1 Genome size and DNA endoreplication pattern of non-generative cells in the antheridia of

2 Chara connivens Salzmann ex A. Braun from Northern Morocco.

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3 °Gargiulo G.M., °Damino R., °° Merzouki A., °Picone R.M., °°Redouan F.Z., °° Boutahar A., and
4 °Crisafulli A.

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5 °Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of
6 Messina, Via F. Stagno D’Alcontres 31, 98166, Messina, Italy; °°Department of Biology, Science
7 Faculty, University of Abdelmaleck Essaadi, Tétouan, Morocco
8
9 Abstract

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10 Data on nuclei, mitotic divisions, chromosomal behaviors, and DNA measurements have been

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11 examined in individuals of Chara connivens collected in a pond from the Tangier-Tetouan Region
12 (Northern Morocco). Image Cytometry of individual nuclei was used to examine the
13 endoreduplication level in germ line and somatic cells of the antheridia. DNA content increased
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14 from 2C to up to 128C in shield cells, manubria and capitular cells. However, a progressive
15 decrease in the amount of DNA after any successive endoreplication cycle was apparent in the
16 antheridial non-generative cells suggesting a possible presence of a DNA remodeling mechanism in
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17 Chara. The 1C-value found in the cells of the filaments at a mature stage of spermiogenesis has an
18 absolute content of 4.47 pg. The obtained data on DNA amount was combined and analyzed with
19 those available on Chara spp. in the literature. An n = 14 chromosome number was confirmed for
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20 this population. Spontaneous anomalies such as laggard chromosomes, formation of micronuclei or

21 amitotic nuclear divisions by gradually pulling apart the nucleus into two parts, were observed in
22 some cells of the antheridial filaments. An attempt to construct and analyze the C. connivens
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23 karyotype was also made.

24
25 Key words: Chara connivens, chromosomes, basic cytogenetics, genome size, endopolyploidy,
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26 karyotype
27 Corresponding author: Gargiulo, G. M. (ggargiulo@unime.it)
28
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29 1. Introduction
30 Basic cytogenetic data, such as classical karyotype analysis and genome size, cannot be considered
31 obsolete and will remain critical because their value is fundamental for planning correct
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32 experimental designs, mainly in evolutionary studies (Leitch et al., 2010; Guerra, 2012; Singh,
33 2018; Gargiulo et al., 2022). The amount of DNA in a nucleus can vary significantly among the
34 somatic cells of an individual because of several mechanisms, but mostly through the

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35 endoreduplication process (Leitch and Dodsworth, 2017). Much data in the literature documents a
36 correlation between the amount of DNA in a cell with several of the phenotypic traits, such as cell

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37 size, nuclear volume, and cell cycle duration, which in turn have been suggested to impact a variety
38 of other morphological and physiological factors (the nucleotypic theory; Bennett, 1971; Bennett,
39 1972; Doyle and Coatet, 2019). Because of the small size of chromosomes and nuclei in the algae,

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40 these data are limited and more often related just to the chromosome number (Godward, 1966).
41 Characeae members represent an exception to this assumption because they possess relatively large
42 nuclei and reasonably easily countable chromosomes (Grant and Proctor, 1972; Gargiulo et al.,
43 2019). The Characeae, comprising six extant genera, is the only family whose members survived

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44 through several periods of diversification and extinction between the upper Silurian and the present

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45 day (Grambast, 1974; McCourt et al., 2017). They are usually considered one of the most
46 morphologically specialized groups of algae (Nishiyama et al., 2018). They show a thallus that
47 differentiates into nodes and internodes with an equisetum-like habitus (Lee, 2008). The two most
common genera are Chara Linnaeus and Nitella C. Agardh, each with several hundred species
48
49
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(Wood and Imahori, 1965; Mouronval et al., 2015).
50 Based on molecular studies, the evolutionary relationship of Charales to embryophytes remains still
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51 unresolved (McCourt et al., 2017). They alternate between the possibility of being the closest living
52 relatives of land plants and conjugating green algae depending on which data are utilized to perform
53 the phylogenetic analysis (Karol et al., 2001; Lewis and McCourt, 2004; Turmel et al., 2006;
54 Nishiyama et al., 2018; Leebens-Macket al., 2019 ).
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55 A good amount of karyological data exists on Characeae with a significant contribution made by
56 species from the Indian sub-continent and Australia (Guerlesquin, 1996; Casanova, 2015,
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57 Subramanian, 2018). Despite this, basic cytogenetics is still far from satisfactory, mainly in cell
58 genome size and endoreduplication patterns. According to McCourt et al. (2017), further
59 karyotypic work on the Characeae is needed. Chromosome numbers vary widely in the genus
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60 Chara, and many species exhibit polyploidy with a chromosome complements up to n = 77

61 (Guerlesquin, 1996). Most numbers were multiples of 7; on these grounds, the base chromosome
62 number was hypothesized to be n = 7 (Bhatnagar, 1983; Guerlesquin, 1984; Grant, 1990; Kapraun,
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63 2007; Casanova, 2015). According to the DNA data Base (Leitch et al., 2019), genome size was
64 reported for eight taxa representing six Chara species (Maszewski and Kołodziejczyk, 1991;
65 Kunachowicz et al., 2001). No data exist for the other genera in the family. Kunachowicz et al.
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66 (2001) reported differences in 1C DNA content between female (7.0 pg) and male (7.4 pg) isolates
67 of Chara tomentosa Linnaeus.

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68 As part of an ongoing study on the basic cytogenetics of charophyte flora from different areas, we
69 studied a population of Chara connivens Salzmann ex A. Braun collected from Tangier-Tetuoan

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70 Region (Northern Morocco). It is a dioicous species with distribution in North Africa, Europe, and
71 South-West Asia; in some countries, it is considered an invasive species, while in others it is a
72 protected species (Rybak and Woyda-Ploszczyca, 2019).

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73 Cytological investigations on Charophyta populations from Morocco, as evident from the literature,
74 are often limited to chromosome numbers, and no data are known on their genome size
75 (Guerlesquin, 1996; Leitch et al., 2019). Karyological information on C. connivens populations
76 from various localities mainly refers to the chromosomal number alone (Tab. 1). The only data on

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77 its karyotype are those from an Iranian population (Noedoost et al., 2016). However, almost all the

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78 populations studied have n = 14 chromosomes (Tab. 1). Polyploidy and aneuploidy were described
79 for some populations (Guerlesquin, 1967, 1996; Trbojević et al., 2020).
80 This paper reports the genome size and endoreplication pattern of cells from the C. connivens
antheridia. The obtained data on the amount of DNA was combined and analyzed with those
81
82
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available in the Plant DNA C-values database on Chara spp. (Leitch et al., 2019 ). An attempt to
83 reconstruct its karyotype and to compare it with those reported in the literature from other
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84 geographical areas was also made.
85
86 2. Methods
87 Plants of C. connivens Salzmann ex A. Braun were collected at a depth of 0.1-0.5 m in a pond
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88 (35°26'18.6"N, 5°23'59.3"W) from Tangier-Tetuoan Region (En Nakhla, Northern Morocco) during
89 the years 2022-2023 (Fig. 1). The samples were determined, and vouchers were deposited in the
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90 herbarium of prof. G.M. Gargiulo at the University of Messina, Italy. The cytometric and
91 cytogenetic analyses were carried out on cells from antheridia. The antheridia were isolated and
92 fixed immediately in the laboratory in 3:1 absolute ethanol-glacial acetic acid (Kapraun and
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93 Gargiulo, 1987) and stored for at least 24 h at 4 °C or utilized directly without fixation according to
94 Gargiulo et al. (2018) and Gargiulo et al. (2019). In both cases, antheridia were hydrolyzed in 5M
95 HCl at 20 °C for 20 min, rinsed in distilled water for about 5 min before staining with a 0.05% Azur
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96 A solution in McIlvaine buffer at pH 4 and squashed on a slide (Gargiulo et al., 2019). The
97 observation was conducted with a microscope Leica Aristoplan and images were grabbed with a
98 digital camera Leica DFC500.
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99 The karyotype was determined by examining at least five clear metaphase plates used for the
100 measurements. To estimate karyotype asymmetry, the Coefficient of Variation of Chromosome
101 Length (CVCL) and the Mean Centromeric Asymmetry (MCA) values were calculated according to

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102 Peruzzi and Eroǧlu (2013). The methods proposed by Paszko (2006) and Huziwara (1962) were
103 also valued. KaryoType software was employed for chromosome identification, size measures and

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104 karyotype asymmetry indices (Altinordu et al., 2016). Haploid idiograms for each studied
105 population were built, pooling the mean absolute length of the long (L) and short (S) arms of
106 chromosome pairs of at least five metaphase plates. When possible, chromosome data on additional

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107 C. connivens populations from other localities were obtained from those reported in the literature or
108 measuring the chromosomes from the original images (Altinordu et al., 2016; Peruzzi et al., 2017)
109 (Tabs 1, 3). In this last case, only good metaphase plates with magnification or scale bar indicated
110 were considered (Peruzzi et al., 2009). An average karyotype (C_con_En Nakhla_Morocco_m) was

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111 constructed for the studied C. connivens population to compare it with those of the other taxa

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112 reported (Tab. 3). The nomenclature of the chromosome follows that recommended by Levan et al.
113 (1964).
114 The DNA amount was estimated by Image Cytometry (IC) on material previously treated for
Feulgen-densitometric measurements using the interphase-peak method (Vilhar et al., 2001). The
115
116
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Feulgen reaction procedures were performed according to Greilhuber and Temsch (2001). The
117 image analysis system was assembled according to Gargiulo et al. (2018). Calibration and
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118 evaluation of the system were performed as described in Vilhar and Dermastia (2002). Our system
119 shows CV= 2.5% for the uniformity test, R2= 0.993 for the linearity test, CV= 1.2% for the shading
120 test and CV= 1.1% and CV= 0.6%, respectively, for noise and drift over time in the stability test.
121
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122 3. Results
123 The Moroccan plants were dioecious from 10 to 40 cm high, and delicate green in color (Fig. 2).
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124 They presented a triplostichous corticated axis to 0.5- 0.8 mm in diameter (Figs 4-6). The stipulodes
125 developed as small dots, hardly visible (Fig. 4). The spine cells were absent or reduced to tiny dark
126 green warts (Fig. 6). The branchlets were totally corticated except the ultimate acute tip. They were
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127 typically incurved in male plants (Figs 2, 3). Gametangia were isolated; the oogonia were up to 1.2
128 mm high, including the coronula (Fig. 5); the antheridia were very large (600-800 µm in diameter)
129 and easily visible to naked eyes on the plants (Figs 2, 3). They originated from a nodal cell by
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130 successive mitosis. The nodal cell, by regular divisions in various planes, gives rise to a complex
131 spherical multicellular structure delimited externally by eight large characteristic shaped cells, the
132 shield cells (Figs 3, 7). Connected to the shield cells (SC), eight longitudinally elongated cells (the
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133 manubria, M) are recognizable internally (Fig. 8). A variable number of capitular cells (C)
134 originated from these last cells (Figs 8, 10). The terminal one (Caf), in turn, produces the mother
135 cells of antheridial filaments (Af, Figs 8-10). Each filament was the result of successive mitotic

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136 divisions of these cells by forming a transverse wall perpendicular to the long axis of the cell (Figs
137 9, 10). As the filaments elongate, the cells all become progressively smaller. The most common

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138 course of this proliferative period was the development of filaments composed of 64 to 128 cells
139 (Fig. 8).
140 Cytokinesis was regular in most of the divisions forming the filaments; however, spontaneous

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141 anomalies were detected in some cells (Fig. 11a, b, c). Obliquely dividing planes originating
142 asymmetrical cells were observed in terminal or intercalary positions in growing filaments. In these
143 cells, the nuclei could differ in size, too (Fig. 11a, c). Telophases indicating the possible presence
144 of spindles parallel to the smallest axis of the cell were also found (Fig. 11b, c). Moreover, some

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145 nuclear morphologies suggesting the processes of amitotic division were evident in some antheridial

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146 cells (Fig. 11b). Formation of micronuclei of different sizes has also been detected (Fig. 12).
147 Karyokinetic anomalies, such as laggard chromosomes, were observed at metaphase and anaphase
148 stages (Fig. 13a, b). Different anomalies could be present in the cells of the same filament. Nuclei
with different sizes, morphology and DNA content were observed in cells of the same antheridium
149
150
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(Figs 14-15). Mature gametes with a spirally or hook-shaped nuclei represented the 1C value (Fig.
151 14a; Tab. 2). While the usually spherical nuclei in both categories of capitular cells, those conjoined
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152 with manubria (Cam) and those with antheridial filaments (CAf), presented a DNA level ranging
153 from 4C to16C and from 2C to 4C, respectively (Fig. 14b-e; Tab. 2). Almost spherical to
154 progressively parallelepiped-shaped nuclei were observed in the manubria showing a DNA level
155 ranging from 32C to 128C (Fig. 14f-h; Tab. 2). Horseshoe-shaped nuclei were characteristic of
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156 mature shield cells; their ploidy level was 64C up to 128C (Fig. 15; Tab. 2).
157 A histogram obtained from Image Cytometry data of nuclei from antheridial cells revealed several
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158 peaks arranged in an endopolyploidy-like fashion resulting in 8 peaks that corresponded from 1C up
159 to 128C values (Fig. 16). However, the ratio between peak positions was irregular between
160 respective peaks. Comparing the expected values with those obtained for each peak showed a
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161 progressive decrease in the DNA amount in the nuclei after any successive endoreplication cycle
162 (Fig. 17). The 1C-value of 4.47 pg was acquired from nuclei of antheridial filaments at a mature
163 stage of spermiogenesis in the studied population. The Chara spp 1C-values in picograms, known
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164 in the literature, are presented in Fig. 18 and Table 2. The coefficient of variation of 1C peak in the
165 Moroccan populations for each measured slide was always under the suggested limit (CVp < 6%).
166 The metaphase plates of all the studied individuals showed a n = x =14 chromosome number (Figs
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167 19a-e; 20a-e). All had a karyotype organization consisting of decreasing chromosomes in size with
168 a centromere presumably localized in the median position (Figs 19a-e; 20a-e; Tab. 3). The haploid
169 idiograms of the studied individuals are showed in Fig. 20a-e. The mean karyotypes value of the

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170 Moroccan population (C_con_En Nakhla_Morocco_m) presented 52.57 ± 10.19 µm total
171 chromosome length, 3.9 ± 1.4 largest to smallest chromosome ratio, and being included in the 1B

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172 classification category of Stebbins (1971).
173 The haploid chromosome number, karyotype features and asymmetric indexes of the studied
174 population, with those obtained from literature and re-examining the original images of other

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175 populations, are reported in Table 3.
176
177 4. Discussion
178 Systemic polyploidy plays a prominent role in determining the dynamics of growth and

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179 differentiation in the cells of an organism (D’Amato, 1984; Leitch and Dodsworth, 2017). In Chara,

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180 changes in genome size by endoreduplication in nodal cells operate to synchronize their
181 developmental behavior to build a complex structure such as the antheridium (Maszewski, 1991).
182 Several rules common to all the investigated Chara species have been found to control the
consecutive developmental stages of their male reproductive structures (Maszewski, 1991;
183
184
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Maszewski and Kołodziejczyk, 1991). Some differences, basically of a quantitative nature, seem
185 present among species due to the diverse numbers of endocycles undergone by various somatic cell
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186 lineages resulting from the haploid initials at the start of antheridial construction. The estimated
187 endocycle sequences in the C. connivens antheridia for the predominant ploidy levels expressed in
188 C parameters for the various cell lineages were similar to those reported for C. tomentosa (Tab. 2;
189 Maszewski, 1991). The mean 1C-value in Chara spp. is 10.42 pg, varying between a minimum of
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190 1.92 pg reported in a population of C. braunii Gmelin (Nishiyama et al., 2018) and a maximum of
191 19.60 pg in a population of C. contraria Kützing f. caespitosa Migula (Maszewski and
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192 Kolodziejczyk, 1991) (Fig. 18; Tab. 2). According to Maszewski (1991), dioecious species with low
193 DNA C-values display higher values of endopolyploidy in antheridia cells than those estimated for
194 monoecious species (Tab. 2). Most monoecious Chara species have chromosome numbers multiple
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195 of those found in dioecious species with n = 14 (Casanova, 2015). C. connivens has a low 1C value
196 (4.47 pg) and a chromosome number n = 14, as reported for the other studied dioecious species
197 (Fig. 18; Tab. 2). This data seems to support the negative correlation between species-specific
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198 haploid levels of DNA (genome sizes) and the attained upper limits of endonuclear polyploidization
199 within antheridial non-generative cells. The finding of the lowest DNA value in a monoecious
200 species such as C. braunii (Nishiyama et al., 2018) opens some doubts about the supposed close
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201 connection of DNA amount with the mono- and dioecious mode of sexuality. According to
202 Nishiyama et al. (2018), there are no known dioecious sister taxa to C. braunii, perhaps due to the

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203 already reduced genome. However, no data are available on the endoreduplication pattern in non-
204 generative cells of this species, so further studies are necessary.

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205 Of interest in C. connivens is that the DNA amount, during each endo-cycle, does not fit the
206 geometrical order which should be found if the total genome is replicated (Fig. 17), suggesting a
207 possible presence of a DNA rearrangement mechanism. DNA remodeling during endoreplication

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208 appears to be a solid developmental characteristic in orchids (Bory et al., 2008; Brown et al., 2017).
209 Additional analyses must be performed to verify this assumption. However, if this hypothesis is
210 confirmed, the antheridia may provide a valid experimental model for such studies because of their
211 functional and structural characteristics and the simplicity of isolating them.

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212 Spontaneous or induced mitotic and cytokinetic anomalies have been reported in antheridial cells of

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213 several charophycean species (Karling, 1927; Mendes, 1946; Gillet, 1960; Guerlesquin, 1967;
214 Sarma and Singh, 1977; Bhatnagar and Johri, 1985; Subramanian, 2018). In C. connivens, we
215 observed spontaneous irregularities in the division of originating asymmetrical cells. In some of
these cases, the nuclei had different sizes. Similar anomalies were reported in other Characeae
216
217
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species (Karling, 1927; Mendes, 1946; Corillion and Guerlesquin,1969). According to Karling
218 (1927), small and large nuclei formation was probably due to the irregular distribution of
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219 chromosomes in anaphase. In the studied population, we observed laggard chromosomes at the
220 metaphase and anaphase stages and micronuclei production, which could justify in part the different
221 chromosome number distribution in the nuclei.
222 Amitosis in Chara is a common occurrence in the Charales somatic cells and was observed by
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223 many workers (Sundaralingam 1947, Gillet 1959, Shen 1967; Pal and Chatterjee, 1989; Maszewski,
224 1991; Vouilloud et al., 2007). The only report of amitosis in antheridial filaments of Characeae was
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225 that of Pal and Chatterjee (1989), who induced the switching over of mitotic division to amitotic
226 division in the antheridial cells by a photosynthesis inhibitor (Diuron). Spontaneous amitosis in
227 antheridial filaments was not reported yet. In our population we observed some nuclear
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228 morphologies that strongly suggest a processes of amitotic division in antheridial cells. However,
229 further observations seem necessary.
230 Polyploid and aneuploid numbers (n = 12, 16, 18, 21, 28) were also documented, during the time, in
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231 C. connivens populations from different localities (Guerlesquin, 1967, 1996; Trbojević et al., 2020).
232 The n = 21 report was observed for a Serbian population collected, in 1983, in a channel near Silver
233 lake (Blaženčić, 2014). No herbarium specimens are available, and no new findings in this locality
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234 have been observed since 1983; however, Trbojević et al. (2020) hypothesized, utilizing
235 unpublished drawings of the Silver lake material, that this old record was the same species as they
236 found in Dulin pond (Serbia). Using the matK gene for DNA barcoding analysis, they noted that the

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237 new population did not cluster with other C. connivens; they advanced the possibility that it may
238 belong to a new, hitherto undescribed species. Unfortunately, no karyological data are available for

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239 this population. Other morphological, karyological and molecular observations on the Serbian
240 populations seem necessary before understanding their taxonomic status and chromosome number.
241 Guerlesquin (1996), in her updated list of chromosome numbers in Characeae, reported n = 28 as

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242 new chromosome data for C. connivens. The information was achieved from Bhatnagar and Johri
243 (1987), who examined an Indian population identified in their study as Chara vulgaris f. contraria
244 (A. Br. ex Kütz.) R.D.W. (= Chara contraria A.Braun ex Kützing). We also believe in this case,
245 that the attribution to C. connivens of the Indian population must be confirmed.

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246 Karyotype analysis relative to the haploid complements of all the studied C. connivens populations

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247 showed the absence of evident karyomorphological variations (Tab. 3). The differences seem
248 attributable to the degree of chromosome contraction as evident by the analysis of the absolute
249 chromosome length values. All the asymmetric indices utilized suggest that the karyotypes are
highly symmetric. Slight differences were detected in the karyotypic formulas among the different
250
251
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populations (Tab. 3). Intrachromosomal (MCA) and interchromosomal (CVCL) values of all the
252 studied individuals also showed heterogeneity within each complement in terms of chromosome
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253 length (Fig. 21a, b). The analysis of plotted CVCL vs. MCA and CVCL vs. CVCI values for each
254 population confirms that the differences between the karyotypes of the distinct chromosome
255 complements are due more to variation in the chromosome sizes than in the position of the
256 centromere. However, to validate our tentative reconstruction of C. connivens karyotype, many
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257 more chromosome observations on different populations, in particular from the area type, seem
258 necessary.
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259 In conclusion, basic cytogenetic information is necessary to reveal Characeae's biological processes.
260 Such data combined with a phylogenetic approach will offer better perceptions of the mechanisms
261 which have shaped the evolution of this exciting group of organisms.
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262
263 Acknowledgments
264 We sincerely thank Prof. Cecilia Totti and Prof. Elisabeth Lambert for their help getting some
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265 difficult-to-find bibliographies. Moreover, we thank Dr. Andreas Polyviou for improving the
266 English of this manuscript. The collaboration between the Italian and Moroccan researchers of the
267 two relative Universities was achieved thanks to an ERASMUS mobility project (ERASMUS+
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268 KA107 – A.A. 2021/2022).

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269 This research did not receive any specific grant from funding agencies in the public, commercial, or
270 not-for-profit sectors. The study was carried out in the framework of the Pietro Castelli Botanical

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271 Garden Aquatic Plant Project, University of Messina.
272
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