularities add together up to reach 5%. Buddleja iresinoides and Castilleja arvensis have high percentages of pollen grains stained (viables) close to 90% and small ranges of variation of size close to 0,7 µm. This fact suggest that polyploid cells (produced through of dyads and triads) which should develop giant pollen grains or “Jumbo grains” were eliminated during the last steps of microsporogenesis. On the other hand, the limit size values of stained pollen grains may mask hyperploids and hypoploid cells as apparently normal and fertile pollen grains. Future studies related to ger- minabaility of pollen grains could clarify the strange behavior of these species that show high percentages of irregularities during the meiosis and low production of sterile pollen grains. CONCLUSIONS Castilleja arvensis is a diploid taxon with n = 12 while the unusual number n = 28 from B. iresinoides suggest that the basic chromosome number for this genus could be less than x = 19. However, that this atypi- cal number could have originated through passage of additional chromosomes from a donor cell to recipi- ent cell by cytomixis or through hybridization process is possible too. In the present study, we have found that cytomixis is a process which is not stage specific and its frequency may vary from species to species which is evi- dent from our results in B. iresinoides where maximum percentage of cytomixis occur during TII, whereas in C. arvensis it is more frequent in pachytene. In addition, this process could cause numerous irregularities that would result in (at the end of meiosis) genetically unbal- anced gametes. Furthermore depending upon the sever- ity of meiotic irregularities it may hamper the reproduc- tive success of species. Cytomixis has been reported here for the first time from both Buddlejaceae and Oroban- chaceae families. ACKNOWLEDGEMENTS This work was made possible by the financial sup- port of Fundacaión Miguel Lillo (Proyecto B-0013/1). We are grateful to the Director of the Genetic Institute of Miguel Lillo Foundation, G. E. Ruiz de Bigliardo for her critical reading of the manuscript. 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