Spongiosarcinopsis terrestris Temraleeva et al., 2018

Temraleeva, Anna, Moskalenko, Svetlana, Mincheva, Elena, Bukin, Yury & Sinetova, Maria, 2018, Spongiosarcinopsis terrestris gen. et sp. nov. (Chlorophyta, Chlorophyceae): a new genus of green algae from gray forest soil, Russia, Phytotaxa 376 (6), pp. 291-300 : 296-299

Spongiosarcinopsis terrestris Temraleeva et al. , gen. et sp. nov. ( Fig. 1A–F )

Type species: — Spongiosarcinopsis terrestris Temraleeva, Moskalenko, Mincheva, Bukin et Sinetova sp. nov.

LM and TEM observations: —Solitary cells are ellipsoidal to spherical when young, 5–10 μm in diameter, covered with thin smooth envelope. Each young cell contains one relatively big nucleus, one parietal chloroplast with one pyrenoid covered with starch envelope and one vacuole ( Fig. 1 A, B ). Solitary cells are derived from zoospores or aplanospores ( Fig. 1 C ). Mature cells in 3-month-old cultures are ellipsoidal, ovoid to irregular in shape, up to 22 μm in max. dimension, mutually combined into diads, tetrads, or packets resulting from desmoschisis; the smooth and homogeneous cell wall in actively growing culture is 0.5 μm thick, and thicken to 5 μm in 6-month-old cultures ( Fig. 1 D–F ). Mature cells remain uninucleate, with one spongy chloroplast possessing one to five pyrenoids in its median thickened part ( Fig. 1 D–E ). Еach pyrenoid is covered by a prominent segmented starch envelope and penetrated by curved thylakoids ( Fig. 2 A–D ). On the cross-sections of mature cells thylakoids are organized in bundles of different thickness which occupy most of the chloroplast area. The color of the algal mass in older cultures changes to orange, red oblong akinetes with a smooth layered cover are observed.

Asexual reproduction is possible by desmoschisis ( Fig. 2 D ) or zoospores and aplanospores ( Fig. 2 E–G ). Aplanosporangia contain eight to 32 spherical aplanospores, each 4.5–6 μm in diameter, covered with a thin smooth cell wall ( Fig. 2 F, G ). Aplanospores differ from mature cells by naked pyrenoids without a starch envelope. Zoospores are naked with two flagella of equal length, ellipsoidal with rounded ends, 7.8–8.1 х 3.6–4.1 μm. After a short moving period, they rapidly become spherical, 5.0–5.2 μm in diameter. The chloroplast is shallow cup-shaped or saucershaped. The pyrenoid is poorly observable. The stigma is small and located in the anterior end of the chloroplast. Sexual reproduction was not observed.

Thus, from genera Chlorosarcinopsis , Chlorochytrium and Chlorosphaeropsis our strain differs by a spongy chloroplast, and from the last - also by the uninuclear vegetative cells. The sarcinoid alga of the genus Spongiococcum also has similar chloroplast type. However, a new genus has naked zoospores without cell wall, in contrast to covered zoospores of genus Spongiococcum .

Habitat: —Gray forest soil.

Distribution:— Russia.

Etymology: —The generic name is derived from spongy type of a chloroplast and ability to a desmoschisis, and the specific epithet means ‘terrestrial’ in Latin.

Cultures: —authentic strain ACSSI 023.

18S rRNA: — Spongiosarcinopsis forms a well-supported (1.00 posterior probability) clade ( Fig. 3 ) within the Protosiphonaceae and contains a total of three OTUs: the studied strain and two isolates, identified as Chlamydomonad sp. (≥99.5% similarity). It is difficult to assume whether strains Pic8/18P-5w and Pic8/18P-3d are of the species S. terrestris or belong to another species of genus Spongiosarcinopsis , since the 18S rRNA marker may be highly conserved for the species separation, and any information about their morphology and secondary structure ITS2 is lacking. The sister taxon of new proposed genus is Chlorochytrium genus, including C. lemnae (type species) and C. hypanicus . All members of Chlorochytrium , but not Spongiosarcinopsis , contain an intron in this gene. All Spongiosarcinopsis strains were 99.9% similar ( Table 2). The representative sister taxa Chlorochytrium were 98.1% similar to genus Spongiosarcinopsis . For some genus from family Protosiphonaceae the low level of intergeneric differences is typical because the similarity in 18S rRNA gene for Protosiphon and Spongiochloris is 98.4–98.6%, for Chlorosphaeropsis and Spongiochloris —98.5–98.6%, for Chlorosphaeropsis and Protosiphon - 99.7%. Alongside with that, the above mentioned taxa have clear differences in morphological organization of thallus, with siphonal in Protosiphon , coccoid in Spongiochloris , and sarcinoid in Chlorosphaeropsis . Since the 18S rRNA gene is very conservative and can distinguish the closely related taxa poorly, we additionally used the ITS2 as a varying marker.

ITS2 Secondary Structure: —The ITS2 sequence of the new algal genus has a length of 263 nucleotides and folds into the usual cloverleaf secondary structure, which shows distinct hallmarks for Chlorophyceae (i) four main helices, (ii) with helix III being the longest, (iii) the presence of a characteristic motif at the apex of helix III, and (iv) a pyrimidine-pyrimidine mismatch in helix II ( Schultz et al. 2005, Fig. 4 ). Unfortunately there is no ITS2 sequence of Chlorochytrium , deposited into the GenBank. Figure 4 also shows all of the CBCs recorded in the conserved regions of Helix II and Helix III of the ITS2 secondary structure of Spongiosarcinopsis , compared to the ITS2 secondary structure of another closely related genus from Protosiphonaceae — Spongiochloris . Spongiosarcinopsis terrestris differs from Spongiochloris spongiosa by two CBCs of conserved ITS2 regions and conserved motif in the 5′ side part near the tip of Helix III ( Fig. 4 ). The lengths of ITS2 S. spongiosa and S. terrestris were 244 and 263 bp, respectively.

Ecology: —The strain ACSSI 023 was isolated from gray forest soil while other two members of the new genus (isolates Pic8/18P-5w, Pic8/18P-3d)—from the park lake. The species of genus Chlorochytrium occur only in fresh water, as well as an algae Pachycladella umbrina . However the most of members of the Stephanosphaerinia clade were isolated from soil, for example the strains of genera Chlorosarcinopsis , Protosiphon , Chlorosphaeropsis , Spongiochloris , and others ( Table 1, Fig. 3 ).

Figures

FIGURE 1. LM micrographs of Spongiosarcinopsis terrestris, strain ACSSI 023. A. Young vegetative cells derived from zoospores. B. Spherical cell in actively growing culture. C. Aplanospores and aplanosporangium. D, E. Mature vegetative cells arranged in diad and tetrad aggregation in 3-month-old cultures. F. Akinetes and the accumulation of secondary carotenoids, which were detected by changes in the color of the algal mass to orange in old cultures (7 months). Scale = 10 μm.

FIGURE 2. TEM images of Spongiosarcinopsis terrestris, strain ACSSI 023.A. Single young ellipsoidal cell just derived from a zoospore or aplanospore. B. Single young sphaerical vegetative cell. Each young cell possess a chloroplast with one pyrenoid covered by a starch envelope. C. Single mature vegetative cell with a chloroplast containing three pyrenoids, each covered with numerous starch plates. D. Thick cell-walled mature cells combined into a diad resulting from desmoschisis. In addition to starch plates around pyrenoids, smaller starch grains are distributed in the chloroplast stroma. E, F Aplanosporangia containing aplanospores covered by thin cell walls. G. Released aplanospores. A = aplanospore; C = chloroplast, N = nucleus, P = pyrenoid, S = starch grains in the chloroplast stroma; SE = starch envelope of the pyrenoid, V = vacuole, W = cell wall. Arrows indicate thylakoids penetrating into the pyrenoid matrix. Scale bar is 1 μm for A–C and 2 μm for D–G.

FIGURE 3. Phylogenetic analysis based on 18S rRNA gene sequences data demonstrating position of Spongiosarcinopsis gen. nov. within the family Protosiphonaceae. The tree is based on Bayesian topology. The support values are given for Bayesian posterior probabilities, and maximum likelihood (BI/ML). The cut-off values for probability and bootstrap are 0.5 and 50%, respectively. Hyphen (-) indicates unsupported node. Our reference strain ACSSI 023 (MF687231) is printed in bold. Black circles indicate which sequences originate from terrestrial (aerophytic/soil), white circles indicate which ones are from aquatic habitats (freshwater), gray circles indicate missing information.

FIGURE 4. Comparison of the ITS2 secondary structures of Spongiosarcinopsis terrestris ACSSI 023 (core structure) and Spongiochloris spongiosa SAG 2469. The CBCs are noted in boxes, CBCs of the conserved regions are marked with arrows, characteristic motifs at the apex of helix III ITS2 are underlined.