Nephrocytium agardhianum Nägeli 1849: 79

Liu, Xudong, Zhu, Huan, Liu, Benwen, Liu, Guoxiang & Hu, Zhengyu, 2017, Phylogeny and morphology of genus Nephrocytium (Sphaeropleales, Chlorophyceae, Chlorophyta) from China, Phytotaxa 319 (1), pp. 84-92 : 86-88

Nephrocytium agardhianum Nägeli 1849: 79 , pl. III: C a–h, l–o

Synonyms: — Nephrocytium naegelii Grun. in Rabenh. 1868, Selenococcùs farcinalis Schmidle & Zachar. 1903 ; incl. var. minus Näg.1849

Holotype: — Nägeli 1849: 79, pl. III: C a–h, l–o. Depicted in Komárek & Fott (1983, pl. 157:1).

Description

Colonies were found to be somewhat asymmetrically oval with 8–16 cells helically embedded ( Fig. 7 ). Cells were cylindrical to oval, obviously bent and asymmetric ( Fig. 7 ). Cell size was 12–25 μm long and 6–13 μm wide. The mucilage envelope beside the mother cell wall was more or less present ( Fig. 11 ). Chloroplasts were single-lobed parietal with a single pyrenoid when young and gradually diffused and filled the whole cell when mature (Figs 8,10). Asexual reproduction was by 4–8 autospores ( Fig. 12 ) that were released by dissolution of the expanded mother cell wall. Sexual reproduction and flagellated stages are unknown. When maintained in cultured dishes, colonies were mostly round with fewer cells (mostly 4) (Figs 8,9). and the mature cells were kidney-shaped to oval, with rounded-acuminate to broadly rounded apices ( Fig. 9 ).

When observed with TEM, the surface of daughter cell walls were found to not be smooth ( Figs 16–18 ) and were sculptured by an irregular network of dense and fine ribs 0.1–0.2 μm long, which were not obviously on the mother cell wall (Figs 16,17). The Oocystis -like ultrastructure of the cell wall was not observed. Thylakoids were observed in stacks of two to five, and none penetrated the pyrenoid matrix, which was surrounded by several starch sheathes (Figs 16,17).

Molecular phylogenetics

We obtained 6 gene sequences and combined these with 92 sequences obtained from GenBank, for a total of 36 included taxa. Nephrocytium agardhianum differed from N. limneticum by 42 of 1623 positions of the 18 S rDNA gene with 97% similarity by BLAST searches. Similarly, the species differed at 51 positions of rbc L cpDNA (95% similarity), and 62 bases of tuf A cpDNA were different (93% similarity). Sequences obtained herein were submitted to GenBank under the accession numbers KY 094106– KY 094111.

The concatenated data set (18 S rDNA, rbc L cpDNA and tuf A cpDNA) contained 3263 characters. The aligned 18 S rDNA data set totaled 1515 characters. Among these, 434 sites (28.6%) were variable and 277 sites (18.3%) were parsimony informative. The aligned rbc L cpDNA data set totaled 1041 characters and 409 sites (39.3%) were variable and 343 sites (32.9%) were parsimony informative. The aligned tuf A cpDNA data set totaled 707 characters with 363 variable sites (51.3%), 310 of which were parsimony informative (43.8%).

The phylogenetic trees constructed by ML and BI had similar topologies and only one of them is presented. The 18 S / rbc L / tuf A combined genes tree ( Fig. 19 ) resolved our two newly isolated strains as a well supported new clade in Sphaeropleales . An uncertained Pseudomuriella sp. (AY195974) and genus Chromochloris were found to be the closest relatives to Nephrocytium with good nodal support.

Figures

FIGURE 19. Phylogenetic tree inferred using concatenated genes of 18S rDNA, rbcL and tufA cpDNA, along with sequences of additional taxa of Sphaeropleales. Bootstrap support from maximum likelihood (ML, constructed by PAUP), Bayesian inference (BI) posterior probabilities and bootstrap support from maximum likelihood (ML, constructed by RAxML) are presented on the nodes, in that order. Values above 0.5 for BI and 50 for ML are shown.

FIGURES 1–12. Light microscopy of Nephrocytium limneticum cultured (1–6), and Nephrocytium agardhianum in the field (7), and cultured (8–12). 1. Young cells in colony. 2. Old cells in colony. 3. Autospores in sporangium. 4. Autofluorescence showing the shape of chloroplasts. 5. Negative stain by ink showing the mucilage envelope. 6. Sporangia in colony. 7 Colony in the field habit. 8. Young cells in colony. 9. Old cells in colony. 10.Autofluorescence showing the shape of chloroplasts. 11. Negative stain by ink showing the mucilage envelope. 12. Autospores in a sporangium. Scale bar 10 μm.

FIGURES 13–18. Transmission electron microscopy of Nephrocytium limneticum (13–15) and Nephrocytium agardhianum (16–18). 13,15. Longitudinal section of a cell, showing the undulated surface of cell wall. 14,17. Cells within the mother cell wall, showing the presence of the pyrenoid and the smooth mother cell wall. 15,18. Details of cell wall. (CW=cell wall, MCW=mother cell wall, P=pyrenoid, St=starch sheath, S=starch grains, t=thylakoids). Scale bar 2 μm (13–14, 16–17), 0.5 μm (15), 0.2 μm (18).