PHYLOGENY AND SYSTEMATICS OF THE GENUS MONOMORPHINA (EUGLENACEAE) BASED ON CYTOPLASMIC SSU rDNA SEQUENCE ANALYSIS

Sylwia Kosmala, Rafał Milanowski, Kamil Brzóska, Małgorzata Pękala, Jan Kwiatowski and Bożena Zakryś

Department of Plant Systematics and Geography, Warsaw University, Al. Ujazdowskie 4,

PL-00-478 Warszawa, Poland

FIGURES

Fig. 1. Light microscope photographs showing an overview of living cells and chloroplast organization for Monomorphina pyrum, Monomorphina aenigmatica and Cryptoglena skujai. (a –  d) Pear-shaped, ovoid cells of the strain M. pyrum ACOI-2778 ending with a sharp, hyaline tail; pellicle has innumerous conspicuous hyaline keels (arrows). (a – c) A single, spherical chloroplast with irregular, small holes visible in young cells (arrowheads). (d) In old cells, the single chloroplast disintegrates into more or less numerous lobes of different size, which appear as separate entities. (e, f) Cells of M. pyrum ACOI-2583. (e) Disintegration of a chloroplast – deep indentations divide the uniform surface of a chloroplast into long lobes. (f) The cross-section of a young cell with a visible, centrally located nucleus and a single parietal chloroplast; small but conspicuous paramylon plate is visible between the chloroplast and the periplast. (g – i) Lengthy/pear-shaped, ovoid cells of M. pyrum ACOI-2266 with prominent (sharp) periplast ribs (arrows). (g) A large nucleus visible in a cross-section of the cell with an orbicular chloroplast located between the nucleus and the periplast. (h) Small perforations visible within the chloroplast (arrowheads). (i) Advanced chloroplast disintegration – numerous lobes appear as separate fragments. (j) Pear/spindle-shaped, distinctly oblate cell of M. pyrum ACOI-2338, with small, noticeable perforations (arrowheads). (k) Two chloroplast lobes, connected to one another by means of narrow bridges (arrowhead) in the cell of M. pyrum ACOI-2801, which appear as two separate structures. A conspicuous paramylon plate is located between the chloroplast and the periplast. (l) Apparent  disintegration of the chloroplast into two lobes inside the pear/spindle-shaped cell of  M. pyrum CCAC-0093. (m) The cell of M. pyrum ACOI-2669 with two spherical structures (haplopyrenoids ?) (arrowheads); a conspicuous, lateral paramylon plate (arrow) is located between the periplast and the chloroplast. (n – o) Cells of M. pyrum AICB-511. (n) A perforated chloroplast and three spherical structures (haplopyrenoids ?) are visible inside the cell (arrowheads). (o) Four spherical structures (haplopyrenoids?) are noticeable in the cell. (p, r) Pear-shaped cells of  M. aenigmatica  CCAP 1261-9 with a hyaline spine at the posterior end. (p) Optical cross-section of the cell with a centrally located nucleus and a parietal, spherical chloroplast with haplopyrenoids present on its inner side (arrowheads); three conspicuous lateral paramylon plates are located between the periplast and the chloroplast (arrows). (r) A pellicle with numerous spiral stripes (arrow) and the parietal chloroplast with numerous small perforations (arrowhead). (s) The cell of Cryptoglena skujai SAG-10.88, resembling a coffee bean, without conspicuous, hyaline posterior tail; a single, parietal chloroplast forms an open cylinder (in the shape of the letter „C”); two lateral shield-shaped paramylon grains are located between the chloroplast and the periplast (arrows). N, nucleus; C, chloroplast. Scale bars, 10 µm.

Fig. 2. Confocal electron microscope photographs showing chloroplast organization of Cryptoglena skujai and of representatives of the genus Monomorphina. (a, b) A single, parietal chloroplast forms an open cylinder (in the shape of the letter „C”) in cells of Cryptoglena skujai. (a) A chloroplast discontinuity (furrow) (arrow) is visible on the ventral side of the cell. (b) Relatively large holes are visible in the chloroplast (arrowhead). (c – e) M. aenigmatica CCAP 1261-9. (c, d) A single, closed (orbicular) chloroplast with numerous, relatively large holes throughout the whole surface (arrowheads). (e) Optical cross-section through the cell showing parietal localization of the chloroplast. (f, g) A chloroplast has irregular, small holes (arrowheads) and its surface is folded following the curvature of periplast ribs in young cells of M. pyrum CCAP-1261/4b. (h, i) Holes of various size and shape (arrowheads) in the chloroplast of M. pyrum ACOI-2583. (j – l) Cells of M. pyrum ACOI-2778. (j, k) Folded surface of the chloroplast with small perforations. (l) Optical cross-section of the cell showing the single, parietally located, spherical, closed chloroplast. (m – p) M. pyrum ACOI-2338. (m) Fragment of the folded surface of a chloroplast with a few, small perforations. (n, o) Numerous, but small perforations of a chloroplast. (p) Optical cross-section of a cell showing the single, parietally located, spherical chloroplast; two shield-like paramylon grains are visible between the chloroplast and the periplast (arrows). (r – t) Surface of the M. pyrum ACOI-2801 chloroplast with visible perforations. (u-x) M. pyrum ACOI-2266. (u) Disintegration of a chloroplast into lobes (ch) connected to one another by means of narrow bridges (arrowhead). (w) Disintegrated chloroplast appears as numerous, separate entities (x) Optical cross-section of a cell showing the single, parietally located, spherical chloroplast (arrow). (y-z) Chloroplast disintegrating inside cells of M. pyrum ACOI-2819. Scale bars, 10 µm

Fig. 3. Confocal electron microscope photographs showing the chloroplast organization of some  representatives of the genus Monomorphina. (a – c) Folded and perforated chloroplast in cells of M. pyrum ACOI-2669. (d-f) M. pyrum AICB-277. (d) A surface fragment of a single disintegrating chloroplast – numerous lobes (ch) connected to one another by means of narrow bridges (arrowheads). (e) Chloroplast with numerous, small perforations. (f) Optical cross-section of a cell showing the single, parietally located chloroplast (arrow). (g – j) M. pyrum AICB-511. (g) Fragment of a folded chloroplast surface with a few small perforations. (h) Progressing chloroplast disintegration – abundant yet tiny openings are visible. (i – j) Beside the small openings, large openings appear (arrowheads). (k – l) Progressing chloroplast disintegration in cells of M. pyrum ACOI-2566. Scale bars, 10 µm

Fig. 4. (Supplemental material). Light microscope photographs showing an overview of living cells and chloroplast organization of Monomorphina pyrum. (a – e) Pear-shaped, ovoid cells of M. pyrum  CCAP-1261/4b ending with a sharp, hyaline posterior tail. (a) pellicle with innumerous conspicuous hyaline keels (arrow). (b, c) Optical cross section of a cell with a centrally located nucleus and a single, parietal chloroplast; paramylon plate (arrow) is visible between the chloroplast and the periplast. (d, e) Surface of a single, parietal and spherical chloroplast with perforations of different sizes (arrowheads); small but conspicuous paramylon plate (arrow) is visible between the chloroplast and the periplast. (f – j) M. pyrum  ACOI-2583. (f) Chloroplast surface with an opening (arrow). (g, h) Optical cross-section of a cell with a centrally located nucleus and a parietal chloroplast; paramylon plates (arrows) are visible between the chloroplast and the periplast. (i) Chloroplast disintegration – deep indentations divide the uniform surface of the chloroplast into long lobes. (j) In old cells the chloroplast disintegrates into more or less numerous lobes of different size, which appear as separate entities; apparent paramylon plate (arrow). (k – o) M. pyrum ACOI-2778. (k, l) A chloroplast with a few small perforations (arrowheads). (m) Optical cross-section of a cell with a centrally located nucleus and a parietal chloroplast. (n) Chloroplast with visible medium-sized opening (arrow). (o) Single chloroplast disintegrating into more or less numerous lobes of different size, which appear as separate entities. N, nucleus; C, chloroplast. Scale bars, 10 µm.

Fig. 5. (Supplemental material). Light microscope photographs showing an overview of living cells and chloroplast organization of Monomorphina pyrum. (a – f) M. pyrum ACOI-2338. (a) Pear/spindle-shaped cell; pellicle with innumerous conspicuous hyaline keels (arrow). (b) Two large paramylon plates are located between the periplast and the chloroplast (arrows). (c) Optical cross-section of a flattened cell viewed from the side; nucleus is visible in the center. (d – f) Chloroplast surface with a visible perforation. (g – l) Subsequent steps of chloroplast disintegration in M. pyrum ACOI-2801 cells. (m – s) M. pyrum ACOI-2819. (m) Pellicle with innumerous conspicuous hyaline keels (arrows). (n – s) Single, parietal chloroplast, perforated in various degrees; conspicuous paramylon plates (arrows) are visible between the chloroplast and the periplast. N, nucleus; C, chloroplast. Scale bars, 10 µm.

Fig. 6. (Supplemental material). Light microscope photographs showing an overview of living cells and chloroplast organization of Monomorphina pyrum. (a – f) M. pyrum AICB-277. (a, b) Pear/spindle-shaped cells ending with a sharp, hyaline posterior tail; pellicle with innumerous conspicuous hyaline keels (arrow). (c) Optical cross-section of a cell with a centrally located nucleus and a single parietal chloroplast. (d – f) Subsequent steps of chloroplast disintegration. (g – l) M. pyrum AICB-511. (g, h) Pear/spindle-shaped cells ending with a sharp, hyaline posterior tail; pellicle with innumerous conspicuous hyaline keels (arrows). (i) Optical cross-section of a cell with a centrally located nucleus and conspicuous paramylon plate (arrow). (j – l) Single, parietal chloroplast with numerous, small perforations. (l) Three circular structures (haplopyrenoids ?) are visible. (m – s) M. pyrum ACOI-2566. (m – p) Pear/spindle-shaped cells ending with a sharp, hyaline posterior tail; pellicle with innumerous, conspicuous hyaline keels (arrowheads); conspicuous paramylon plates located under the pellicle (arrows). (r) Side view of a cell. (s) Optical cross-section of a cell with a centrally located nucleus and conspicuous paramylon plates (arrows). N, nucleus; C, chloroplast. Scale bars, 10 µm.

Fig. 7. (Supplemental material). Light microscope photographs showing an overview of living cells and chloroplast organization of Monomorphina pyrum, Monomorphina aenigmatica and Cryptoglena skujai. (a – f) M. pyrum ACOI-2669. (a, b) Circular structures (haplopyrenoids ?) are visible on the chloroplast (arrowheads). (c) Optical cross-section of a cell with a centrally located nucleus and a single parietal chloroplast; two, small but conspicuous paramylon plates (arrows) are visible between the chloroplast and the pellicle. (d – f) Chloroplast with large perforations (arrowheads). (g – l) M. pyrum ACOI-2266. (g) Lengthy/pear-shaped cell ending with a sharp, hyaline posterior tail; pellicle with innumerous, conspicuous hyaline keels (arrow). (h, i) Optical cross-section of a cell with a centrally located nucleus and a single parietal chloroplast. (j, k) Young cells with small chloroplast perforations (arrowheads). (l) Chloroplast disintegration - numerous lobes, appearing as separate fragments. (m – p) M. aenigmatica CCAP-1261-9. (m, n) Optical cross-section of a cell with a  parietal, spherical chloroplast and haplopyrenoids present on its inner side (arrowheads); three conspicuous lateral paramylon plates are located between the periplast and the chloroplast (arrows). (o, p) Chloroplast with numerous, small perforations. (r, s) Resembling a coffee bean, a cell of Cryptoglena skujai SAG-10.88, without a conspicuous, hyaline posterior tail; one parietal chloroplast forms an open cylinder (in the shape of the letter „C”); two lateral, shield-shaped paramylon grains located between the chloroplast and the periplast (arrows). N, nucleus; C, chloroplast. Scale bars, 10 µm.

Fig. 8. (Supplemental material). Light microscope photographs showing an overview of live, swimming cells of Monomorphina pyrum. (a, b) Pear-shaped, ovoid cells of M. pyrum. (a) strain CCAP-1261/4b. (b) strain ACOI-2778. (c) Lengthy/pear-shaped, ovoid cells of M. pyrum ACOI-2266. (d, e) Pear/spindle-shaped, distinctly oblate cells of M. pyrum. (d) strain ACOI-2338. (e) strain ACOI-2801. (f-i) Pear/spindle-shaped, slightly oblate cells of M. pyrum. (f) strain ACOI-2669. (g) strain AICB-277. (h) AICB-511. (i) strain M. pyrum ACOI-2566. Scale bars, 10 µm.

Fig. 9. The phylogenetic tree of the 18S rDNA sequence obtained by Bayesian inference (model TrNef + G). Numbers at the essential nodes show posterior probabilities of the tree bipartitions as well as the bootstrap values/decay indices obtained for the main clades by MP analysis and bootstrap values obtained by NJ and ML analysis (model GTR+I+G). Branches leading to nodes with support of less than 50% are collapsed. For strains of M. pyrum respective symbols from culture collections were given.