A New Cryptic Species of the Genus Mychonastes (Chlorophyceae, Sphaeropleales)

A new species of green coccoid algae, Mychonastes hindakii sp. nov., was isolated from the River Moscow (Russia, Moscow). The taxon is described using morphological and molecular methods. Mychonastes hindakii sp. nov. belongs to the group of species of the genus Mychonastes with spherical single cells joined with mucilaginous, irregularly shaped stalks. A comparison of ITS2 rDNA sequences and its secondary structures combined with the compensatory base changes approach confirms the separation between Mychonastes hindakii and other species of the genus. Mychonastes hindakii sp. nov. represents a cryptic species that can only be reliably identified using molecular data.

The algal genus Mychonastes comprises unicellular or colonial algae with spherical, ovoid, or ellipsoidal cells with or without a mucilage envelope. Each cell contains 1-4 discoid or mostly cup-shaped chloroplasts without a pyrenoid. Colonies consist of 4-64 or more cells. Cells are in colonies that are connected by gelatinous strands formed by cell wall remnants of the mother cell [11]. Originally, it was described as a monotypic genus with M. ruminatus Simpson and Van Valkenburg as its type [12]. Later, three species were added [13,14]. Then, Krienitz et al. [11] revised the genus based on morphological observations and SSU and ITS rRNA sequence analyses, described eight new species and transferred the species from the genus Pseudodictyosphaerium Hindák (=Dictyosphaeriopsis Hindák) to Mychonastes. Recently, Patova et al. [15] described another new species from a cold-water stream in the subpolar Urals.
The members of the genus Mychonastes are widely distributed throughout the world inhabiting lotic and lentic waters, soils, and aerial habitats [11,16]. For instance, the type species, M. ruminatus, was isolated from the brackish water of Chesapeake Bay (Maryland, USA) [12]. Although M. homosphaera was described from freshwaters, it is considered one of the most common green algal species in soils [16,17]. Actually, strain M. homosphaera QUCCCM70, isolated from desert soil in Qatar, showed a high antioxidant capacity [18]. Additionally, Mychonastes can dominate in freshwater picoplankton of lakes [19]. Selected strains of some Mychonastes species (e.g., M. afer, M. homosphaera and M. spp.) are a potential feedstock for biodiesel and feed production [18,[20][21][22].
The aim of this article is to report our observations on the Mychonastes strain isolated from the Moscow River (Moscow, Russia) and describe it as a new species.
The aim of this article is to report our observations on the Mychonastes strain isolated from the Moscow River (Moscow, Russia) and describe it as a new species.

Results
Cells of the strain M323 are small, spherical, and commonly in colonies connected by strands with mucilaginous envelopes. Stalks between cells are visible with light microscopy. The cellular and colony morphologies of the isolate suggested that it is related to the genus Mychonastes ( Figure 1).    [3], which have cells in colonies, joined by stalks. Species in this group can be unambiguously distinguished by the presence of CBCs (Compensatory Base Changes) in ITS2 sequences [3]. Analysis of the ITS2 rDNA of strain M323 allowed the recognition of the new lineage on the ITS2 rDNA tree (Figure 3).    This strain forms its own branch with high values of Bayesian posterior probability and ML bootstrap (1/81). The ITS2 sequence data show that M. hindakii is included in a clade that consists of two morphologically different species: M. jurisii and M. huancayensis Krienitz et al. [11]. This cluster also included two unidentified symbiotic strains LbS_7 [23] and LBMa-1 [24].
Here  This strain forms its own branch with high values of Bayesian posterior probability and ML bootstrap (1/81). The ITS2 sequence data show that M. hindakii is included in a clade that consists of two morphologically different species: M. jurisii and M. huancayensis Krienitz et al. [11]. This cluster also included two unidentified symbiotic strains LbS_7 [23] and LBMa-1 [24].
Distribution: To date, Mychonastes hindakii has been observed in this type locality. At the time of collection, the pH was 7.03, the temperature was 19.9 • C, and the specific conductance was 347 µS/cm. To date, Mychonastes hindakii, except the type locality, was also found widespread in Europe. Molecular signatures of ITS1 of this species were spotted in 31 lakes of Europe in Austria, France, Germany, Hungary, Italy, Poland, Romania, Spain, and Sweden [25]. Some characteristics of the lakes where M. hindakii was found by molecular signatures are presented in Table 1.

Discussion
Currently Molecular data demonstrated that the Mychonastes clade represents a monophyletic lineage within Chlorophyceae, which is clearly differentiated from other evolutionary lineages containing small-celled coccoid green algae [11]. Like many other green algae, morphological convergence and high levels of morphological plasticity complicate the application of traditional classification methods in this genus. For morphological delineation of species in the genus, features such as form and size of cells, colony-forming or solitary cells, presence or absence of mucilaginous stalks and their branching patterns (regular or irregular), presence or absence of mucilaginous envelope are usually used [11]. However, most of these features are variable in culture. Under culture conditions, most colonial forms can disintegrate, thus presenting challenges for their differentiation not only from Mychonastes species with solitary cells, but from other single small-celled, spherical or ellipsoidal chlorophytes such as Choricystis (Skuja) Fott, Meyerella Fawley and K. Fawley, or Nannochloris Naumann [26][27][28][29][30]. The ITS2 sequence data did not support discrimination between the colonial and single-celled Mychonastes species. It is considered that the shape of mucilaginous stalks might have some taxonomic value but it seems that their shape can significantly vary in culture. For instance, Hindák [31] illustrated a strain of Mychonastes jurisii that has colonies with both regular mucilaginous stalks (such as in M. timauensis) and irregular ones. Based on these data, Krienitz et al. [11] stated that only molecular data can provide conclusive evidence for the characterization and separation of species of the genus Mychonastes.
The ITS2 region has an important role in rRNA processing because it contains specific cleavage sites for further elimination. The process of excising ITS2 in the RNA transcript requires the formation of the correct ITS2 secondary structures, which are conserved across most eukaryotes [32,33]. ITS2 secondary structures are often used for delimiting biological species based on the presence of compensatory base changes (CBCs) [34,35]. The CBC species concept states that two organisms/strains whose ITS2 sequences differ by even a single CBC in the conserved regions of Helix II and Helix III represent two different biological species [34]. According to Müller et al. [36] and Wolf et al. [37], the presence of any CBC in the whole ITS2 molecule is sufficient for distinguishing species. The CBC concept was successfully used for species delimitation in different and independent groups of algae such as cryptophytes [38,39], diatoms [40], eustigmatophytes [41] and green algae [42]. Comparing the secondary structure of ITS-2, compensatory base changes are found to discriminate new species in the Mychonastes clade too [11,15].
All Mychonastes taxa are characterized by variability in the length and shape of the ITS2 secondary structure. M. hindakii has a 367 bp ITS2 molecule length. It is the longest in comparison with relative species M. jurisii (319 bp) and M. huancayensis (328 bp), but shorter than M. frigidus and M. pusillus (383 bp). To take into account the difference in length and a high degree of variability, we used the secondary structure alignment for CBCs search and unrooted ITS2 tree recovering. Comparison of the ITS2 secondary structures of M. hindakii with other species with known ITS2 rDNA sequences revealed a number of CBCs with all studied taxa, including highly conserved areas of Helix III (Figure 4).  (Table 2).     Mychonastes hindakii clustered with colonial M. jurisii and unicellular M. huancayensis on the unrooted ITS2 tree and formed an individual branch (Figure 3). The clade, consisting of two morphologically different species, was also recovered in previous ITS2 analyses [11,15], which contradicts the importance of cell organization in the systematics of Mychonastes. Morphologically, Mychonastes hindakii is most similar to M. jurisii. This species was originally described as Dactylosphaerium jurisii Hindák [31], however, since the type species of Dactylosphaerium, D. sociale Steinecke is multiplied by zoospores and has chloroplasts with a pyrenoid, D. jurisii was transferred to Pseudodictyosphaerium [43]. Later, Krienitz et al. [11]  There is one additional important difference between these species; unlike the famous and widespread M. jurisii, the second sister species, M. huancayensis, is known only from Peru [11].
The cluster of the described species also included two unidentified strains LbS_7 and LBMa-1, which are symbiotic for a different group of protists. The strain LbS_7 was isolated from primmorphs of the endemic Baikal Lake freshwater sponge Lubomirskia baicalensis Pallas (Lubomirskiidae, Porifera). This strain has a simple morphology with solitary cells, such as M. homosphaera, which is not enough for the assignment of this strain to any described species [23]. Molecular data, obtained from the strain, show crucial differences from other strains, and probably, it represents a new species, which has not been described yet. The second strain, LBMa-1, is a photobiont of Stentor polymorphus (Müller) Ehrenberg (Heterotrichea, Ciliophora). Cells of this strain in culture were solitary, without a mucilaginous covering, lacking inter-connecting threads between cells, and spherical or ovoid (2.8-3.4 vs. 4.1-4.8 µm) [24]. This strain does not have significant differences in the nucleotide composition of ITS2 rDNA and its secondary structure from M. jurisii, thus, it refers to this species.
Other colonial species with nucleotide sequence data, Mychonastes afer, M. ovahimbae and M. pushpae, have the same cell diameter range (2-5 µm), which overlaps with the cell diameter range of M. hindakii (3-4.4 µm), but they differ considerably from M. hindakii by the ITS2 sequences, secondary structure, and CBC number ( Table 2 Moreover, Mychonastes hindakii is similar to M. racemosus but the latter species has a greater cell diameter range (2.5-6 µm) and larger colonies (10-80 µm). The main morphological distinction of Mychonastes hindakii from M. timauensis is that, in the latter species, the cells in colonies are connected by mucilaginous, regularly shaped, broad and flat rhomboid stalks, while in M. hindakii, the stalks are irregularly shaped. Additionally, M. timauensis colonies are larger (8-60 µm). As with the previous taxa, M. racemosus and M. timauensis also differ from new species by CBCs; nine with the first and six with the latter species, respectively. Among species without nucleotide sequence data, Mychonastes hindakii can be compared with M. minusculus and M. lacunaris. Mychonastes minusculus, however, has smaller cells (1.8-3.7 µm in diameter) than M. hindakii. In contrast to M. lacunaris, M. hindakii has irregularly shaped mucilaginous stalks. Additionally, M. hindakii is somewhat similar to the specimens reported by Hindák from a fishpond in Western Slovakia and identified as Dictyosphaerium botrytella Komárek and Perman forma [44]. The mature cells of Slovak specimens were spherical to spherical-oval, 4-6 × 3.5-5 µm. Unfortunately, the true taxonomic identity of those Slovak specimens cannot be resolved. It is very likely that they represent a separate, yet undescribed species but they definitely do not belong to D. botrytella in terms of cell shape and size. Now, D. botrytella is a member of Mychonastes [11].
Our analysis confirms the widespread distribution of M. hindakii across European water bodies. Molecular signatures of the ITS1 of this species were found in 31 lakes of Europe in Austria, France, Germany, Hungary, Italy, Poland, Romania, Spain, and Sweden [25]. The new species tolerates a wide range of environmental parameters. Mychonastes hindakii, like other species of the genus, can be unambiguously identified only with molecular data.

Study Area
The sample was collected from the Moscow River (55 • 47 28 N, 37 • 24 53 E, 123 m above sea level) on 28 July 2019. The studied area is located in the central part of the East European Plain, in Moscow city, at the territory of the natural monument, Park Serebryany Bor. The climate of Moscow city is moderately continental with an average February temperature of −6.7 • C and an average +19.2 • C in July. Annual precipitation is 600-800 mm [45]. The Moscow River has a longitude of 473 km, and the water basin is nearly 17,600 km 2 . The river accounts 61% of the snow supply, up to 27% of the underground water supply, and 12% of the rain supply [46].

Culturing
The sample was placed immediately in the field in a sterile Waris-H medium [47]. Monoclonal strain was established by examination of micro-pipetted single cells under an inverted microscope. Non-axenic unialgal cultures were maintained in Waris-H liquid medium at 10 • C in a growth chamber with a 12:12 h light:dark photoperiod with 50-100 mmol photons m -2 s -1 . Morphological features were investigated after cultivation on 3N BBM, WC, BG-11 liquid and agar mediums [16,48].

Light Microscopy
Light microscopy observations and photography were carried out using a Zeiss Scope A1 microscope (Carl Zeiss AG, Oberkochem, Germany) and Nikon Eclipse80i (Nikon, Tokyo, Japan) with optics of Nomarski (DIC), equipped with an oil immersion objective (Plan-Apochromat 100x/1.40 Oil DIC M27) with magnification up to 1000×. Cells were stained with 0.1% methylene blue solution and 1.0% ink solution for the determination of the mucilage structure. Cultures were observed in exponent and stationary growth phases.
Light micrographs were taken with an AxioCam ERc 5s Rev.2 and Nikon Digital Sight Ds-2Mv cameras.

Extraction of DNA and Amplification
The total DNA of the monoclonal culture was extracted using InstaGeneTM Matrix according to the manufacturer's protocol. Fragments of SSU-ITS1 rRNA (2076 bp) were amplified using pairs of primers: 18S-F [49] and 18L [50] for SSU rRNA fragments, and SR10 [51] and ITS2_broad R [25] for the 3 -end of SSU, including the V9 region, and ITS1 rRNA fragments. Fragment of partial 5.8S-ITS2-partial LSU rDNA (753 bp) was amplified using a pair of primers crITS_03F, crITS_05R [52].
Amplification of all studied fragments was carried out using the premade mix Screen-Mix (Evrogen, Russia) for the polymerase chain reaction (PCR). The conditions of amplification for SSU rRNA, V9 SSU-ITS1 rRNA, and 5.8S-IST2-LSU rDNA fragments were: an initial denaturation of 5 min at 95 • C, followed by 35 cycles at 94 • C for denaturation (30 s), 52 • C for annealing (30 s) and 72 • C for extension (50-80 s), and a final extension of 10 min at 72 • C. The resulting amplicons were visualized by horizontal agarose gel electrophoresis (1.5%), colored with SYBR Safe (Life Technologies, Carlsbad, CA, USA). Purification of DNA fragments was performed with the ExoSAP-IT kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's protocol. All studied fragments were decoded from two sides using forward and reverse PCR primers and the Big Dye system (Applied Biosystems, Foster City, CA, USA), followed by electrophoresis using a Genetic Analyzer 3500 sequencer (Applied Biosystems, Foster City, CA, USA). For phylogenetic analysis fragments were used separately, containing only partial SSU rDNA, and only ITS2 rDNA.

Phylogenetic Analysis of SSU rDNA Data
Sequences were edited manually and assembled using BioEdit v7.1.3, and MegaX [53]. Newly determined sequences and GenBank sequences of 43 other chlorophytes from different morphological groups were included in the alignment. Additionally, two species, Ulothrix zonata (F. Weber et Mohr) Kützing and Gloeotilopsis planctonica M.O.P. Iyengar and Philipose, were chosen as outgroup taxa. Sequences were aligned using global SILVA alignment for rRNA genes in the SINA v1.2.11 [54]. jModelTest ver. 2.1.10 [55], using the Bayesian information criterion (BIC), indicated that the TrN model of nucleotide substitution, with Gamma (G) distributed rates across sites and a proportion of invariable sites (I), was the most appropriate evolutionary model for the SSU rDNA alignment. Maximum likelihood phylogeny (ML) was constructed using MegaX [53] with the Subtree-Pruning-Regrafting tree rearrangements algorithm (SPR). The bootstrap analysis with 1000 replicas was used. Bayesian Inference (BI) analysis was conducted with MrBayes-3.2.5 [56], using SYM+G+I model. Three "hot" and one "cold" Markov chains were run for 15 × 10 6 cycles in two repetitions with the selection of each 100th generated tree. Phylogenetic tree and posterior branching probabilities were obtained after discarding the first 25% to produce estimate parameter models of nucleotide substitutions and likelihood. Viewing and editing of trees were carried out in the programs FigTree (ver 1.4.2) and Adobe Photoshop CC (19.0).

Internal Transcribed Spacer 2 Annotation, Secondary Structure Modeling, Alignment and Phylogeny
For the annotation of the ITS2 sequence, we used ITS2-Annotation [57]. This tool uses HMMer [58] to annotate ITS2 sequences with Hidden Markov Models (HMMs) [59]. It returns, by definition of the ITS2, the sequence between the conserved 5.8S and 28S (or equivalent) rRNA funding region. The secondary structure of the ITS2 rDNA region was modeled using the Predict a Secondary Structure Web Server (http://rna.urmc.rochester.edu; accessed on 21 November 2020). This server combines four separate prediction and analysis algorithms: calculating a partition function, predicting a minimum free energy (MFE) structure, finding structures with maximum expected accuracy, and pseudoknot prediction.
The construction of the model accounted for the presence of the pyrimidine-pyrimidine unpaired section in the second helix at the seventh position [60], as well as the length and nucleotide composition of the spacers in the core of the model defining the helix boundaries [33]. Detail features of the ITS2 secondary structures of the studied species, such as the presence of helices Ib and IIIb, were also checked using secondary structures of other Mychonastes species [11]. The resulting secondary structure was visualized in PseudoViewer3 [61]. The ITS2 sequences were aligned according to the secondary structure by ClustalW algorithm [62] in 4SALE [63]. Delimitations of species secondary structures of Mychonastes strains were checked for compensatory base changes (CBCs) according to Coleman [34,35] and Müller et al. [36] in comparison with species described by Krienitz et al. [11] and Patova et al. [15]. For the phylogenetic analysis, the sequence part from the sequence-structure alignment (as obtained by 4SALE) was further processed using the same programs and methods as described above for the 18S data, except for the model of nucleotide substitution-HKY+G+I.

Blast Search of Barcoding Data (ITS1)
To assess the distribution of described species, we carried out a blastn search of its internal transcribed spacer 1 sequence in the database, containing data of metabarcoding studies of 218 European freshwater lakes presented by Boenigk et al. [25] In this study, we used a 300 nt region of ITS1-5.8S rDNA as a representative for delineation of Mychonastes species. The search was carried out in all 436 SRA experiments with program megablast and 5000 target sequences. We considered only matches with 99% similarity, i.e., sequences with no more than two changes (substitution, indel) in comparison with the initial sequence.