Polulichloris henanensis HY.

Song, Huiyin, Zhang, Qi, Liu, Guoxiang & Hu, Zhengyu, 2015, Polulichloris henanensis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid green alga, Phytotaxa 218 (2), pp. 137-146 : 140-142

Polulichloris henanensis HY. Song, Q. Zhang, GX. Liu & ZY. Hu, sp. nov. ( Figs. 1 , 2 )

Vegetative cells solitary, uninucleate. Young cells irregularly egg-shaped or ellipsoidal, 2.98‒3.70 × 3.95‒5.67 μm; mature cells ellipsoidal, 4.86–5.84 × 6.08–8.20 μm. Cell have parietal, cup-shaped chloroplast with a pyrenoid surrounded by starch envelope. Asexual reproduction via 2 to 8 elliptical or irregularly egg-shaped autospores, 5.71‒8.70 × 6.90‒9.28 μm. Sexual reproduction not observed.

Type: — CHINA. Henan Province: microbial biofilm on surface soil in Zhoukou, 33° 48’ 40.02” N, 114° 28’ 20.80” E, elevation: 56 m a.s.l., H. Y. Song, February 2013 (holotype: FACHB!, fixed specimen shy053 deposited in the Freshwater Algae Specimen Station, Institute of Hydrobiology, Chinese Academy of Sciences. Reference strain: living culture (ex-holotypus), accession no. FACHB-1765, deposited in FACHB: http://algae.ihb.ac.cn/).

Light and electron microscopy: — Polulichloris henanensis shares general morphological characteristics with the Chlorella -like green microalgae: unicellular, elliptical, parietal plastid with a pyrenoid surrounded by a starch envelope. Young cells are ellipsoidal or irregularly egg shaped, 2.98–3.70 × 3.95–5.67 μm in size. When mature, the cells are ellipsoidal or broadly ellipsoidal and 4.86–5.84 × 6.0–8.20 μm. Chloroplasts are parietal and cup-shaped, sometimes occupying most of the cell. The pyrenoid is barely visible by light microscope but is well developed in most cells and surrounded by a starch envelope composed of 2–4 plates; with a few thylakoid bands transecting the pyrenoid matrix ( Fig. 2 ). The alga reproduces by 2, 4, or 8 asexual autospores. The autosporangium of P. henanensis strain FACHB-1765 was typically elliptical. Autospores were elliptical or irregularly egg-shaped. Sometimes, a single relatively large autospore and several smaller autospores were produced within a single sporangium; some autospores within a sporangium were almost equal in size. The autospores were discharged through an aperture.

Molecular phylogeny: —The 18 S rDNA and rbc L gene sequences were obtained from strain FACHB-1765, and the sequenced lengths were 1686 and 1248 bp, respectively. The phylogenetic position of P. henanensis was inferred by analyzing the 18 S rDNA and rbc L DNA sequences. The 18 S rDNA and the18 S rDNA + rbc L alignments consisted of 1637 and 2721 characters, respectively. The corresponding ML and Bayesian topologies were consistent for these clades, and the best ML trees for 18 S rDNA and 18 S rDNA + rbc L are shown in Figures 3 and 4 , respectively, with Bayesian posterior probability ( BPP) and bootstrap support values ( BP) indicating branch support. According to the phylogenetic tree based on 18 S rDNA ( Fig. 3 ), FACHB-1765 was positioned on a solitary branch nested within the Watanabea clade ( Trebouxiophyceae, Chlorophyta), likely sister to a well-supported clade including Phyllosiphon arisari ( FJ 829884) and the uncultured strain ( AM 260450) (1.00 / 98). However, it differed from P. arisari ( FJ 829884) by 126 of 1692 positions of the 18SrDNA gene and Blast searches resulted in hits of 93% similarity to P. arisari . The topology of the phylogenetic tree derived from analysis of the concatenated 18 S rDNA + rbc L sequences ( Fig. 4 ) was consistent with the tree based on 18 S rDNA ( Fig. 3 ). We did not find rbc L sequence data for Phyllosiphon in the NCBI database; the phylogenetic tree based on 18 S rDNA + rbc L does not include Phyllosiphon . Strain FACHB-1765 was in a supported sister position with the genus Desertella (0.99 / 71) in the Watanabea clade. The phylogenetic tree based on ITS ( Fig. S1 ) is available online as supplementary material.

Figures

FIGURE 1. Morphology of Polulichloris henanensis strain FACHB-1765.A–G: young vegetative cells. H, I: mature vegetative cell. J‒O: autosporangium. P: vegetative cells and liberation of autospores. Scale bars: A‒D = 2 μm, E–P = 5 μm.

FIGURE 2. Ultrastructure of Polulichloris henanensis. C: chloroplast, P: pyrenoid, SE: starch envelope, S: starch grains. Cell of P. henanensis with a parietal and cup-shaped chloroplast, pyrenoid bisected by a few thylakoid bands, and starch envelope composed of 2–4 plates surrounding the pyrenoid. A. Young cell. B–D. Vegetative cell.

FIGURE 3. Phylogenetic position of Polulichloris henanensis within class Trebouxiophyceae (Chlorophyta), based on 18S rDNA sequences. The analysis was based on reduced alignment with an outgroup formed by the chlorophycean species Chlamdomonas rosae. The tree was inferred using PAUP*4.0 with the TrNef + I + G evolutionary model. Numbers at branches correspond to MrBayes posterior probabilities (BPP)/maximum likelihood (ML) bootstrap values. Values below 0.95 BPP and 50% ML bootstrap support are not shown. Scale bar shows estimated number of substitutions per site.

FIGURE 4. Phylogenetic position of Polulichloris henanensis within class Trebouxiophyceae (Chlorophyta), based on 18S rDNA + rbcL sequences. The analysis was based on reduced alignment with an outgroup formed by the chlorophycean Chlamydomonas bilatus. The tree was inferred using PAUP*4.0 with the GTR + I + G evolutionary model. Numbers at branches correspond to MrBayes posterior probabilities (BPP)/maximum likelihood (ML) bootstrap values. Values below 0.95 BPP and 50% ML bootstrap support are not shown. Scale bar shows estimated number of substitutions per site.