Ultrastructure and phylogeny of Parietochloris toyamaensis sp. nov. and P. bilobata (Trebouxiophyceae)

Abstract We describe Parietochloris toyamaensis sp. nov. and verify the taxonomic status of P. bilobata (originally described as Neochloris and once combined with Ettlia), using ultrastructural and molecular analyses. P. toyamaensis was isolated from soil collected in Toyama, Japan. It had a parietal chloroplast with pyrenoids that were discontinuously covered with starch segments and penetrated more or less in parallel by thylakoid membranes, and reproduced by forming naked, somewhat dorsoventral, biflagellate zoospores. Two contractile vacuoles were in line lengthwise, located in the median ventral side of the zoospores, as in the type P. alveolaris. The basal apparatus components included a single microtubule in the dexter root. P. toyamaensis was separately resolved from other members of the genus in the 18S rDNA and ITS2 trees. Since the authentic strain ASIB V141 of P. bilobata (stored as Ettlia) has been lost, ASIB V143 was used as a reference in place of it. P. bilobata ASIB V143 was resolved in the ‘Parietochloris sensu stricto’ clade but not ‘Lobosphaera’ clade in the 18S rDNA tree, so taxonomically the combination of N. bilobata with Parietochloris as P. bilobata by Andreyeva was confirmed. In zoospores of the strains of P. bilobata, contractile vacuoles were located in the ventral side, as in P. alveolaris. The taxonomic relationship between P. bilobata and P. grandis, and evaluation of the unique position of contractile vacuoles in dorsoventral zoospores as one of the key characters of Parietochloris were discussed. Highlights Characterization of the species of Parietochloris by using ultrastructural and molecular data. Proposal of Parietochloris toyamaensis sp. nov. Verification of phylogenetic position of Parietochloris bilobata.


Introduction
described the genus Neochloris with the type N. aquatica, in which vegetative cells were multinucleate, chloroplasts were parietal, hollow-spherical with pyrenoids, and reproduction occurred by forming naked, biflagellate zoospores. In addition to multinucleate species, uninucleate members were also described in the genus (Arce & Bold, 1958;Bold, 1958;Deason & Bold, 1960;Groover & Bold, 1969;Archibald & Bold, 1970;Vinatzer, 1975). Two different approaches were proposed to improve the classification of Neochloris. Komárek (1989) restricted Neochloris to the species with multinucleate cells and classified uninucleate ones in the genus Ettlia, recognizing the importance of the difference in the nuclearity (uni-or multinucleate condition) of vegetative cells. Ettl & Gärtner (1995) accepted the generic idea, and listed seven species in Ettlia. On the other hand, Watanabe & Floyd (1989) published that the species of Neochloris consisted of three groups differentiated not only by nuclearity but also by ultrastructural features, namely basal body orientations (directly opposed (DO), clockwise (CW), counterclockwise (CCW)) and by the type of zoospore surroundings (naked, or thin-walled that was once regarded as naked by some authors). Based on these key characteristics, they defined three genera, as follows: Neochloris sensu stricto in the Sphaeropleales, Chlorophyceae for the multinucleate species having naked zoospores with DO basal bodies; Chlorococcopsis in the Chlorococcales, Chlorophyceae for the uninucleate members producing thin-walled zoospores with CW basal bodies (this genus was later synonymized with Ettlia sensu (Deason et al., 1991) that was amended to bear generic diagnostic features of Chlorococcopsis); and Parietochloris in the Trebouxiophyceae for the uninucleate species producing naked zoospores with CCW basal bodies. Because Ettl & Gärtner (1995) reserved the use of these ultrastructural features for taxonomy, the uninucleate genus Ettlia sensu Komárek (Ettl & Gärtner, 1995) resulted in a mixture of species producing zoospores of different types of surroundings and basal body orientations. The three species of Neochloris (N. alveolaris Bold, N. pseudoalveolaris Deason & Bold, N. cohaerens Groover & Bold) were consequently classified as either Ettlia (Komárek, 1989;Ettl & Gärtner, 1995) or Parietochloris (Watanabe & Floyd, 1989;Deason et al., 1991).
To solve such confusion in classifying the Neochloris species, molecular phylogenetic analysis was useful. Analysing 18S rDNA sequence data, Lewis et al. (1992) supported separating the members of Neochloris at the rank of order or class according to differences in the ultrastructural traits, as used by Watanabe & Floyd (1989). However, later phylogenetic studies revealed that the Parietochloris species belonged to two separate lineages: (1) 'Parietochloris sensu scricto (= s.s.)' clade including the type P. alveolaris (Bold) (Neustupa et al., 2011;Maltsev et al., 2018).
In August 2007, we isolated a coccoid green alga (NIES-3574) from farm soil collected near the campus of the University of Toyama in Japan. The alga was preliminarily identified as a member of Parietochloris, but it remained unknown whether it belonged to the 'Parietochloris s.s.' or 'Lobosphaera' clade. To classify this strain, we examined the phylogenetic position by molecular analyses, and compared its morphological and ultrastructural traits with members of the 'Parietochloris s.s.' clade and two strains of P. bilobata (Vinatzer) Andreyeva (1998). Taxonomically P. bilobata was first established as Neochloris bilobata by Vinatzer (1975), then informally synonymized with N. alveolaris (Watanabe, 1983), transferred to Ettlia (Komárek, 1989), and now currently accepted as P. bilobata. The three living cultures of N. bilobata (V141, V143, T58) stored in ASIB as Ettlia (Gärtner, 1996) will henceforth be referred to as the genus Parietochloris. The authentic strain of P. bilobata ASIB V141 is unavailable in the culture collection (Neustupa et al., 2011), so we are unable to identify to which of the two clades of this genus it belongs. The first goal of this paper is to describe a new species Parietochloris toyamaensis Shin Watanabe, N.Mezaki & Nakada for NIES-3574. The second is to verify the phylogenetic position of P. bilobata by selecting a proper reference strain from ASIB V143 and T58. We also discuss common ultrastructural features of Parietochloris sensu stricto.

Materials and methods
The soil sample was collected from a farm in Toyama, Japan (36.6963N, 137.1855E). Suspension of the soil sample with distilled water was scattered on agar plates with a 9:1 + V medium that contained 9 parts Bold's Basal Medium (Bischoff & Bold, 1963), 1 part soil extract (Starr & Zeikus, 1993), 10 µg l −1 vitamin B1, 0.1 µg l -1 vitamin B12 and 0.1 µg l -1 biotin. The plates were maintained at 20-23°C under a 12:12 h (L:D) photoregime. Cultures of the isolated organism Parietochloris sp. NIES-3574, P. alveolaris UTEX 836 and two strains of P. bilobata ASIB V143, T58 were maintained in test tubes containing the same agar media or AF-6 (Kato, 1982; modified as in Andersen et al., 2005) under the similar conditions. LM was carried out using a CH-2 microscope (Olympus, Tokyo, Japan) or AXIO ImagerM2 (Zeiss, Tokyo, Japan) equipped with differential interference contrast at ×20, ×40, ×100 magnification. Light micrographs were obtained using a DP20 digital camera (Olympus, Tokyo, Japan) or Axiocam305 colour (Zeiss, Tokyo, Japan). Zoospore production and release were promoted by transferring mature cells on dried slants onto a new agar plate with an excess of liquid medium, and zoospores were collected after the beginning of light period on the following day. For LM of the zoospores of Parietochloris sp. NIES-3574, slides were prepared using narrow tape methods (Nakada, 2018). For TEM, zoospores were fixed by adding 5% glutaraldehyde (GA) contained in a mixture of growth medium and 0.1 M sodium cacodylate buffer (pH 7.2) to the zoospore suspension to make a final concentration of 1% GA and kept for 1 h at room temperature. Fixed cells were collected on a Millipore membrane filter (0.8 µm pore size) and embedded in 1.5% agar. After washing with buffer, zoospores were post-fixed with 1% OsO 4 for 1 h at 4°C, then washed with distilled water and stained with 1% aqueous uranyl acetate overnight at 4°C. Dehydration was carried out in an acetone series. Samples were embedded in Epon-Araldite and cured at 60°C for 48 h. Serial sections were cut on an Ultracut-E ultramicrotome (Reichert Jung, Germany), and stained with uranyl acetate for 5 min or TI Blue solution (Nisshin EM, Tokyo, Japan) in 50% methyl alcohol for 10 min, and with lead citrate for 3 min. TEM was performed on an H-7650 (Hitachi, Tokyo, Japan).
To construct an 18S rDNA tree, the sequence data of Parietochloris sp. NIES-3574, P. bilobata ASIB V143, T58 were assembled with those of 35 species of Trebouxiophyceae and aligned as per Nakada et al. (2008) (Mendeley Data: https://data.mendeley.com/ datasets/7sn5jf8z67/1). Two species of Pyramimonas were used as an outgroup. Phylogenetic analyses were performed using three methods: Bayesian inference (BI), maximum likelihood (ML) and neighbour joining (NJ). BI was conducted using MrBayes v3.2.5 (Ronquist et al., 2012). Markov Chain Monte Carlo analysis (MCMC) was performed for 2 000 000 generations in length. Trees were sampled every 100 generations to yield 20 000 trees, of which the initial 5000 were discarded as the burn-in phase. The remaining trees were used to construct a 50% majority rule consensus tree. The ML and NJ trees were produced using PAUP v4.0b10 (Swofford, 2002) with TrN+I+G as the best-fit model of DNA substitution selected by the Akaike information criterion (AIC) in Modeltest v3.04 (Posada & Crandall, 1998). The ML analysis was executed by heuristic search, with stepwise approach with 10 random additions of taxa and tree bisection-reconnection branch-swapping algorithm to find the best tree. Bootstrap values in the ML were determined using the fast stepwise addition with 1000 replications; those in NJ were calculated from 1000 replications.
The secondary structure of ITS2 transcripts of Parietochloris sp. NIES-3574, P. alveolaris UTEX 836, the two strains of P. bilobata ASIB V143, T58 and 'Ettlia' pseudoalveolaris (Deason & Bold) Komárek NV-5 were modelled using mFold (Zucker, 2003). These ITS2 RNA sequence data were assembled with those of P. grandis CAMU MZ-Ch5 and two strains of Parietochloris sp., CCALA 1082, 1084. ITS2 data of P. pseudoalveolaris UTEX 975 were not available. The data matrix was aligned using LocARNA  and manually refined according to identical conservative base positions in the helices (Mendeley Data). The sequences were analysed using the BI, ML and maximum parsimony (MP) methods. The BI was conducted under 1 000 000 MCMC generations with same procedures used for 18S rDNA analysis. The ML was performed with a heuristic search under K80+G selected by the hierarchical likelihood ratio tests (Posada & Crandall, 1998). The MP tree was also performed using a heuristic search. Bootstrap values in the ML and MP were obtained with 1000 data replications. Predicted secondary structures were visualized in VARNA (Darty et al., 2009).

Molecular phylogeny
The 18S rDNA tree topologies obtained from the three methods roughly resembled each other with some minor differences. The BI tree is representatively shown in Fig. 1. In the tree, two clades of 'Parietochloris s.s.' and 'Lobosphaera' were separately resolved from each other. The strains Parietochloris sp. NIES-3574 (described as Parietochloris toyamaensis in the figures) and Chlorophyta sp. GIL-4a formed a sister that was a first offshoot at a deep region of the robust clade of 'Parietochloriss.s'. Then P. alveolaris UTEX 836 derived second with only a moderate support value in NJ. A clade comprising P. pseudoalveolaris UTEX 975, P. grandis CAMU MZ-Ch5 and two strains of P. bilobata ASIB V143 and T58 was robust, in which a trifurcate branch consisting of the three latter strains was supported with low statistical values.

LM and TEM
Parietochloris sp. NIES-3574 -Vegetative cells were subspherical to spherical, 20−28 μm diameter at mature (Figs 3−5). The cell wall was thin without a partial thickening. The chloroplast was parietal and cup-shaped in young cells. As cells grew, the choroplast became hollowspherical to cover most of the cell sphere, lobed to assume a dumbbell shape, or deeply incised variously (Figs 3−5). In young cells and autospores, a single pyrenoid was located at the bottom of cup-shaped chloroplast, starch segments were sometimes poorly accumulated and thylakoid membranes scarcely developed (Figs 6, 7,). In mature cells, two or three pyrenoids were observed in the chloroplast lobes, and the presence of pyrenoids was easily recognizable by LM because many starch segments were discontinuously distributed around the pyrenoids. Cells were uninucleate during vegetative cell growth. Reproduction occurred by forming autospores and zoospores. Mature cells divided into autospores of dyad (Fig. 3), tetrad (Fig. 4) or more (Figs 5, 7). The zoospores were naked, not covered with rigid cell wall, assuming fusiform to raindrop shapes as a whole (Fig. 8). In the TEM the zoospores were dorsoventral with relatively swollen dorsal side, and straight-lined ventral side, 1.5-2 × 6-8 μm in size (Fig. 9). The chloroplast covered the posterior end of the cell, extending anteriorly along the dorsal side. The pyrenoid was not observed in the present TEM. The nucleus was located just posteriorly to the basal bodies. In the median to anterior ventral side of the cell, two contractile vacuoles were present in line lengthwise (Fig. 9). The eyespot was rod-like, located anteriorly in the chloroplast (Fig. 8). Many adhesive vesicles secreted by exocytosis (Melkonian & Peveling, 1988) were present in the zoospore cytoplasm (Figs 9-11, 14-16), but not seen in the vegetative cells and autospores. The two basal bodies were inserted at almost right angles to the longitudinal axis of the cell (Figs 10-12), arranged in counterclockwise orientation, and overlapped at their most proximal ends (Figs 13, 16). The cruciate microtubule rootlet system (Fig. 10) alternately comprised the dexter root consisting of one microtubule and the sinister root of four microtubules forming the three-over-one configuration (Figs 14, 15). In the transition region between the basal body and flagellum, the ratio of lengths of the distal to proximal cylinders was roughly 1:1 (Fig. 10). A fibre of nucleus-basal body connection (nbbc: rhizoplast) extended downward from the basal body to the nucleus (Figs 11, 12, 16).
Parietochloris alveolaris UTEX836 -LM features of vegetative cells and zoospores were described by Bold (1958) and Ettl & Gärtner (1995), and some TEM information was presented by Gromov & Gavrilova (1987). Taxonomically important ultrastructural features obtained from UTEX836 are shown here. The pyrenoid was penetrated with several thylakoid membranes that passed largely in parallel at regular intervals (Figs 17, 18). Zoospores were naked and dorsoventral. The chloroplast extended along the dorsal side, posteriorly containing the pyrenoid (Fig. 18). The nucleus was located in the anterior region of cell. Two contractile vacuoles were present in line lengthwise in the median to anterior ventral side. The basal apparatus components included the counterclockwise basal bodies and the cruciate root system (Fig. 19), identical to those of Parietochloris sp. NIES-3574 except for the two microtubules in the dexter root (Fig. 20).
Parietochloris bilobata ASIB V143, T58 -LM features of vegetative cells and zoospores were originally described as Neochloris by Vinatzer (1975). Because the authentic strain ASIB V141 was unavailable, this study examined the remaining ASIB V143 isolated by Vinatzer from soil in Dolomites, South Tyrol (Italy) in 1975 and ASIB T58 by Trenkwalder from soil in Brixen, South Tyrol (Italy) in 1975(as Ettlia in Gärtner, 1996, as substitutes for the original. The morphology of the vegetative cells and zoospores from both strains were identical to each other and concordant with the original description of N. bilobata, although smaller cell sizes are noted for V143 in the culture collection catalogue (Gärtner, 1996). In the TEM, the pyrenoids of both strains were covered with many discontinuous starch segments and traversed in parallel by thylakoid membranes at regular intervals, which differed in numbers and appearance in ASIB V143 and T58 (Figs 21, 22).
Zoospores were fusiform, narrow ellipsoidal or raindrop-shaped with acute anterior and rounded posterior ends in LM (Vinatzer, 1975) and dorsoventral with swollen dorsal, and straight-lined or slightly concaved ventral sides in TEM (Fig. 23). The nucleus was located in the anterior region of the cell. The chloroplast   Fig. 3. Mature vegetative cell with hollow-spherical chloroplast (empty arrowhead), two divided chloroplast (arrowhead). Autospores forming dyad, in each of which chloroplast is lobed or dividing (arrow). P, pyrenoid. Fig. 4. Mature vegetative cell with bilobed chloroplast (arrowhead). Autospores forming tetrad (arrow), and mother cell liberating autospores (empty arrowhead). P, pyrenoid. Fig. 5. Mature vegetative cell with lobed, parietal chloroplast (arrowhead). Mother cell containing more than eight autospores (arrow). P, pyrenoid. Fig. 6. Cross section of young vegetative cell. Pyrenoid (P) is poorly surrounded by starch segments and thylakoid membranes are not developed. Ch, chloroplast; Mt, mitochondria; N, nucleus; S, starch segment. Fig. 7. Cross section of autospores in mother cell. Pyrenoids (P) are discontinuously covered with starch segments (S) and poorly penetrated with thylakoid membranes (arrows). N, nucleus. Fig. 8. Light micrograph of zoospore. Eyespot (E) is located anteriorly in chloroplast. Fig. 9. Longitudinal section of zoospore, including cross section of basal body (bb). Nucleus (N) is located at anterior end, and contractile vacuoles (CV) are in line lengthwise, located at median ventral side. Many adhesive vesicles (arrowheads) distribute in cytoplasm. Ch, chloroplast; Mt, mitochondria. Figs 10−12. Consecutive sections of anterior end zoospore, including longitudinal sections of basal body (bb). Arrowheads, adhesive vesicles; df, distal connecting fibre; N, nucleus; nbbc, nuclear basal body connection; sr, longitudinal section of sinister root microtubule. Fig. 10. Distal and proximal cylinders (arrows) at transitional region between basal body and flagellum. Fig. 13. Longitudinal section of two basal bodies shifted in counterclockwise orientation and overlapped at most proximal ends. Dexter (dr) and sinister (sr) roots are associated with basal bodies. Fig. 14. Cross sections of a single microtubule of dexter root (dr) and four of sinister (sr) arranged in three-over-one configuration. Arrowheads, adhesive vesicles; bb, basal body. Fig. 15. Cross and longitudinal sections of a single microtubule of dexter root (dr) at dexter side of basal bodies (bb). extended from the posterior end to almost the anterior end to cover the dorsal side. The pyrenoid was at a nearly central position and the eyespot was at the most anterior end of the chloroplast (not shown). The contractile vacuoles were in line lengthwise at the median ventral side. Many adhesive vesicles were present in the cytoplasm, often swelled or ruptured due to the fixing procedures. The basal apparatus components were identical to those of Parietochloris alveolaris UTEX 836.

Discussion
As shown in the present 18S rDNA trees, Parietochloris sp. NIES-3574 belongs to the 'Parietochloriss.s.' clade and is resolved closer to P. alveolaris UTEX 836 than to other species. Parietochloris sp. NIES-3574 is clearly distinguished from P. alveolaris UTEX 836 in having a longer ITS2 sequence and different secondary structure of its transcript. Ultrastructurally, the d-root in Parietochloris sp. NIES-3574, which consists of a single microtubule, is conspicuous among the species of Parietochloris. In the Trebouxiophyceae, all organisms examined so far possess two microtubules in the d-root (Bakker et al., 1997), except for P. cohaerens (Watanabe & Floyd, 1989) of the 'Lobosphaera' clade and Coleochlamys apoda Korshikov (as Fusochloris perforata Floyd, Shin Watanabe & Deason in Floyd et al., 1993) in the Microthamniales, in which one or two microtubules were observed. Based on the molecular and phenotypic features, we propose Parietochloris toyamaensis sp. nov. for NIES-3574 as follows.

Parietochloris toyamaensis
Shin Watanabe, N. Mezaki, Nakada sp. nov. (Figs 3-16) DIAGNOSIS: Cells broad ellipsoidal, subspherical when young, spherical, 20-28 µm diameter at mature. Chloroplast parietal, cup-shaped, dumbbell shaped, often deeply incised variously, with one or a few pyrenoids. Pyrenoid covered with discontinuous starch segments and traversed by several thylakoid membranes. Cells uninucleate. Reproduction by autospores and zoospores. Zoospores naked, biflagellate, dorsoventral with chloroplast in dorsal side. Nucleus anterior. Two contractile vacuoles in line lengthwise at median to anterior ventral side. Microtubular root system consisting of one microtubule in dexter side to basal body and four microtubules in sinister side. HOLOTYPE: NIES-50022, a metabolically inactive cryopreserved specimen, derived from culture strain NIES-3574. ETYMOLOGY: The specific epithet toyamaensis is after the locality 'Toyama', where the soil sample was collected. LOCALITY: The soil sample was collected from a farm in Toyama, Japan (36.6963N, 137.1855E). Note: The phylogenetic position is supported by the 18S rDNA and ITS2 sequence data.
Among the three strains of Parietochloris bilobata (V141, V143, T58) in ASIB (as Ettlia in Gärtner, 1996), the V143 was found to be smaller in cell size than the authentic V141. However, we confirmed here that the morphology of this strain is concordant with the original description of N. bilobata by Vinatzer (1975), including cell sizes and other traits. The strain V143 was isolated from the same locality where V141 was obtained and identified as N. bilobata by the same author. So, it was reasonable to use V143 as a reference strain in place of V141. The present examination clarified that P. bilobata ASIB V143 is a member of the 'Parietochloris s.s.' clade. Thus, taxonomically, we confirm the validity of the combined taxon P. bilobata (Vinatzer) Andreyeva (1998).
In this study, six strains of 'Bilobata' subclade were found to be very closely related phylogenetically. It is also revealed that ASIB V143 and T58 differ only slightly from each other in ultrastructure. Previously, Maltsev et al. (2018) suggested that P. grandis CAMU MZ-Ch5 and Parietochloris sp. CCALA 1082 may represent a single species when morphological characters are matched because of their similar ITS secondary structure. Thus, the close relationships of the strains within the 'Bilobata' subclade could be a future taxonomic topic. However, in considering this issue, the anterior position of contractile vacuoles in the zoospores of P. grandis CAMU MZ-Ch5 needs to be reconfirmed, as these vacuoles have been observed in the ventral side of zoospores in other species of the genus. If the contractile vacuoles are located anteriorly, P. grandis CAMU MZ-Ch5 would be clearly distinguished even from P. bilobata V143, which has very close sequences of 18S rDNA and ITS2. Conversely, if the contractile vacuoles are ventral, it would confirm this as a highly common trait of the 'Parietochloriss.s.' clade, and consequently could be regarded as a characteristic of the genus Parietochloris. These results will also address the taxonomic relationship between P. grandis CAMU MZ-Ch5 and P. bilobata.

Disclosure statement
No potential conflict of interest was reported by the authors.

Supplementary information
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