Taxonomic revision of Chloromonas nivalis (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting Chloromonas species

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting Chloromonas species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of C. nivalis; however, strains with such vegetative cells in snow-inhabiting species of Chloromonas have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several C. nivalis strains. The morphological data demonstrated that one North American strain could be identified as C. nivalis, whereas three other strains should be re-classified as C. hoshawii sp. nov. and C. remiasii sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of C. nivalis was phylogenetically separated from the Austrian and Japanese specimens previously identified as C. nivalis based on zygote morphology.


Introduction
During the snow melt season, snowfields in polar regions and snowpacks in mountainous areas are sometimes stained green, red, or other colors. These events are typically caused by PLOS  blooms of cold-adapted microalgae [1][2][3]. In green snow, species belonging to the genus Chloromonas Gobi (Volvocales, Chlorophyceae) are generally dominant [2,3]. The genus contains at least 16 snow-inhabiting species [4][5][6][7] in addition to approximately 130 mesophilic morphological species [8,9], all of which are unicellular, green, and biflagellate. Taxonomic studies based on consecutive light microscopy (LM) of field-collected materials from North America revealed that several previously described snow species of nonmotile chlorococcalean algae, such as Scotiella Fritsch spp., are actually identical to the zygotes of snow-inhabiting Chloromonas species [10][11][12][13].
Among the snow-inhabiting Chloromonas species, C. nivalis (Chodat) Hoham et Mullet was considered cosmopolitan because of the world-wide distribution of cysts morphologically identified as zygotes of this species [formerly classified as Scotiella nivalis (Chodat) F.E. Fritsch] based on studies of North American material [1,11]. The species was generally identified solely on the basis of zygote morphology [1,[14][15][16][17] according to the species concept of previous studies [11,18]. This reliance on morphology possibly arises from the difficulty of inducing vegetative cell production from field-collected zygotes of snow-inhabiting Chloromonas. Very recently, Scotiella tatrae Kol was transferred to C. nivalis and reduced to a subspecies as C. nivalis subsp. tatrae Procházková et al., based on morphological and molecular data obtained from field-collected materials from Austria and Slovakia [19,20]. Recent molecular data also demonstrated that field-collected cysts morphologically identified as C. nivalis zygotes are composed of at least four distinct lineages or species, one of which is considered conspecific with the strains of C. miwae (Fukushima) Muramoto et al. [6]. In addition, it has been demonstrated that correct species identification of snow Chloromonas species is possible based on light and electron microscopy of asexual life cycles of cultures, as well as by molecular phylogenetic analyses [5]. Vegetative cells without eyespots and of inverted teardrop shape have been reported once in North American material of C. nivalis [11]. Subsequently, several strains from public culture collections were designated as C. nivalis [21][22][23], possibly based on such vegetative cell morphology. To date, three strains designated as C. nivalis (CCCryo 005-99, UTEX SNO66 and UTEX SNO74) have been examined by molecular phylogenetic analyses or LM of asexual reproductive cell morphology [19,[23][24][25][26]. However, detailed descriptions of vegetative cell morphology in these strains have not been provided. In addition, molecular phylogenetic analyses suggest that these strains were not monophyletic [6,19].
Therefore, in the present study, we carried out taxonomic re-examination of strains designated as C. nivalis using detailed morphological and molecular analyses. The data demonstrate that one North American strain not previously studied could be identified as C. nivalis, whereas the other strains should be re-classified as C. hoshawii Matsuzaki et al. sp. nov. and C. remiasii Matsuzaki et al. sp. nov. We also document phylogenetic relationships between the North American strain of C. nivalis and previously examined specimens of C. nivalis zygotes.
The strain UTEX SNO74 was excluded from further analyses because light microscopic and molecular data demonstrated that it has been replaced with a species of Trebouxia (Trebouxiales, Trebouxiophyceae) (S1 Text; S2 Table;

Morphological observations
Light and epifluorescence microscopy were performed using a BX51 microscope (Olympus Corp., Tokyo, Japan) equipped with Nomarski differential interference optics. Transmission electron microscopy (TEM) was performed as described previously [5] using a JEM-2010 transmission electron microscope (JEOL, Tokyo, Japan). Cells in actively growing 5-to 12-day-old cultures were investigated. In addition, we carried out LM of cultures at 1, 2 and 3 months after inoculation to detect the production of cell aggregates resulting from repeated divisions of daughter cells retained within the parental cell wall [5,29].

Molecular analysis
For molecular analysis, we used nucleotide sequences of nuclear-encoded 18S and 26S ribosomal DNA (rDNA), chloroplast-encoded ATP synthase beta subunit (atpB), P700 chlorophyll a apoprotein A2 (psaB) and the large subunit of RuBisCO (rbcL) genes, and internal transcribed spacer 2 (ITS2) region of nuclear rDNA. Sequences from five snow-inhabiting strains and of the 12 mesophilic ones (S3 Table) were determined as described previously [6,30] using newly designed specific primers (S4 Table).
For multigene phylogeny, we used four strains examined in this study (CCCryo 005-99, CCCryo 047-99, UTEX SNO66 and UTEX SNO71) as well as 28 operational taxonomic units examined in previous studies [6,9] (S3 Table). All belong to the genus Chloromonas sensu Pröschold et al. [31] or the Chloromonadinia clade [32]. The mesophilic strains (S3 Table) were treated as the outgroup according to previous results [9,24,25]. The 18S and 26S rDNA, atpB and psaB gene sequences were aligned as described previously [5,33,34]. In addition, only the first and second codon positions of the nucleotides in the atpB and psaB were used for phylogenetic analyses. This was because the third nucleotide positions of the codons had an unusual base composition and markedly higher substitution rates than the 18S and 26S rDNA and the first and second codon positions of the atpB and psaB genes [6,33,[35][36][37]. The combined 5,497-bp data matrix of the regions was subjected to Bayesian inference (BI), maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ) analyses as described in previous studies [9,33] except that IQ-TREE v. 1.4.3 [38] was used in ML analysis instead of PAUP 4.0b10 [39]. In each analysis, identical sequences were reduced to a single operational taxonomic unit. Since rbcL gene substitutions in Chloromonas are unusual and may result in artifacts [33,40], we did not concatenate the rbcL gene sequences with the data matrix.
For comparison of the previously published sequence data from field-collected cysts identified as C. nivalis zygotes, we performed single-gene phylogenetic analyses using 18S rDNA or rbcL gene sequences as described above. In addition, we set three partitions (first, second, and third codon positions) for BI and ML analysis of rbcL gene sequences according to a previous study [4]. Additional operational taxonomic units were selected from previous studies [19,20,41] and shown in S3 Table. Substitution models for each phylogenetic analysis are described in S5 Table. The data matrices used in the present study are available from TreeBASE [42] (matrix accession number S22105).
Methods for annotation and prediction of secondary structures of nuclear rDNA ITS2 region were described in a previous study [5]. For detecting compensatory base changes (CBCs), the ITS2 sequences were aligned on the basis of sequence-structure analysis [43] using 4SALE [44,45].

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) (http://iapt-taxon.org/nomen/main. php), and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

Morphological observation
Light and epifluorescence microscopy (Figs 1 and 2) demonstrated that the strains could be subdivided into three morphological species (C. hoshawii, C. nivalis, and C. remiasii) based on differences in cell shape and size, chloroplast morphology, presence of eyespots, number of zoospores formed within the parental cell wall during asexual reproduction, and presence of cell aggregates (aggregates of 16 or more cells resulting from repeated divisions of daughter cells retained within a parental cell wall [5,29]) in culture (Table 1). In C. nivalis strain UTEX SNO71, vegetative cells had an inverted-teardrop shape with a prominent posterior tail ( Table 1). In addition, cell aggregates resulting from repeated divisions of daughter cells retained within the parental cell wall [5,29] were produced in fresh (5-to 12-day-old) cultures as well as in old (almost or more than one-month-old) cultures of C. remiasii (S3 Fig). In contrast, such cell aggregates were not observed in the other two species (Table 1). Sexual  reproduction or hypnospore formation was not observed in the three species. All three species failed to grow at 20˚C after two weeks of cultivation, as described in previous reports of other species of snow-inhabiting Chloromonas [5,6,46]. TEM (Fig 3) showed that each cell of the three species possessed a nucleus, and a cupshaped chloroplast without pyrenoid matrices (Fig 3A, 3C and 3E). As in other snow-inhabiting Chloromonas species [5], mitochondria and Golgi bodies were present mainly between the nucleus and chloroplast. Several small vacuoles with crystalline content were observed in the cytoplasm of the three species (Fig 3A-3F). Tangential sections of C. nivalis showed the chloroplast profiles to be almost elongate in shape (Fig 3B). In contrast, chloroplasts of C. hoshawii and C. remiasii were generally angular in shape (Fig 3D and 3F). LM surface views of the chloroplasts correlated with TEM images; the chloroplasts appeared to be composed of elongate platelets or angular discs (Figs 1C, 1D, 1G, 1H, 1K, 1L and 2A-2C). The eyespot of C. remiasii was comprised of a single layer of electron-dense globules (Fig 3G). Such structures were not seen in C. hoshawii or in C. nivalis, even under TEM.

Molecular phylogenetic analyses
Phylogenetic analyses (based on the sequences of 18S and 26S rDNA, and the first and the second codon positions of atpB and psaB), revealed four robust monophyletic groups of snowinhabiting Chloromonas species (A-D) resolved with 1.00 posterior probabilities (PP) in BI and 92-100% bootstrap values (BV) in ML, MP and NJ analyses (Fig 4). Chloromonas nivalis strain UTEX SNO71 and both C. remiasii strains (CCCryo 005-99 and CCCryo 047-99) were included within groups C and D, respectively, whereas C. hoshawii strain UTEX SNO66 was positioned outside of the four groups and therefore represents an independent lineage. Group  In the present multigene phylogenetic tree, the four robust monophyletic groups and one independent lineage of C. hoshawii were subdivided into two large clades: one composed of groups A and C together with C. hoshawii (1.00 PP in BI and 94% and 73% BV in ML and NJ analyses, respectively), and the other constructed of groups B and D (1.00 PP in BI and 73-92% BV in ML, MP and NJ analyses). Within the former clade, phylogenetic relationships among groups A and C and C. hoshawii were not resolved.
Further comparison of phylogenetic relationships between C. nivalis strain UTEX SNO71 and field-collected C. nivalis zygote specimens examined in previous studies [19,20,41] was performed by single-gene phylogenetic analyses using 18S rDNA and rbcL sequences (S4 and S5 Figs). Both trees reconstructed the monophyletic groups B-D which were robustly resolved in the multigene phylogenetic tree (Fig 4), but statistical support values for monophyly were lower. Group A in Fig 4 was recovered only in the rbcL-based tree (S5 Fig). In the 18S rDNAand rbcL-based trees (S4 and S5 Figs), the Austrian C. nivalis zygote specimen (P24/DR4 [19,20]) and the Slovak C. nivalis subsp. tatrae zygote specimen (LP01 [20]) were positioned within group B and formed a small robust clade (1.00 PP in BI and >89% BV in ML, MP and NJ analyses). This subclade was sister to the Japanese C. nivalis zygote specimens (Gassan-B and Hakkoda-3 [6]) with 1.00 PP in BI and 74-83% and 83-94% BV in ML, MP and NJ analyses in 18S rDNA-and rbcL-based tree, respectively. In addition, the two Japanese C. nivalis zygote specimens (Gassan-NIV1 and Gassan-NIV2 [41]) were positioned outside of groups A-D in the phylogenetic tree of rbcL sequences (S5 Fig). However, C. nivalis strain UTEX SNO71 was included within group C in 18S rDNA-and rbcL-based trees, and was phylogenetically separated from the Austrian, Japanese and Slovak C. nivalis zygote specimens.

Comparative molecular analyses
To verify separation of C. remiasii and C. chenangoensis, which were sister to each other (Fig  4), we compared the secondary structures of the nuclear rDNA ITS2 region. The predicted secondary structures (S6 and S7 Figs) possessed four helices, a U-U mismatch in helix II (S6 and S7 Figs, arrowheads), and the YGGY motif on the 5 0 side near the apex of helix III (S6 Fig and  S7 Fig, boldface). All these features are common structural hallmarks of eukaryote nuclear rDNA ITS2 secondary structures [47][48][49][50]. In C. remiasii and C. chenangoensis, at least two CBCs were detected near the apex of helix III encompassing the YGGY motif (the most conserved region of nuclear rDNA ITS2 secondary structures [48,49]) (Fig 5A). In addition, we estimated the uncorrected p-distances in nuclear-encoded 18S and 26S rDNA, and in chloroplast-encoded atpB and psaB genes, for C. remiasii and C. chenangoensis. The nucleotide differences between the two species were much larger than those between snow-inhabiting C. hohamii and C. tenuis, and also between mesophilic C. chlorococcoides (H. Ettl et K. Schwarz) Matsuzaki et al. and C. reticulata (Goroschankin) Gobi, each pair being sister species previously delineated by morphological and molecular data [5,51] (Fig 5B).

Discussion
Zygotes or cysts morphologically identified as C. nivalis have been reported from various localities of the world [1,11,18]. However, motile vegetative cells directly obtained from such dormant cells have never been reported [11,19]; the partial life cycle (from vegetative cells to zygotes) of C. nivalis was observed only in North American field-collected material [11]. The type locality of Pteromonas nivalis Chodat (the basionym of C. nivalis) is in the French Alps [52]; however, the original species description lacks information on motile vegetative cells, and neither a strain nor sequences are available. Recent robust molecular data indicated that Japanese field-collected cysts morphologically identical to the North American C. nivalis zygotes (= P. nivalis and S. nivalis [11]) contain multiple species [6]. Thus, C. nivalis should be circumscribed by the vegetative morphology reported from the North American material [11].
The light microscopic features of the North American strain UTEX SNO71, which has not been examined in previous studies, were consistent with those of North American C. nivalis [11] with respect to cell shape, chloroplast morphology, and the number of zoospores formed within the parental cell wall (Table 1; S2 Text). Thus, we consider the strain UTEX SNO71 as C. nivalis, although zygotes were not observed in our study. Contrary, the vegetative morphology of strains previously designated as C. nivalis (CCCryo 005-99 and UTEX SNO66) [19,23,24] differed from that of strain UTEX SNO71 (Table 1). In addition, these three strains were phylogenetically well separated from each other (Fig 4). Therefore, based on morphology and phylogeny of vegetative cells, we re-classified strains CCCryo 005-99 and UTEX SNO66 as C. remiasii and C. hoshawii, respectively.
LM and TEM showed that chloroplasts of C. hoshawii and C. remiasii lack pyrenoids (Figs 1E, 1I and 3C-3F), and the species are robustly positioned within Chloromonadinia clade (Fig  4). These characteristics correspond to both traditional [8,53] and phylogenetically revised [31] concepts of the genus Chloromonas. Among the snow-inhabiting species of the genus, C. hoshawii resembles C. chenangoensis and C. pichinchae in having an ellipsoidal or elongate- Taxonomic revision of Chloromonas nivalis strains ovoid vegetative cell with rounded anterior and posterior ends, and a chloroplast which appeared to be composed of angular discs and had no eyespot (Figs 1E and 2B; S2 Text; S6  Table) [5,8,10,25]. However, C. hoshawii differs from C. pichinchae in that it does not produce cell aggregates in old cultures (S2 Text; S6 Table) [5]. Maximum cell width is less than 10 μm in C. hoshawii, whereas vegetative cell width of C. chenangoensis is up to 17.5 μm (S2 Text; S6 Table) [5,25]. Furthermore, the phylogenetic position of C. hoshawii strain UTEX SNO66 is separated from those of C. chenangoensis strain UTEX SNO150 and C. pichinchae strain UTEX SNO33 (Fig 4). On the other hand, C. remiasii is very similar to C. alpina Wille in possessing an ellipsoidal vegetative cell with rounded anterior and posterior ends, and a chloroplast seemingly composed of angular discs and having an eyespot (Figs 1I, 1K and 2C; S2 Text; S6 Table) [8,53,54]. However, C. remiasii differs from C. alpina (of which no sequences are available) in cell size (10.2-15.6 μm wide × 18.2-30.8 μm long vs. 4-7 μm wide × 9-12 μm long, respectively; S6 Table) [8,53,54]. Although the present phylogenetic results demonstrate that C. remiasii is sister to C. chenangoensis (Fig 4), C. remiasii can be distinguished from C. chenangoensis in having an eyespot on the chloroplast and in producing cell aggregates in culture (Figs 1K and 2C; S3 Fig; S2 Text; S6 Table) [5,25]. In addition, the two species had at least two CBCs in the most conserved region of nuclear rDNA ITS2 secondary structures (Fig 5A). The CBCs correlate with the separation of biological species, according to [49]. Furthermore, genetic differences in the four genes between these two species were much larger than those between snow-inhabiting C. hohamii and C. tenuis, or between mesophilic C. chlorococcoides and C. reticulata, each pair being sister species delineated by morphological and molecular data [5,51] (Fig 5B). Therefore, the separation of C. remiasii and C. chenangoensis was supported by morphological and molecular data, and apparently they have different patterns of geographic distribution (Arctic Svalbard vs. Arizona, USA [21,22,23,25]).
Although neither C. hoshawii nor C. remiasii could grow at 20˚C, a comparison of their vegetative morphology with that of mesophilic Chloromonas species was performed: The mesophilic species C. enteromorphae (Brabez) Gerloff et H. Ettl, C. eumaculata P.C. Silva, C. gutenbrunnensis Wawrik, and C. granulata (L.Ş. Péterfi) Gerloff et H. Ettl resemble C. hoshawii and C. remiasii in that the cells are ovoid to ellipsoidal with rounded anterior and posterior ends, and they have a cup-shaped chloroplast seemingly composed of angular discs [8,53]. However, C. hoshawii differs from the mesophilic species by the lack of an eyespot on the chloroplast (Figs 1G and 2B) [8,53,[55][56][57][58]. The eyespot of C. remiasii is positioned in the anterior third of the cell, whereas those of C. eumaculata and C. gutenbrunnensis are located near the equator or in the posterior third of the cell, respectively [8,53,55,58]. The cell wall of C. granulata is quite swollen; this trait was not observed in vegetative cells of C. remiasii (Figs 1I and 2C) [8,53,56]. The nucleus of C. remiasii is positioned in the middle of the protoplast (Figs 1I  and 2C), whereas the nucleus is in the posterior third of the cell in C. enteromorphae [8,53,57]. Moreover, the vegetative cells of C. remiasii are smaller than those of C. enteromorphae (up to 30.8 μm long vs. up to 44 μm long, respectively) ( Table 1) [57]. Thus, C. hoshawii and C. remiasii represent two new morphological species of the genus Chloromonas.
Molecular phylogenetic analyses (Fig 4; S4 and S5 Figs) demonstrated that the North American strain morphologically assignable to C. nivalis from North America is phylogenetically separated from Austrian, Japanese and Slovak field-collected zygote specimens earlier identified as C. nivalis [6,19,20,41]. Therefore, taxonomic re-examination of the latter specimens should be carried out based on their vegetative morphologies. In addition, scanning electron microscopic features of the zygotes might also help their taxonomic revision [20]. Although no one has successfully induced the production of motile vegetative cells from field-collected zygotes of snow-inhabiting Chloromonas under controlled laboratory conditions [10][11][12][13]19,23], our recent study provided a practical method for molecular identification of such zygotes by using data obtained from accurately identified cultures [6]. Thus, further taxonomic studies of cultured snow-inhabiting Chloromonas are required to reveal the correct affiliation of field-collected cysts currently identified as C. nivalis zygotes.
Vegetative cells solitary, having two flagella, without a prominent anterior papilla. Cells ellipsoidal or elongate-ovoid; 4.9-9.3 μm wide and 13.8-18.6 μm long. Cells with a central nucleus and a single cup-shaped chloroplast. Chloroplast seemingly composed of angular discs, showing irregular incisions on the surface, without an eyespot and pyrenoids. Asexual reproduction by formation of generally two or four zoospores, with rotation of the protoplast before the first cell division. Cell aggregates not observed in culture.
Holotype: Specimen TNS-AL-58946 deposited at TNS (National Museum of Nature and Science, Tsukuba, Japan); material consists of resin-embedded vegetative cells from strain UTEX SNO66.
Etymology: The species epithet hoshawii is in honor of Dr. Robert W. Hoshaw who contributed greatly to the taxonomy of green algae (e.g. [59,60]). He participated in collection of material from which the authentic strain of this species was isolated [21].
Etymology: The species epithet remiasii is in honor of Dr. Daniel Remias, who has contributed greatly to the ecology and physiology of snow-inhabiting microalgae (e.g. [19,26,61]).
Remarks: A previous study [23] suggested relationship between the strain CCCryo 005-99 and field-collected cysts or zygotes, both of which were collected at the same location in Svalbard. The cysts resemble North American C. nivalis zygotes in having spindle-shaped cell with several longitudinal, slightly helical ridges on the cell wall extended partially to the poles. Since molecular data of the cysts are not available and sexual reproduction of C. remiasii has not been observed, we could not confirm this possible relationship.  Table).  [19,20], and Gassan-NIV1 and Gassan-NIV2 [41], respectively) and the Slovak C. nivalis subsp. tatrae zygote specimen (LP01 [20]) are shadowed in black.   Table. Substitution models applied to respective data matrices of the present phylogenetic analyses (Fig 4; S4 and S5 Figs).