The single celled green alga Coccomyxa antarctica Shunan Cao & Qiming Zhou, sp. nov. was isolated from the Antarctic torrential lichen Usnea aurantiacoatra (Jacq.) Bory. It is described and illustrated based on a comprehensive study of its morphology, ultrastructure, ecology and phylogeny. C. antarctica is a lichenicolous alga which has elongated cells and contains a parietal chloroplast as observed under the microscope. C. antarctica is clearly different from other species by phylogenetic analysis (ITS rDNA and SSU rDNA sequences), also it differs from its phylogenetic closely species C. viridis by its larger cell size.

Lichen epiphyte, morphology, TEM, phylogeny

Lichens, the typical symbiosis, generally consist of one fungal partner and its photosynthetic partner alga (usually a green alga or a cyanobacterium). With the development of research techniques, many other eukaryotic (Wilkinson et al. 2015, Spribille et al. 2016) and prokaryotic microbes (Aschenbrenner et al. 2016) have been observed in concurrence with lichen thalli besides the mycobiont and photobiont partners, such as lichenicolous fungi (Edwards et al. 2017, Asplund et al. 2017) and algae (Gustavs et al. 2017).

The green algae of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta) are distributed worldwide and can be found in both aquatic and terrestrial habitats, in free living and symbiotic status (Malavasi et al. 2016). The species of Coccomyxa can be lichenicolous algae or lichenised photosynthetic partners in lichens (Malavasi et al. 2016). Historically, the taxonomy of this genus has been problematic. Originally a total of 14 free living species, 13 lichenised species and six lichen epiphytic species were summarised by Jagg (1933) based on morphology. Recently, a total of seven species has been distinguished, since the morphological characters of the unicellular green algae Coccomyxa vary in different environments and a DNA-based identification approach was proposed by Darienko et al. (2015). Subsequently, an improved method based on phylogenetic and ecological features was used for delimiting the species of this genus and 27 species scenario were recognised (Malavasi et al. 2016). The combination of ecological and DNA sequences data seems to be effective in distinguishing the Coccomyxa species.

In this current study, an epiphytic green alga was isolated from the Antarctic lichen Usnea aurantiacoatra (Jacq.) Bory. It will be demonstrated that this green alga is new to science based on the comprehensive analysis approach including morphology, ultrastructure, ecology and phylogeny.

We examined the algal strain (Ua6) isolated from Antarctic lichen Usnea aurantiacoatra using both morphological identification and molecular markers. The isolated alga Ua6 was observed with elongated cells (4–7 µm wide and 8–12 µm long), whose cell wall was thin and smooth, each cell contained a parietal chloroplast (Figure 1); no pyrenoid was observed within their chloroplast using transmission electron microscopy (Figure 2). The alga strain Ua6 appeared to have a shorter growth cycle when cultured in PDA medium than that in BBM medium, but no significant morphological differences were observed from the cells cultured in PDA and BBM mediums (Figure 1).

Figure 1. 

Morphology of Coccomyxa antarctica Shunan Cao & Qiming Zhou, sp. nov. a–d cultured in BBM medium; e-f cultured in PDA medium. Scale bars: 10 μm.

Figure 2. 

Ultrastructure of Coccomyxa antarctica Shunan Cao & Qiming Zhou, sp. nov. a–c cultured in BBM medium; d-f cultured in PDA medium. a, b mature autosporangium c, d Cup-shaped chloroplast e, f vegetative cell. Key: Ch: chloroplast; Cw: cell wall; Mit: mitochondria; N: nucleus; S: starch granules; Th: thylakoids. Scale bars: 0.5 μm(a, d); 0.2 μm (b, c, e, f).

The phylogenetic analysis of both ITS rDNA and SSU rDNA supported that the isolated green alga Ua6 was an undescribed Coccomyxa species. For the ITS rDNA, the sequences of Coccomyxa clustered as six subgroups. The newly isolated green alga Ua6, C. viridis, C. avernensis, Coccomyxa sp. Clade M, Clade N and Clade KL clustered as a subgroup, was supported with a bootstrap value 100, but the new species Ua6 was clearly different from the other species in this subgroup according to the branch length. For the SSU rDNA, the sequences of Coccomyxa clustered as five subgroups. The newly isolated green alga Ua6 also showed a close relationship with C. viridis, C. avernensis, Coccomyxa sp. Clade K, Clade L, Clade M and Clade N as a well-supported subgroup with the bootstrap value 100. Furthermore, the SSU rDNA sequence of Ua6 was clearly distinguished from the other species.

According to the comprehensive study of both morphological and phylogenetic analysis, the isolated single cell green algae Ua6 is a newly reported species and here described as new:

Coccomyxa antarctica Shunan Cao & Qiming Zhou, sp. nov.

Figures 1, 2

Holotype

Preparation FACHB-2140, Freshwater Algae Culture Collection, the Institute of Hydrobiology (FACHB-Collection) represented here by Figure 1d.

Type locality

Antarctic, Fildes Peninsula, on stone (62°12.70'S, 58°55.70'W), 44 m a.s.l., Isolated from the Antarctic lichen Usnea aurantiacoatra (d-B1, BIRDS ID: 2131C0001ASBM100063) on 5^th May 2014.

Diagnosis

The vegetative cells are ovoid to ellipsoidal, asymmetrical, 4–7 µm wide and 8–12 µm long; some cells were sub-sphaeroidal in BBM medium, without mucilaginous sheath. Cell wall smooth, double in ultrastructures. Protoplast with single central cell nucleus, filled with lipid droplets. Chloroplast parietal, with starch granules in interthylacoidal spaces, without pyrenoid. Reproductive cells were not observed. It looks morphologically similar to other Coccomyxa species but differs from other species of Coccomyxa in ITS rDNA (Table 1 & Figure 3a) and SSU rDNA (Table 1, Figure 3b).

Figure 3. 

The NJ tree based on ITS rDNA (a) and SSU rDNA (b) sequences phylogenetic analyses. The sequences obtained by the authors were exhibited in bold font. The clades referred to Malavasi et al. (2016).

Habitat

Epiphytic green alga, living with lichen Usnea aurantiacoatra in harsh environments (Antarctic).

The morphological and ultrastructure characters indicate that the isolated green alga Ua6 is a Coccomyxa species, which is characterised by ovoid to ellipsoidal single cells. The isolated strain Ua6 is morphologically similar to the other Coccomyxa species, but different from the phylogenetic closely related species C. viridis by its larger cell size (4–7 µm wide and 8–12 µm long vs 1.8–3.6 µm wide and 4.7–8.4 µm long) (Hodač 2015). However, the morphological characters are not stable and non-credible as they change under different environments or culture conditions. For example, the cell shape is significantly dependent on culture conditions (Tsarenko and John 2011) and the mucilaginous sheaths are highly dependent on nutrient availability which is the key trait in separating Coccomyxa and Pseudococcomyxa (Darienko et al. 2015).

Since the molecular barcode provides a more stable and informative tool in identification and classification of the species of Coccomyxa (Darienko et al. 2015, Malavasi et al. 2016), both the ITS rDNA and SSU rDNA phylogenetic analyses were applied in the current study. The results supported the observation that the single cell green alga Coccomyxa antarctica sp. nov. is different from the other reported species of Coccomyxa, indicating that it is a species new to science.

Furthermore, species of Coccomyxa have been reported as photobionts of lichen genera Baeomyces, Dibaeis, Icmadophila, Lichenomphalia, Micarea, Multiclavula, Nephroma, Orceolina, Peltigera, Placynthiella, and Solorina in earlier studies (Poulsen et al. 2001, Smith et al. 2009, Wirth et al. 2013, Gustavs et al. 2017), but not Usnea. The authors’ earlier studies also revealed that the photosynthetic partner of the Antarctic lichen U. aurantiacoatra was Trebouxia jamesii (Hildreth and Ahmadjian) Gärtner (Cao et al. 2015b, Cao et al. 2017); as a result, the isolated green alga Coccomyxa antarctica sp. nov. is not lichenised alga, but a lichen epiphytic alga.

Samples Information and Data were issued by the Resource-sharing Platform of Polar Samples (http://birds.chinare.org.cn) which was established by the National Science & Technology Infrastructures Polar Research Institute of China (PRIC) and the Chinese National Arctic & Antarctic Data Centre (CN-NADC). We are grateful to the Chinese Arctic and Antarctic Administration for their help in carrying out the project in the Great Wall Station during the 30^th Chinese National Antarctic Expedition (2012GW03003). This research was supported by the Natural Science Foundation of Shanghai (No. 16ZR1439800) and National Infrastructure of Natural Resources for Science and Technology Program of China (No. NIMR-2017-8).