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FIGURE 8 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 8. Cultured material of Cladophoraceae sp. 2 (Rhizoclonium minutissimum). (A, B) Morphology of filamentous thalli. (C) Filamentous thalli with rhizoid-like structure. (D, E) Spindle- and band-shaped chloroplasts with pyrenoids (arrowheads). (F) Autofluorescence of chloroplasts. (G) Cells stained with DAPI, showing nuclei. (H) Cells stained with Lugol's iodine, showing the pyrenoids. (I) Mature sporangia with spores and vegetative cells. Arrowhead points to a mature sporangium. (J) Detail of a hapteroid attachment cell. Scale bar = 20 μm (A–C, G–I); Scale bars = 10 μm (D–F, K).

FIGURE 10 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 10. Cultured material of Cladophoraceae sp. 3 (Rhizoclonium umbraticum). (A, B) Morphology of filamentous thalli. (C) Tip of filamentous thalli. (D) Filamentous thalli with rhizoidal structures. (E–G) Spindle- and band-shaped chloroplasts. (H) Autofluorescence of chloroplasts. (I) Cells stained with DAPI, showing nuclei. (J) Cells stained with Lugol's iodine, showing the pyrenoids. (K) Detail of hapteroid attachment cell. Scale bars = 10 μm (A, B, D, F–H, K); Scale bars = 20 μm (C, E, J).

FIGURE 7 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 7. Field-collected specimens of Cladophoraceae sp. 2 (Rhizoclonium minutissimum). (A) Field-collected specimens. (B, C) Morphology of field-collected specimens. (D, E) Filamentous thalli with non-septate rhizoids. (F) Tip of filamentous thalli. (G–I) Spindle- and band-shaped chloroplasts. Arrowhead points to a pyrenoid. (J) Autofluorescence of chloroplasts. (K) Cells stained with DAPI, showing nuclei. (L) Cells stained with Lugol's iodine, showing pyrenoids. (M, N) Pyrenoids observed by transmission electron microscopy. Pyrenoids have starch plates. (M) Three or two thylakoid membranes traversing a pyrenoid. (33) One thylakoid membrane. Scale bar = 5 mm (A); Scale bars = 40 μm (B, C); Scale bars = 20 μm (D–G, K, L); Scale bars = 10 μm (H–J); Scale bars = 0.5 μm (M); Scale bars = 1 μm (N).

FIGURE 5 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 5. Field-collected specimens of Cladophoraceae sp. 1 (Rhizoclonium fractum). (A, B) Field-collected specimens. (C–E) Range of morphological types in field-collected specimens. (E) Arrowhead pointing to rhizoidal structure. (F, G) Cells of field-collected materials. (H, I) Spindle- or band-shaped chloroplasts. Arrowheads indicate pyrenoids. (J, K) Autofluorescence of chloroplasts. (L) Cells stained with DAPI, showing nuclei. (M) Cells stained with Lugol's iodine, showing the pyrenoids. (N) Polypyramidal pyrenoid observed by transmission electron microscopy. Scale bar = 5 mm (A, B,); Scale bars = 20 μm (C–F, L, M); Scale bar = 10 μm (G–K).; Scale bar = 0.5 μm (N).

FIGURE 4 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 4. Maximum likelihood (ML) tree for Cladophorales developed using large subunit ribosomal RNA gene (LSU) rDNA sequences. ML bootstrap values (>50) and Bayesian inference posterior probabilities (>0.90) are indicated at branches. Scale bar = 0.02 substitutions per nucleotide site. Asterisks indicate the type species of each genus.

FIGURE 6 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 6. Cultured material of Cladophoraceae sp. 1 (Rhizoclonium fractum). (A–D) Morphologies of growing filamentous thalli. (A, B) Some of the thalli have rhizoidal structures. (E, F) Chloroplasts with many starch granules. (G) Autofluorescence of chloroplasts. (H) Cells stained with DAPI, showing nuclei. (I) Cells stained with Lugol's iodine, showing the pyrenoids. Scale bar = 20 μm (A–C, E–G); Scale bars = 40 μm (D); Scale bars = 10 μm (H, I).

FIGURE 3 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 3. Maximum likelihood (ML) tree for Cladophorales developed using the small subunit ribosomal RNA gene (SSU) rDNA sequences. ML bootstrap values (>50) and Bayesian inference posterior probabilities (>0.90) are indicated at the tree branches. Scale bar = 0.01 substitutions per nucleotide site. Asterisks indicate the type species of each genus.

FIGURE 1 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 1. Fields material of Cladophoraceae spp. (A, B) Cladophoraceae sp. 1 and 2 growing on mangroves. Arrow heads point to (C) Cladophoraceae sp. 3 growing at shady covered conduits.

FIGURE 2 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 2. Map showing the collecting site of Cladophoraceae spp. (1) Tanotsu, Fukuoka, Fukuoka Pref., (2) Kunimikoujiro, Unzen, Nagasaki Pref., (3) Ki-ire, Kagoshima, Kagoshima Pref., (4) Atake River, Tanegashima Is. Kagoshima Pref., (5) Kesaji, Higashi, Okinawa Pref., (6) Oura, Nago, Okinawa Pref., (7) Lake Man, Naha, Okinawa Pref., (8) Miyara, Ishigaki, Okinawa Pref. ○: Cladophoraceae sp. 1 (= Rhizoclonium fractum), O: Cladophoraceae sp. 2 (= R. minutissimum), Δ: Cladophoraceae sp. 3 (= R. umbraticum).

FIGURE 9 in Two new green algae, Rhizoclonium fractum sp. nov. and R. umbraticum sp. nov., from tropical and subtropical brackish waters of Japan

FIGURE 9. Field-collected specimens of Cladophoraceae sp. 3 (Rhizoclonium umbraticum). (A) Morphologies of field-collected specimens. (B, C) Filamentous thalli with non-septate rhizoids. (D–F) Spindle- and band-shaped chloroplasts with pyrenoids (arrow heads). (G, H) Autofluorescence of chloroplasts. (I) Cells stained with DAPI, showing nuclei. (J) Cells stained with Lugol's iodine, showing pyrenoids. (K, L) Pyrenoids observed by transmission electron microscopy. Pyrenoids with starch plates. (K) A thylakoid membrane; (L) Three thylakoid membranes traversing a pyrenoid. Scale bars = 80 μm (A–C); Scale bars = 10 μm (D–H); Scale bars = 40 μm (H, J); Scale bars = 0.5 μm (K, L).

FIGURE 4 in Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

FIGURE 4. Phylogenetic tree of the 18S rDNA sequences from Rhizoclonium genus members and relatives. The Bayesian inference (BI) posterior probabilities and bootstrap support based on maximum-likelihood (ML) analyses are shown on the nodes. Values> 0.50 are shown for BI and> 50 for ML.

FIGURE 2. Rhizoclonium pachydermum. A–B. Gross morphology. C in Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

FIGURE 2. Rhizoclonium pachydermum. A–B. Gross morphology. C. Autofluorescence showing the reticulated structure of the chloroplasts. D. Gametangia, where the cell is empty after liberating the gametes through a lateral pore. The arrowhead indicates a domed pore. E. Autofluorescence showing the number of nuclei. Scale bars: A–D = 50 µm, E= 100 µm.

FIGURE 1. Rhizoclonium pachydermum. A. Entire filament. Note the long, almost unbranched filaments. B. Basal region. Note the basal branches and the holdfast. C. Long, almost unbranched filaments. D in Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

FIGURE 1. Rhizoclonium pachydermum. A. Entire filament. Note the long, almost unbranched filaments. B. Basal region. Note the basal branches and the holdfast. C. Long, almost unbranched filaments. D. Showing the number of nuclei. Scale bars: A, C = 500 µm B, D = 100 µm.

FIGURE 5 in Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

FIGURE 5. Phylogenetic tree of the ITS2 sequences of members of Rhizoclonium and Cladophora. The Bayesian inference (BI) posterior probabilities and bootstrap support based on maximum-likelihood (ML) analyses are shown on the nodes. Values> 0.50 are shown for BI and> 50 for ML.

FIGURE 3. A–D in Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

FIGURE 3. A–D. Rhizoclonium riparium HB1302. A–B. Filament with rhizoidal cells. C. The reticulated structure of the chloroplasts. D. Autofluorescence showing the number of nuclei. E–H. Rhizoclonium hieroglyphicum HB1303. E–F. Structure of cells. G. Autofluorescence showing the reticulated structure of the chloroplasts. H. Autofluorescence showing the number of nuclei. Scale bars: A, B, D, E, G, H = 50 µm. C, F = 25 µm.