Euchlorocystis marina sp. nov. (Oocystaceae, Trebouxiophyceae), a New Species of Green Algae from a Seawater Shrimp Culture Pond

: Oocystaceae is a cosmopolitan family of green algae with distinct morphology and ultrastructure. Most of the reported species in this family are freshwater species, and there are few marine species reported. In this study, we describe a new marine species of Oocystaceae, Euchlorocystis marina sp. nov. based on material collected from a seawater shrimp culture pond in Zhanjiang, China. An integrative approach, including phylogenetic analyses of 18S rDNA, light microscopy, and transmission electron microscopy, was used for the taxonomic study of the strains. Morphological observation results showed that it has a multilayer thick cell wall, multiple pyrenoids in the chloroplast, and usually 2–16 cells forming a colony in the extended mother cell wall. These features are morphologically similar to the genus Euchlorocystis and are distinguished from other taxa of the family Oocystaceae. The 18S rDNA phylogenetic trees revealed that the strains and Euchlorocystis subsalina formed an independent clade in Oocystaceae with robust support. However, horseshoe-shaped chloroplasts and rounder cells morphologically distinguished it from Euchlorocystis subsalina . Apart from the morphology, the direct comparison of sequences also supported that they were distinct species. The discovery and description of the new species enriches the marine species record of the family Oocystaceae.


Introduction
Oocystaceae is a cosmopolitan family of green algae with distinct morphology and ultrastructure. It has formed unique and complex biological characteristics and ecological environment adaptability in the course of long-term evolution. Some Oocystaceae species, owing to their high nitrogen and phosphorus removal ability [1,2], high CO 2 -tolerance [3], simple growth requirements, and adaptability to various environmental conditions [4], have shown great potential in improving the aquaculture water environment [1,4,5], wastewater treatment [2,6], carbon emissions [3], and biofuel [7].
Oocystaceae are widely distributed, with records and reports all over the world. The early morphological classification assigned the Oocystaceae family to the Chlorophyceae class [8,9], but molecular studies confirmed that this family was a monophyletic group in the Trebouxiophyceae class [10][11][12][13]. Štenclová et al. [14] conducted a molecular phylogenetic study on 54 Oocystaceae species in 2017, and divided Oocystaceae into three subfamilies and five clades. Recently, five new genera were defined in the family Oocystaceae, including Planctonemopsis [15], Quadricoccopsis [16], Euchlorocystis, Densicystis [17], and Reticulocystis [18]. Currently, 96 genera and 304 species of Oocystaceae have been recorded in AlgaeBase [19]. However, few marine species of this family have been recorded.
As a new member of the Oocystaceae family, the genus Euchlorocystis was defined by Liu et al. [17] in 2018. The genus Euchlorocystis has multiple pyrenoids, distinguishing it from Oocystis. It also has a multilayered cell wall and trough-shaped chloroplast, which is different from the Oonephris genus. In addition, the cell size of Euchlorocystis and the number of chloroplasts per cell were used as the main morphological markers that distinguish the genus Eremosphaera. Currently, only one taxonomically accepted species of Euchlorocystis is listed in AlgaeBase [19]. In this study, we isolated a new strain of algae from a mariculture pond of shrimp in Zhanjiang, Guangdong Province, China, and successfully cultured it in the laboratory. Morphological and phylogenetic analyses identified this strain as a new species, Euchlorocystis marina sp. nov., belonging to the Oocystaceae (Trebouxiophyceae, Chlorophyta). The results will increase the understanding of the genus Euchlorocystis and enrich records of marine species of Oocystaceae.

Morphological Observations
Cells are solitary or in 2-16 cell colonies within a thin, hyaline mucilaginous envelope or within an expanded mother cell wall ( Figure 1). Cells are round, oval, or slightly reniform, ranging in size from 11.3 to 16.6 µm long and 6.3 to 10.3 µm wide. Cell walls are thick, sometimes stratified (Figure 1b,c), and smooth, without thickening polars. Mother cell walls are extended according to the number and arrangement of daughter cells and are usually an irregularly round shape, with or without tapered thickening ends (Figure 1b,d). The number and shape of chloroplasts are difficult to discern on the microscopic level. When observed with TEM, a horseshoe-shaped chloroplast was observed, which occupied most of the cell volume (Figure 2a,d). Asexual reproduction was by 2-4 autospores released by a rupture of the mother cell wall. These autospores could be propagated again independently inside the mother cell (Figure 1e,g). The asynchronous cell division led to the size difference between the daughter cells ( Figure 1i) and the formation of multilayer mother cell walls (Figure 2e,f). and Reticulocystis [18]. Currently, 96 genera and 304 species of Oocystaceae have been recorded in AlgaeBase [19]. However, few marine species of this family have been recorded. As a new member of the Oocystaceae family, the genus Euchlorocystis was defined by Liu et al. [17] in 2018. The genus Euchlorocystis has multiple pyrenoids, distinguishing it from Oocystis. It also has a multilayered cell wall and trough-shaped chloroplast, which is different from the Oonephris genus. In addition, the cell size of Euchlorocystis and the number of chloroplasts per cell were used as the main morphological markers that distinguish the genus Eremosphaera. Currently, only one taxonomically accepted species of Euchlorocystis is listed in AlgaeBase [19]. In this study, we isolated a new strain of algae from a mariculture pond of shrimp in Zhanjiang, Guangdong Province, China, and successfully cultured it in the laboratory. Morphological and phylogenetic analyses identified this strain as a new species, Euchlorocystis marina sp. nov., belonging to the Oocystaceae (Trebouxiophyceae, Chlorophyta). The results will increase the understanding of the genus Euchlorocystis and enrich records of marine species of Oocystaceae.

Morphological Observations
Cells are solitary or in 2-16 cell colonies within a thin, hyaline mucilaginous envelope or within an expanded mother cell wall ( Figure 1). Cells are round, oval, or slightly reniform, ranging in size from 11.3 to 16.6 μm long and 6.

Molecular Phylogeny
Sequencing of the 18S rDNA of the new strain produced a 1720 bp sequence, introns were excluded. The final 18S rDNA sequences alignment of 29 taxa with 1633 base positions were used for the phylogenetic tree construction, and Prasiola mexicana (Prasiolaceae) was chosen for the outgroup. The ML and NJ analyses yielded a similar topology and only the ML tree was presented in Figure 3. The 18S rDNA phylogenetic trees consistently recovered that the newly isolated strain and Euchlorocystis subsalina formed an independent clade with high support (99/100 for ML/NJ) ( Figure 3). Direct comparison showed that the 18S rDNA of the new strain had 41 differences (97.5% identity) to Euchlorocystis subsalina (Table 1).

Molecular Phylogeny
Sequencing of the 18S rDNA of the new strain produced a 1720 bp sequence, introns were excluded. The final 18S rDNA sequences alignment of 29 taxa with 1633 base positions were used for the phylogenetic tree construction, and Prasiola mexicana (Prasiolaceae) was chosen for the outgroup. The ML and NJ analyses yielded a similar topology and only the ML tree was presented in Figure 3. The 18S rDNA phylogenetic trees consistently recovered that the newly isolated strain and Euchlorocystis subsalina formed an independent clade with high support (99/100 for ML/NJ) (Figure 3). Direct comparison showed that the 18S rDNA of the new strain had 41 differences (97.5% identity) to Euchlorocystis subsalina (Table 1).

Discussion
Members of the family Oocystaceae are common in freshwater, but they are rarely found in seawater. Currently, only a few marine species in this group are taxonomically accepted, such as Oocystis submarina, Oocystis marina, and Euchlorocystis subsalina. Some other species, due to their extensive adaptation, are also reported to exist in saline or semisaline conditions [1, 20-24], but they are taxonomically considered freshwater species. In

Discussion
Members of the family Oocystaceae are common in freshwater, but they are rarely found in seawater. Currently, only a few marine species in this group are taxonomically accepted, such as Oocystis submarina, Oocystis marina, and Euchlorocystis subsalina. Some other species, due to their extensive adaptation, are also reported to exist in saline or semi-saline conditions [1,[20][21][22][23][24], but they are taxonomically considered freshwater species. In this study, a new member of this family was found in a mariculture pond of shrimp in China. An integrative taxonomic study including morphological and phylogenetic analyses identified it as a new species, Euchlorocystis marina sp. nov., belonging to the genus Euchlorocystis (Oocystaceae).
The genus Euchlorocystis was originally erected by Liu et al. [17] in 2018. It has morphology similar to the well-known genus Oocystis. As a type of the family Oocystaceae, Oocystis has always been controversial in taxonomy. Komárek and Fott [8] listed 28 species with an additional 16 species designated as incompletely described or difficult to identify. Hindák [25] assigned to the genus Oocystis only those forms without a pyrenoid and listed eight species for the genus. Those forms included in the genus Oocystis with a pyrenoid were classified in the genus Oocystella [26]. The chloroplasts of Euchlorocystis mature cells have multiple pyrenoids, a feature that easily distinguishes them from Oocystis, Oocystella, and other Oocystaceae members without or with one pyrenoid. Oonephris species were described as having a central pyrenoid and sometimes more than one pyrenoid within the chloroplast [8]. According to Liu et al. [17], Euchlorocystis was morphologically different from Oonephris in terms of chloroplast shape and layered cell walls. We believe that the asynchronous cell division present in Euchlorocystis is also a distinctive feature to distinguish from Oonephris. Eremosphaera were also described as having more than one pyrenoid [8]. Liu et al. [17] suggested that Euchlorocystis and Eremosphaera could be distinguished by cell size and the number of chloroplasts per cell. In addition, the number of cells within colonies and whether the division of daughter cells within the mother cell is synchronized can also distinguish Euchlorocystis from Eremosphaera. In conclusion, the asynchronous cell division may be one of the most distinctive features for distinguishing Euchlorocystis from other taxa of the family Oocystaceae.
Euchlorocystis subsalina was originally described by Liu et al. [17] based on the materials collected from a semi-saline lake, Qinghai Lake. It is a type of the genus Euchlorocystis and is the only taxonomically accepted Euchlorocystis species reported currently. The new species presented here, Euchlorocystis marina, was collected from a seawater shrimp culture pond in Zhanjiang, China. It has a multilayer thick cell wall, multiple pyrenoids in the chloroplast, and usually 2-16 cells forming a colony in the extended mother cell wall. These features are morphologically similar to the genus Euchlorocystis, and are distinguished from other taxa of the family Oocystaceae. Most importantly, however, the 18S rDNA phylogenetic trees revealed that Euchlorocystis marina and Euchlorocystis subsalina formed an independent clade in Oocystaceae with robust support. However, horseshoe-shaped chloroplasts and rounder cells morphologically distinguish it from Euchlorocystis subsalina ( Table 2). Apart from the morphology, the molecular results of this study also supports that they are distinct species. The discovery of Euchlorocystis marina may imply more hidden species of the genus Euchlorocystis that have yet to be discovered. More new species within this genus should be collected and sequenced in the future.   Rosowski, 1973) until single colonies were obtained. Then, dividual colonies were cultivated in a seawater medium (Zhanshui 107-13) at pH = 8.0 with the following nutrient compositions: KH 2 PO 4 (8 mg·L −1 ), NaNO 3 (80 mg·L −1 ), ferric citrate (50 mg·L −1 ). The cultures were carried out in a 500 mL Erlenmeyer flask containing 300 mL medium at 25±1 • C and 30 µmol·m −2 ·s −1 of continuous light (T8 LED lamps, white light).

Morphological Observation
Microscopic observation and photographing of algal cells were performed using an OLYMPUS BX53 Light microscope.
For ultrastructure, algal cells were collected by centrifugation (3000 rpm, 5 min) and washed three times with PBS buffer. Then, the cells were fixed with 2.5% glutaraldehyde solution for 2 h at 4 • C, and post-fixed with 1% OsO 4 in 0.1 M phosphate buffer for 2 h at 4 • C. The fixed materials were dehydrated with a graded acetone series and embedded in Spurr resin. Uranium acetate and lead citrate were used to stain the final sections. The samples' slice preparation and staining were performed by Guangdong Medical University (Zhanjiang, China). The algal slices were examined and photos taken by a JEOL JEM-1400 transmission electron microscope.

Molecular Analyses
Total genomic DNA of algae was extracted with the DNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer's protocol. The PCR cycling conditions were as follows: 5 min initial denaturation at 95 • C; 35 cycles of denaturation at 95 • C for 30 s, annealing at 55 • C for 30 s, and extension at 72 • C for 40 s; a final extension of 10 min at 72 • C. The 18S rDNA was amplificated using Echl18SF (ACTGTGAAACTGCGAATGG) and Echl18SR (TAGGTGGGAGGGTTTAGG) primers. The PCR products were sequenced by Shanghai SaiHeng Biotechnology Co., Ltd., Shanghai, China. The assembled sequences were submitted to GenBank under the accession number OM413748.
The new 18S rDNA sequences were aligned with gene sequences downloaded from GenBank of 28 representative species. Phylogenies were estimated by Clustalx 1.8 and Mega 5.0 software with maximum likelihood (ML) and neighbor-joining (NJ) methods. Kimura 2-parameter model was selected as best-fit model. Bootstrap analysis was performed with 1000 replicates of the dataset to estimate statistical reliability.