New Species of Chloroidium (Trebouxiophyceae, Chlorophyta) from East Asia

Chlorella-like green algae that reproduce only asexually by immotile autospores or motile zoospores are the most typical inhabitants of non-aquatic environments. They have a simple morphology that hampers their differentiation, but algae of such habit represent a diverse array of lineages, which are mostly in the classes Chlorophyceae and Trebouxiophyceae. One of these lineages is the order Watanabeales (Watanabea clade; Trebouxiophyceae), which comprises 10 genera that share a distinct mode of reproduction through unequally sized autospores. Most of these genera account for a few species that are rarely recorded in nature. In contrast, the genus Chloroidium is one of the most species-rich and widely distributed members of the order. Three strains of coccoid green alga were isolated during a study of soil algae in the temperate monsoon climate zone of Asia. These strains are described here as a new species, Chloroidium orientalis. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiate these strains from known members of the genus.


Introduction
Coccoid spherical and ellipsoid (Chlorella-like) green algae reproduce only asexually by immotile autospores or motile zoospores. These organisms are frequent and abundant inhabitants of terrestrial and aquatic (mostly freshwater) environments. This morphotype is typical of many photobionts of lichens as well. They have a simple morphology that hampers taxonomic differentiation, but algae with such a habit represent a diverse array of lineages belonging to the classes Chlorophyceae and Trebouxiophyceae according to the molecular phylogenetic data [1][2][3][4][5][6]. One of these lineages is the Watanabea clade in the Trebouxiophyceae, which predominantly comprises terrestrial microalgae that are currently classified in 10 genera. Most of them (seven of the 10) were described based on mainly molecular phylogenetic data and include only a few species with scarce records from nature. Morphological identification of genera and infrageneric taxa comprising Watanabea is difficult and could only be achieved with the establishment of their exact molecular phylogenetic affiliation.
Recently, the Watanabea clade was defined as the order Watanabeales based on the results of molecular phylogenetic analyses and shared morphological features, especially of unequally sized autospores [7]. The Watanabeales include the genera Calidiella Darienko and Pröschold, Chloroidium Nadson, Jaagichlorella Reisigl, Kalinella J. Neustupa

Results
We examined three algal strains isolated from soil (Primorsky Territory, Russia) and concrete wall biofouling (Jilin Province, China) using both phenotypic features and molecular markers. The phylogenetic analysis of concatenated SSU rDNA and ITS rDNA revealed that these strains represent an undescribed Chloroidium taxon. Hereafter, we refer to these strains as a new species, Chloroidium orientalis.
Differs from other Chloroidium species by the following set of morphological characters: a distinct pyrenoid with several starch grains; autosporangia with three types of autospore sizes (equal small, equal large, and unequal small and large). Also, distinct genetically by differences in the ITS2 sequence and hCBCs in the conservative part of ITS2. Etymology: The species epithet "orientalis" is based on the name of the geographic region (East Asia), where it was found.
Holotype: Exsiccatum number VLA-CA-0963, a dried biomass of unialgal population was deposited in the Herbarium, Federal Scientific Center of East Asian Terrestrial Biodiversity, Vladivostok, Russia. Gene sequence: DNA sequences obtained from clonal strains of Chloroidium orientalis were deposited in GenBank under accession no. MZ558750-MZ558752.  Etymology: The species epithet "orientalis" is based on the name of the geographic region (East Asia), where it was found.
Holotype: Exsiccatum number VLA-CA-0963, a dried biomass of unialgal population was deposited in the Herbarium, Federal Scientific Center of East Asian Terrestrial Biodiversity, Vladivostok, Russia. Gene sequence: DNA sequences obtained from clonal strains of Chloroidium orientalis were deposited in GenBank under accession no. MZ558750-MZ558752.

Phylogenetic Analyses
The 18S rRNA gene sequence divergence between all Chloroidium species was low and did not exceed 1-2% (Table S1). The divergence was sufficiently higher for the ITS1-5.8S-ITS2 region (5-29%; Table 1). ITS sequences in the strains differed from those in other species for more than 11%, while the difference between C. ellipsoideum and C. lichenum and C. engadinensis and C. viscosum was sufficiently low, 5.05 ± 0.67% and 8.34 ± 0.96%, respectively. The results of the sequence comparisons suggested that we were likely dealing with a new species. To test this hypothesis, we accessed the phylogenetic position of our strains in the genus Chloroidium using a dataset based on analyses of Darienko et al. [9] (Table S2). The dataset included 61 Chloroidium accessions representing 9 described species and our isolates. It was 2471 nt long, and of these 332 characters were parsimony-informative. The overall topologies of our unrooted ML and BI trees were similar to that presented by Darienko et al. [9] (Figure 3). For all but the species C. lichenum, represented by more than one sequence, respective generic clades were resolved, C. sacharophyllum, C. viscosum, C ellipsoideum, and C. arboriculum Darienko and Pröschold. Our strains formed a highly supported (100/1.00) clade that was resolved as a sister lineage to C. engadinensis with moderate support (70/0.98). This lineage was a part of the moderately supported clade (93/0.99) formed by C. antarcticum Darienko, Lukešová and Pröschold, the C. ellipsoideum/C. lichenum subclade, and C. viscosum. The relationship between these entities remained largely unresolved. One more Chloroidium intrageneric clade united long-branched C. arboricolum, C. laureanum, and C. lobatum (Figure 3).

Phylogenetic Analyses
The 18S rRNA gene sequence divergence between all Chloroidium species was low and did not exceed 1-2% (Table S1). The divergence was sufficiently higher for the ITS1-5.8S-ITS2 region (5-29%; Table 1). ITS sequences in the strains differed from those in other species for more than 11%, while the difference between C. ellipsoideum and C. lichenum and C. engadinensis and C. viscosum was sufficiently low, 5.05 ± 0.67% and 8.34 ± 0.96%, respectively. The results of the sequence comparisons suggested that we were likely dealing with a new species. To test this hypothesis, we accessed the phylogenetic position of our strains in the genus Chloroidium using a dataset based on analyses of Darienko et al. [9] ( Table S2).
The dataset included 61 Chloroidium accessions representing 9 described species and our isolates. It was 2471 nt long, and of these 332 characters were parsimony-informative. The overall topologies of our unrooted ML and BI trees were similar to that presented by Darienko et al. [9] (Figure 3). For all but the species C. lichenum, represented by more than one sequence, respective generic clades were resolved, C. sacharophyllum, C. viscosum, C ellipsoideum, and C. arboriculum Darienko and Pröschold. Our strains formed a highly supported (100/1.00) clade that was resolved as a sister lineage to C. engadinensis with moderate support (70/0.98). This lineage was a part of the moderately supported clade (93/0.99) formed by C. antarcticum Darienko, Lukešová and Pröschold, the C. ellipsoideum/C. lichenum subclade, and C. viscosum. The relationship between these entities remained largely unresolved. One more Chloroidium intrageneric clade united long-branched C. arboricolum, C. laureanum, and C. lobatum (Figure 3).

ITS2 Secondary Structure
To analyze relationships between the new species and its sister taxon, C. engadinensis (Figure 3), we compared their ITS2 secondary structures. Figure 4 illustrates the proposed base pairing in ITS2 of C. orientalis strains and C. engadinensis. The spacer had a four-helical domain structure with five single-stranded regions, which is typical for most eukaryotes. Approximately 82% of the nucleotides were involved in the formation of these hairpin loops. The terminal part of helix I and all of helix IV were the most variable domains in these species. Due to this variability, it was somewhat difficult to access homology in these regions between C. orientalis and C. engadinensis models (Figure 4). We analyzed ITS2 secondary structures in our strains to detect CBC/hCBC events that may mark speciation and found an hCBC in the basal part of the helix III that differentiated C. orientalis from C. engadinensis (G•U > G-C, 102-197 pair (position 33 in the barcode alignment, see below). In addition, our strains also differed in an hCBC and one CBC in more-variable helix I (G•U > G-C, 38-51 pair, and A-T > G-C, 39-50 pair, respectively), which were not included in the list of conservative ITS2 barcoding positions.
The conserved regions of ITS2 were transformed into a unique numerical code using the ITS2/CBC approach described by Darienko et al. [9,13] (Figures 4 and 5). C. orientalis added two ITS2 ribotypes (1CORI and 2CORI) and two unique barcodes (BC-10a and BC-10b) in addition to 25 ribotypes and 22 barcodes, which were revealed earlier in the ITS2 secondary structure of Chloroidium members [9]. The sistership of C. orientalis and C. engadinensis was supported by a number of unique CBCs/hCBCs in the barcode alignment (

Discussion
We isolated three strains of Chlorella-like coccoid green alga and examined them using an integrative approach including light and confocal microscopy and molecular phylogenetics. The results of the 18S rDNA sequence comparisons (Table S1) revealed affinity of these strains to the genus Chloroidium. Using molecular characteristics (phylogenetic reconstructions and secondary structure models) and phenotypic features, we found evidence to describe our strains as a new species, Chloroidium orientalis. The new species is characterized by a relatively long branch in the phylogenetic tree (Figure 3), reflecting a large number of substitutions differentiating it from the rest of the genus, as well as elevated p-distances (see Results). Most of these molecular characters were contributed by the divergent ITS rDNA sequences (Table 1).
Phylogenetic analyses resolved C. orientalis as a sister of C. engadinensis in a clade comprising five species out of 10 genus members (Figure 3). Close affinity between C. orientalis and C. engadinensis was supported by not only significance thresholds, but also a number of shared marker substitutions in the conserved domains of ITS2 (Figure 4). Despite these synapomorphic characters, unique marker CBCs and hCBCs differentiated C. orientalis from C. engadinensis, as well as two ribotypes of C. orientalis from each other ( Figure 5).
C. orientalis is characterized by ellipsoid to spherical cells, the presence of unequally sized autospores, and parietal band-shaped to cup-shaped chloroplasts with a pyrenoid (Figures 1 and 2), and these features correspond to the diagnosis of the genus Chloroidium.
Although the diagnosis stresses that the cup-shaped chloroplast is not typical for the genus representatives, at least two species (C. lobatum and C. laureanum, former Parachloroidium members) have chloroplasts of such type [14]. The set of morphological characters described in C. orientalis does not fit diagnoses of any known species. Although most Chloroidium taxa have ellipsoid cells, species with spherical cells are also members of the genus [9]. In this respect, the new species is intermediate between those characterized exclusively by ellipsoid cell shape and former members of the genus Parachloroidium with spherical cells. Unlobed chloroplasts (Figure 1) are not typical for Chloroidium, and this feature sets C. orientalis apart from the rest of the genus. In most species, the chloroplasts are deeply lobed (C. ellipsoideum, C. lichenum, C. antarcticum, C. lobatum, and C. laureanum) or have a wavy margin (C. arboriculum, C. saccharophilum, C. engadinensis, and C. viscosum). Another feature of C. orientalis that is rare in the genus is a distinct pyrenoid with starch grains. Similar pyrenoids were described in C. lichenum and C. ellipsoideum, which are members of the same clade ( Figure 3), but these species differ from C. orientalis in chloroplast morphology.
The most striking difference between C. orientalis and other species of Chloroidium is the mode of reproduction. Like other taxa in the Watanabeales, the genus members reproduce through unequally sized autospores in small and large autosporangia [9]. In addition to that C. arboriculum and C. viscosum produce small autospores of equal size [8,9]. However, only C. orientalis produces three types of autospores: autospores of equal small size, autospores of equal large size, and autospores of unequal size (one large broadly ellipsoidal and 3-7 small narrowly ellipsoidal) (Figures 1 and 2). Even a greater diversity of autospore types was described in C. antarcticum, which has autospores of equal small size with narrowly ellipsoidal shape and three types of autospores of unequal size: (1) in autosporangia with 4-8 spores containing large and small autospores of the same broadly ellipsoidal cell-shape; (2) in sporangia with 4-16 cells containing one large broadly ellipsoidal cell and other small narrowly ellipsoidal cells, and (3) in sporangia with two large and two small broadly ellipsoidal cells. It was hypothesized that variability and flexibility in autospore formation may be advantageous under fluctuating environmental conditions that are typical for non-aquatic habitats where Chloroidium occurs [9].

Strain Origin, Culture Conditions and Light Microscopy
Three strains of coccoid green alga were isolated from soil samples and concrete wall biofouling collected in the temperate monsoon climate zone in the Primorsky Territory, Russia (one sample) and in Jilin Province, China (two samples). Sampling was carried out using standard methods [15]. The strains were isolated using the micro-pipette method [16] and cultured in liquid nutrient medium Waris-H [17]  The morphology of vegetative and reproductive cells was examined using an Olympus BX 53 light microscope equipped with Nomarski DIC optics and Olympus DP27 digital camera. Cultures were repeatedly examined throughout the lifecycle stages, i.e., in cultures of different ages since transfer.
For confocal microscopy algae were fixed in FAA (3.7%: formaldehyde: 50% ethanol: 5% acetic acid) for 20 min, then rinsed twice and counterstained with DAPI (4,6-diamidino-2-phenylindole, Molecular Probes Inc., Eugene, OR, USA) at the final concentration of 5 µg/mL. After another rinse of samples, the fluorescence was detected with LSM 710 LIVE confocal laser scanning microscope (Carl Zeiss, Germany) at the Instrumental Centre of Biotechnology and Gene Engineering of FSCEATB FEB RAS. DAPI fluorescence detected at 410-497 nm and autofluorescence of chloroplasts was recorded in the additional emission channel after 600 nm using Plan-Apochromat 63x/1.40 Oil DIC M27 objective with digital zoom. 3D files of the captured images were recorded and analyzed with ZEN microscope software.

Phylogenetic Analysis
Maximum likelihood (ML) analysis was carried out using PAUP 4.0b10 [27]. Bayesian inference (BI) was performed using MrBayes 3.1.2 [28]. To determine the most appropriate DNA substitution model for our datasets, the Akaike information criterion (AIC; [29]) was applied with jModelTest 2.1.1 [30]. MEGA v.7.0.26 [31] was used to estimate interspecific pairwise distances (p-distances). ML analysis was done using heuristic searches with a branch-swapping algorithm (tree bisection-reconnection). In BI, four runs of four Markov chains were carried out for 2 million generations, sampling every 100 generations for a total of 20,000 samples. Convergence of the two chains was assessed, and stationarity was determined according to the 'sump' plot with the first 5000 samples (25%) discarded as burn-in; posterior probabilities were calculated from trees sampled during the stationary phase. The robustness of the ML trees was estimated by bootstrap percentages (BP; [32]) and posterior probabilities (PP) in BI. BP < 50% and PP < 0.95 were not considered. MLbased bootstrap analysis was inferred using the web service RAxML version 7.7.1 (http: //embnet.vital-it.ch/raxml-bb/; accessed on 15 July 2021; [33]).
The Mfold web server (http://www.unafold.org/mfold/applications/rna-foldingform.php; accessed on 20 July 2021; [34]) was used with the default settings to generate the folding pattern of ITS2 ribosomal RNA secondary structure. An ITS2 model was constructed based on models proposed by Darienko et al. [9]. For the ITS2/CBC approach, the conserved regions of ITS2 were extracted and analyzed following Darienko et al. [9].

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. In addition, the data that support the findings of this study are openly available in GenBank (see Table S2).