Background

Background on chromosome counting in Zygnema

Zygnematophyceae are the immediate sister group to land plants, thought to have colonized land 550 million years ago (Wodniok et al., 2011; Leebens-Mack et al., 2019). Data on nuclear DNA content are available for Zygnema (Čertnerová, 2021; Feng et al., 2021 and 2023), and genome sizes for different strains of Zygnema spp. have been established, but these show great variation (Feng et al., 2023). Chromosomes of Zygnema have previously been illustrated by transmission electron microscopy (Bakker and Lokhorst, 1987), although without giving actual numbers. The actual chromosome numbers of Zygnema vary drastically, ranging from 14 to 82 (Kurssanow, 1911; Miller 1973; Guiry et al., 2022). Thus, it is unclear if polyploidization has happened. Traditional counting methods established for higher plants are hampered in green algae by pectins [massive homogalacturonan accumulations have been reported by Herburger et al. (2019)] and arabinogalactan proteins in the cell wall. Even though numerous protocols have been established for chromosomal staining in green algae (Godward, 1948; Wittmann, 1965; Prasad and Godward, 1966; Staker, 1971; Gerlach, 1977; Fujii and Guerra, 1998), they still face difficulties handling Zygnema spp., as their chromosomes are small, sticky, and mostly only able to be counted in their mid- to late-prophase. Furthermore, conventional staining methods lead to over-staining of the cell wall and DNA-dense areas in the cytoplasm known as karyoide in Zygnema, Charophyta (Kopetzky-Rechtperg, 1934).

Modification of the methods from Wittmann (1965), Staker (1971), and Fujii and Guerra (1998)

For all three Zygnema ssp., two different modified staining procedures were used: (1) acetocarmine (Figure 1A–1E) and (2) haematoxylin (Figure 1A, 1F–1L). To achieve full mitotic synchronization in Zygnema, a light/dark cycle (10:14 h) was applied following Staker (1971), i.e., with a longer dark period than our standard cultivation cycle (light/dark, 16:8 h).

Figure 1. Summary of the experimental procedure for acetocarmine (B–E) and haematoxylin (F–L) staining in Zygnema circumcarinatum.(A) Erlenmeyer flask with young culture; (B) 8-hydroxyquinoline prefixation; (C, F) fixation in Carnoy’s fluid, illustrating the gradual bleaching from time 0, 1, and 12 h (from left to right); (D) staining with acetocarmine; (E) projected Z-stack rendered by Helicon Focus software used for counting the chromosomes (circle: chromosomes marked with green crosses in ImageJ; inset: symbolic representation of individual images for Z-stacks); (G) algal biomass mounted in acetic acid and squished between two slides; (H) slides dipped in liquid nitrogen; (I) HCl treatment; (J, K) aceto-haematoxylin-iron alum staining; (L) light microscopic image used for quantifying the number of chromosomes (circle). Scale bars = 10 µm.

For the staining of the chromosomes, Staker (1971) used propriocarmine, which was replaced with 1% acetocarmine in the current protocol; also, the Zygnema filaments were not chopped or randomized, which would lead to complete destruction. Moreover, prior to the fixation in Carnoy’s fluid, cells were treated with 8-hydroxyquinoline to depolymerize microtubules, resulting in sticky and condensed metaphase chromosomes (Bukhari, 2004). Haematoxylin staining by Wittmann (1965) was used, but the treatment with chloralhydrate and slide heating steps was omitted and replaced with hydrolysis by 5 N HCl (Fujii and Guerra, 1998), resulting in contrast improvement between chromosomes and cytoplasm. Changes only occurred with the algal filaments being placed between two microscopic slides before liquid nitrogen treatment with a freeze-shattering method (Wasteneys et al., 1997); the HCl treatment time was reduced to 10 min to assure the integrity of the Zygnema filaments, which disintegrate after prolonged treatment.