Coccomyxagreatwallensis sp. nov. (Trebouxiophyceae, Chlorophyta), a lichen epiphytic alga from Fildes Peninsula, Antarctica.

A single-celled green alga Coccomyxagreatwallensis Shunan Cao & Qiming Zhou, sp. nov., isolated from a specimen of Antarctic lichen Psoromahypnorum (Vahl) Gray, is described and illustrated based on a comprehensive investigation of morphology, ultrastructure, ecology and phylogeny. The cells of C.greatwallensis are ovoid to long ellipsoidal and measured 3-5 µm × 6-12 µm. The new species has distinct ITS rDNA and SSU rDNA sequences and differs from the phylogenetic closely related species C.antarctica, C.arvernensis and C.viridis in cell size, distribution and habitat.

Introduction

The coccoid green algal genus Schmidle (1901) is well known for its diversified ecological habitats and worldwide distribution. of this genus has been reported as free living (Blanc et al. 2012), endophytic (Tremouillaux-Guiller et al. 2002; Zuykov et al. 2014) and lichen photobionts (Zoller and Lutzoni 2003; Muggi et al. 2010). can survive under extremely harsh environments, such as in the spent fuel cooling pond of a nuclear reactor (Rivasseau et al. 2016), in a highly acidic lake (pH~2.6) (Hrdinka et al. 2013), as well as in polar regions (as low as -88 °C) (Blanc et al. 2012).

Based on the mucilaginous colonies’ structure, cell length and width variability details, Jaag (1933) delimited 33 species of this genus, including 14 free-living, 13 lichenised and 6 lichen epiphytic species. Subsequently, only seven species were well recognised based on some morphological characters, such as cell shape and size, chloroplasts numbers and mucilage properties (Ettl and Gärtner 1995). However, the morphological characters of depend on the culture conditions, for example, salinity influenced the phenotypic plasticity significantly (Darienko et al. 2015) and nutrient availability influenced the presence of mucilaginous sheaths (Malavasi et al. 2016). As the instability of morphological features led to a problematic morphological delineation of the genus , a DNA-barcode based method has been developed and seven distinct species were subdivided (Darienko et al. 2015). Malavasi et al. (2016), combining morphological characters, ecological features and DNA sequences of , recognised 27 species scenarios. Subsequently, Shunan Cao & Qiming Zhou, 2018 was described as a new epiphytic species living with lichen on King George Island (Cao et al. 2018)

Currently, 28 species scenarios have been accepted, amongst which Rivasseau, Farhi & Couté, 2016, , Jaag, 1933, T. Darienko & T. Pröschold, 2015, E. Acton, 1909 and undescribed spp., belonging to Clade I, Clade KL and Clade N according Malavasi et al. (2016), are the eight epiphytic species scenarios. Meanwhile, the species and are also reported as lichen photobionts. The other lichenised species scenarios include Schmidle, 1901, Chodat, 1909, Chodat, 1913 and Clades A, D and F (Malavasi et al. 2016). The species , , and Mainx, 1928 show the Antarctic distribution, amongst which is the only free living one (Holm-Hansen 1964; Darienko et al. 2015; Borchhard et al. 2017; Cao et al. 2018).

The Fildes Peninsula undergoes a typical sub-Antarctic oceanic climate with relatively high precipitation (89%) with 56–64 mm rainfall, wind blowing from west through northwest with a speed of 6.8–7.4 m/s and the average temperature ranging from 0.5–1.8 °C in summer (Yang et al. 2013). About 127 lichen species have been recorded in Fildes Peninsula (http://www.aari.aq/KGI/Vegetation/lst_lichens.html). The lichen (Vahl) Gary, one of the four spp. found in this region, is characterised by its squamulose thallus without secondary products, dull brown discs, apothecia margin without or with very short hairs (Øvstedal and Smith 2001). Both cyanobacteria and green have been reported as photosynthetic partPageBreakners of (Holien and Jørgensen 2000; Øvstedal and Smith 2001; Wirtz et al. 2003; Ekman et al. 2014).

In the current study, a lichenicolous single cell green alga was isolated from . Based on the comprehensive analysis approach, including morphology, ultrastructure, ecology and phylogeny, the green alga is demonstrated to be new to science.

Material and methods

Isolation and culture

The lichen specimen (collection No. 274) of was collected from Fildes Peninsula, King George Island, Antarctica () during the 30th Chinese National Antarctic Research Expeditions in summertime (1 February 2014–15 March 2014). The specimen was kept in the Resource-sharing Platform of Polar Samples which includes samples of Biology, Ice-snow, Rock, Deep-space and Sediment (BIRDS ID 2131C0001ASBM100076) at 4 °C till the isolation was processed.

A single algal cell was obtained following a modified aseptic isolation procedure (Cao et al. 2018). The isolations, cultured on a petri-dish with PDA and BBM medium in an illumination incubator (4 °C, 12 hr light/12 hr dark), were deposited in the Freshwater Culture Collection at the Institute of Hydrobiology (FACHB) as an open collection (FACHB-2139).

Light and electron microscopy

For observing and photographing the algal cultures, compound microscopes Nikon Eclipse 80i and Nikon ACT-1 V2.70 were used.

After fixing with 2.5% glutaraldehyde buffer, the algal cells were used for transmission electron microscopy (TEM). The procedures and reagents (including 2.5% glutaraldehyde buffer) used followed Cao et al. (2018). The 70 nm cell sections, cut by a Leica EM UC6 ultramicrotome and stained with 3% uranyl acetate and lead citrate, were observed using a Jeol JEM1230 transmission electron microscope at 80–120 kV. The micrographs were captured using iTEM software by an Olympus SIS VELETA CCD camera.

DNA extraction, amplification, sequencing and analysis

A modified CTAB method (Cao et al. 2015) was used to extract the alga genomic DNA. Primer pairs NS1, NS4; NS3, NS6; NS5, NS8 (White et al. 1990) and primer pair ITS5, O2 (Cao et al. 2015) were used to amplify the SSU rDNA and ITS rDNA, respectively. A 50 µl volume PCR reaction was selected, PCR application and products verification followed Cao et al. (2015) and double-stranded PCR products were sequenced by an ABI3730XL sequencer.

SEQMAN programme within Lasergene v.7.1 software (DNASTAR Inc.) was selected to check the double-directional ITS rDNA and SSU rDNA sequences. These two regions were overlapped into one single contig and the flanking regions were trimmed off. The sequence representing the new species was submitted to GenBank (MF465899).

ClustalW algorithm, including in MEGA 7 (Kumar et al. 2016), was performed to align the sequences with default parameters (Higgins et al. 1994) and then adjusted manually. The Neighbour-Joining (NJ) was selected to calculate the ITS phylogenetic structures, PageBreakas well as Maximum Likelihood (ML) method for SSU sequences. Pairwise distances of ITS rDNA and SSU rDNA sequences were calculated using MEGA 7. A 1000 resamplings bootstrap was tested for the reliability of the inferred trees. In total, 42 sequences, which have been confirmed by Malavasi et al. (2016), were retrieved from GenBank (Table 1).

Table 1.

spp. sequences used in the present study.

Species	Collection No.	GenBank No.
		ITS rDNA	SSU rDNA
Clade B*Coccomyxa sp.	GA5a	AB917140	AB917140
Clade D*Coccomyxa sp.	CCAP 216/24	FN298927	FN298927
	CCAP 812/2A	HG972992	HG972992
Clade E*Coccomyxa sp.	IB-GF-12	–	KM020052
Clade E*Coccomyxasubellipsoidea	CCAP 812/3	HG972972	HG972972
Clade H*Coccomyxa sp.	KN-2011-U5	HE586557	–
Clade I*Coccomyxa sp.	KN-2011-T3	HE586515	HE586515
	KN-2011-T1	HE586550	–
Clade K*Coccomyxa sp.	KN-2011-C4	HE586508	HE586508
Clade L*Monodus sp.	UTEX B SNO83	–	HE586506
Clade M*Monodus sp.	CR2-4	HE586519	HE586519
Clade N*Coccomyxaviridis 3	CAUP H5103	HG973007	HG973007
	SAG 2040	HG973004	HG973004
Coccomyxa actinabiotis	216-25	FR850476	FR850476
	KN-2011-T4	HE586516	HE586516
Coccomyxa antarctica	FACHB-2140	MF465900	MF465900
Coccomyxa arvernensis	SAG 216-1	–	HG972999
	Wien C19	HG973000	HG973000
Coccomyxa dispar	SAG 49.84	HG972998	HG972998
Coccomyxa elongata	CAUP H5107	HG972981	HG972981
	SAG 216-3b	HG972980	HG972980
Coccomyxa galuniae	CCAP 211/97	FN298928	FN298928
	SAG 2253	HG972996	HG972996
Coccomyxagreatwallensis sp. nov.	FACHB-2139	MF465899	MF465899
Coccomyxa melkonianii	SCCA048	KU696488	KU696488
Coccomyxa onubensis	ACCV1	HE617183	HE617183
Coccomyxa polymorpha	CAUP H5101	HG972979	HG972979
	KN-2011-T2	HE586514	HE586514
Coccomyxa simplex	CAUP H 102	HE586504	HE586504
	SAG 216-2	HG972989	HG972989
Coccomyxa solorinae	SAG 216-12	HG972987	HG972987
	SAG 216-6	HG972988	HG972988
Coccomyxa subellipsoidea	SAG 216-7	HG972976	HG972976
	Wien C20	HG972975	HG972975
	CAUP H5105	HG972974	–
Coccomyxa vinatzeri	ASIB V16	HG972994	HG972994
Coccomyxa viridis	SAG 216-14	HG973002	HG973002
	SAG 216-4	HG973001	HG973001
Elliptochloris bilobata	SAG 245.80	HG972969	HG972969
Hemichloris antarctica	SAG 62.90	HG972970	HG972970

* Clades referred after Malavasi et al. (2016).

Results

Shunan Cao & Qiming Zhou sp. nov.

Figures 1 , 2

Figure 1.

Shunan Cao & Qiming Zhou, sp. nov., light microphotographs. Cells cultured in BBM medium (a, b) and in PDA medium (c, d). Scale bar: 10 µm.

Figure 2.

Ultrastructure of Shunan Cao & Qiming Zhou, sp. nov. in PDA medium; note: chloroplast (2), plastoglobuli (2), cell wall (3), starch granules (4), thylankoids (5) and mitochondria (6). Scale bar: 0.2 µm.

Holotype.

Strain FACHB-2139, Freshwater Culture Collection, the Institute of Hydrobiology (FACHB-Collection) (Fig. 1a).

Type locality.

Antarctic, Fildes Peninsula, on soil (), 40 m a.s.l.; isolated from the Antarctic lichen (collection No. 274, BIRDS ID: 2131C0001ASBM100076) on 14 February 2014.

Habitat.

Epiphytic green alga, living with lichen in Sub-Antarctic climate.

Description.

Single-celled green alga, ovoid to long ellipsoidal, asymmetrical, measured 3–5 µm × 6–12 µm, some cells nearly rounded in nutrient-rich PDA mediumPageBreak; cells without mucilaginous sheath (Fig. 1). Cell wall smooth, three layers in ultrastructures. Protoplast filled with lipid droplets. Chloroplast parietal, without pyrenoid and with starch granules in the inter thylacoidal spaces. One nucleus in the central part of the cell present. Reproductive process not observed (Fig. 2).

Molecular analyses

The pairwise distance analysis of ITS rDNA sequences shows that the overall mean distance is 0.171±0.015. The pairwise distance between our algal strain FACHB-2139 and the other species of ranged from 0.253 to 0.022, of which PageBreak shows the minimum distance with our isolate of 0.022 followed by sp. Clade N of 0.030 (Suppl. material 1: Table S1). The pairwise distance analysis of SSU rDNA sequences shows that the overall mean distance is 0.017±0.002. In addition, the pairwise distance between alga strain FACHB-2139 and the other species of ranged from 0.025 to 0.001, amongst which both and show the minimum distance of 0.001 with our sample (Suppl. material 1: Table S1). That indicated that alga FACHB-2139 is closely related to and .

For the ITS rDNA, all the sequences clustered into one group supported with bootstrap value 100 and within , six subgroups have been clustered. The alga FACHB-2139 together with , , , spp. of clade KL, Clade M and Clade N clustered as a subgroup, were supported with a bootstrap value 100; but the newly isolated strain FACHB-2139 differs from the other species clearly, no well supported clade for FACHB-2139 and species mentioned above were formed (Fig. 3a). In the SSU rDNA phylogenetic result, the sequences of clustered into five subgroups and the alga FACHB-2139, , , , spp. Clade K, Clade L, Clade M and Clade N lay in the same subgroup whose bootstrap value was 97. Though alga strain FACHB-2139 and formed a monophyletic group, this clade was supported by a low bootstrap value 53, which indicated that these two species were insufficiently supported statistically (Fig. 3b).

Figure 3.

The NJ tree based on ITS rDNA (a) and the ML tree based on SSU rDNA (b) sequences phylogenetic analyses. The sequences marked with clade A–N referred after Malavasi et al. (2016).

Diagnosis

Morphologically, our sample FACHB-2139 can be distinguished from its phylogenetically close congeners (1.8–3.6 µm × 4.7–8.4 µm) and (3–4 µm × 6–8 µm) (Müller 2005, Hodač 2015) by its larger cells and from (4–7 µm × 8–12 µm) by its smaller cells (Cao et al. 2018). The cell sizes of the above species were recorded when cultured in BBM medium. In addition, both and are lichenised or are epiphytic species and have not been recorded as an Antarctica distribution.

Our molecular and morphological analyses indicate that algal isolate FACHB-2139 represents a new species which we named Shunan Cao & Qiming Zhou sp. nov.

Discussion

Shunan Cao & Qiming Zhou sp. nov., isolated from Antarctic squamulose lichen , is one of the species, which is characterised by ovoid to ellipsoidal single cells. The usage of molecular barcode provides an effective and stable tool to identify and classify the species of (Darienko et al. 2015; Malavasi et al. 2016). In the current study, both ITS and SSU rDNA were used and a comparison with the closely related species had been listed in Suppl. material 3:Table S3. The minimum pairwise distance was calculated between and using ITS rDNA sequences, but the bootstrap value, which was lower than 50, did not support these two species as a monophyletic group. A similar result was also obtained using SSU rDNA sequences.

Though some species could be the photosynthetic partner of lichens (Honegger and Brunner 1981), due to the lichen mycobiont’s selectivity to its photobiont partner, one photobiont group occurs within relative stable lichen groups (Tschermak-Woess 1988; Cao et al. 2015); for example, is known as the photobiont of lichenised ascomycots belonging to (i.e. Müll. Arg., Willd. and Ach.), ( Pers., Clem., Hertel and Gyeln.), ( Trevis.), ( Redhead, Lutzoni, Moncalvo & Vilgalys), ( Fr.) and ( R.H. Petersen) (Poulsen et al. 2001; Smith et al. 2009; Wirth et al. 2013), as well as the basidiomycots belonging to ( Quél.) (Jaag 1933; Zoller and Lutzoni 2003). In addition, is optionally lichenised with the fungus Gilenstam, H. Döring & Wedin () (Muggi et al. 2010). Furthermore, there is also evidence to support the photosynthetic partner of Antarctic lichen is cyanobacteria or the green Printz (Brodo et al. 2001; Øvstedal and Smith 2001; Wirtz et al. 2003; Ekman et al. 2014) but not the species of . We therefore conclude that the newly described green alga is an epiphytic alga of lichen .