Delineation of six species of the primitive algal genus Glaucocystis based on in situ ultrastructural characteristics

The field of microbiology was established in the 17th century upon the discovery of microorganisms by Antonie van Leeuwenhoek using a single-lens microscope. Now, the detailed ultrastructures of microorganisms can be elucidated in situ using three-dimensional electron microscopy. Since the availability of electron microscopy, the taxonomy of microscopic organisms has entered a new era. Here, we established a new taxonomic system of the primitive algal genus Glaucocystis (Glaucophyta) using a new-generation electron microscopic methodology: ultra-high-voltage electron microscopy (UHVEM) and field-emission scanning electron microscopy (FE-SEM). Various globally distributed Glaucocystis strains were delineated into six species, based on differences in in situ ultrastructural features of the protoplast periphery under UHVEM tomography and in the mother cell wall by FE-SEM, as well as differences in the light microscopic characteristics and molecular phylogenetic results. The present work on Glaucocystis provides a model case of new-generation taxonomy.

the electron beam are transmitted [3][4][5] . On the other hand, scanning electron microscopy (SEM) can reveal the characteristics of the entire cell surface, globally, but conventional SEM does not have sufficiently high resolution to observe ultrastructures in detail 10,11 . Recently, two types of new-generation EM, ultra-high-resolution (UHR) field-emission (FE)-SEM and ultra-high-voltage electron microscopy (UHVEM), have introduced a new paradigm in the field of biology [10][11][12][13][14] . Despite this, in some recent studies of microalgal and protozoan taxonomy, the utility of molecular approaches continues to be overemphasised [15][16][17][18] .
UHR FE-SEM enables ultrafine observations of the entire cell surface even at low accelerating voltages (LV); it also allows in situ surface ultrastructures in numerous cells to be observed all at once [10][11][12] . Our recent study using LV FE-SEM unveiled species diversity within the flagellate glaucophyte genus Cyanophora, identifying three new species 10 . However, LV FE-SEM cannot be applied to examine a protoplast enclosed by a cell wall, as in the coccoid glaucophyte genus Glaucocystis 19,20 . For this, UHVEM, which enables in situ 3D ultrastructural observation by thick-section tomography using an ultra-high accelerating voltage, can be used 5 . Recently, 3D UHVEM tomography revealed morphological diversity in terms of the 3D ultrastructure of the protoplast periphery using three divergent strains of Glaucocystis 13,14 . Thus, undescribed species of this genus are expected to be delineated morphologically among strains distributed across the globe, based on new-generation EM characteristics.
Here, we aimed to delineate morphologically and phylogenetically different Glaucocystis species based on the combination of several types of microscopy, including 3D UHVEM tomography and LV FE-SEM, combined with molecular phylogenetic results, from 10 globally distributed strains labelled G. nostochinearum Itzigs. ex Rabenh. (1866) 21,22 and three newly established strains of Glaucocystis (Supplementary Table 1). A new taxonomic system of Glaucocystis species delineated using new-generation EM is described in this report (Table 1).

Results
Light microscopy. Using LM on the 13 strains (Supplementary Table 1), two Glaucocystis species were identified based on the traditional taxonomic system 19,23,24 (Supplementary Table 2): G. nostochinearum and G. oocystiformis Prescott (1944) 23 (see Supplementary Note). Moreover, we found differences that could contribute to species delineation within G. nostochinearum in our new taxonomic system ( Field-emission scanning electron microscopy. The cell wall of Glaucocystis is composed of cellulose filaments and has the highest cellulose I α crystallite content of all organisms [25][26][27][28] . The cellulose filament structure derived from this alga was previously examined by TEM and several types of spectroscopy [25][26][27][28][29][30] . However, FE-SEM was not yet used to reveal the in situ ultrastructural surface of the Glaucocystis colony or mother cell wall. Using LV FE-SEM, we detected cellulose filaments of the mother cell wall on the surface of Glaucocystis colonies (Fig. 2). The fibrils on the colony surface were essentially identical in shape among the strains examined, but two types of filament arrangements were recognised. The entire colony surface generally exhibited a gauze fabric-like appearance, with small spaces between fibrils, in G. geitleri E.G.Pringsh. ex Tos.Takah. & Nozaki sp. nov., G. nostochinearum, G. oocystiformis and G. miyajii Tos.Takah. & Nozaki sp. nov. (Fig. 2a,b,d,e). On the other hand, the fibrils were tightly arranged, with no spaces between them, over nearly the entire colony surface  Mother cell wall extension  prominent  prominent  not prominent  not prominent  not prominent  not prominent   Gauze fabric-like appearance  of mother cell wall  present  present  absent  absent  present  present   Cell numbers within a colony 2-4, generally

Ultra-high-voltage electron microscopy and ultrathin-section transmission electron microscopy.
Recent reports 13,14 using UHVEM tomography clearly revealed the 3D ultrastructural features of the plasma membrane and the flattened vesicles at the protoplast periphery in three strains or species of Glaucocystis (G. geitleri strain SAG 229-1, G. nostochinearum strain SAG 16.98 and G. incrassata strain SAG 229-2), even though the protoplast was tightly enclosed by a cell wall. In addition, the 3D ultrastructures of the protoplast periphery in these three strains are diverse and can be classified into three types (periphery types A, B and C) 13,14 . Since these three strains were found to represent three different species (G. geitleri strain SAG 229-1 of type A, G. nostochinearum strain SAG 16.98 of type B and G. incrassata strain SAG 229-2 of type C), they were assigned as authentic strains for these species (see below).
To examine the peripheral 3D ultrastructure of protoplasts in the other three Glaucocystis species, we observed various regions of mature vegetative cells in three strains representing the three species (G. oocystiformis strain 126, G. miyajii strain Thu10 and G. bhattacharyae strain 118, designated here as the authentic strains for the three species; see below) by UHVEM and tomography, as well as ultrathin section TEM (Table 1; Fig. 3; Supplementary Videos 1-3; Supplementary Fig. 3). The protoplast periphery of these species was similar to that in the former three species examined previously. The flattened vesicles were leaflet-like in shape, lacked a plate-like interior structure, and were distributed throughout the entire protoplast periphery just underneath the single-layered plasma membrane (except for the region near basal bodies), but they did not completely enclose the protoplast periphery to form small spaces between the vesicles at the protoplast periphery. In addition, based upon the present UHVEM tomography, G. oocystiformis strain 126 was assigned to periphery type A, whereas G. miyajii strain Thu10 and G. bhattacharyae strain 118 were assigned to periphery type C. Based on the native 3D ultrastructural features of the protoplast periphery established by previous and present studies using UHVEM tomography 13,14 , the three periphery types are evident and distinguishable from each other, even based on ultrathin-section TEM alone. Thus, peripheral protoplast types were determined in other strains based on ultrathin-section TEM alone; each of the six species exhibited only a single periphery type, despite being composed of more than one strain. G. nostochinearum (periphery type B) and G. miyajii (periphery type C) were clearly distinguished from each other based on the difference in the periphery type, although they were indistinguishable under LM alone (Table 1; Figs 1 and 4; Supplementary Fig. 1).

Molecular phylogenetic analyses.
The phylogenetic tree of the concatenated plastid gene sequences ( Supplementary Fig. 5) demonstrated that 13 Glaucocystis strains could be subdivided into six phylogenetic groups [four robust monophyletic groups and two independent operational taxonomic units (OTUs)], which are essentially equivalent to the G1-G6 groups recognised previously 31 . These six groups corresponded to the six species delineated by our comparative morphological analysis (Table 1).
In the phylogenetic tree, basal phylogenetic relationships were robustly resolved (with bootstrap values of 82-100%; Supplementary Fig. 5); G. geitleri and G. incrassata occupied the most and second most basal positions, respectively, whereas the other four species (G. nostochinearum, G. oocystiformis, G. miyajii and G. bhattacharyae) represent a large robust monophyletic group (crown lineage), supported by bootstrap values Internal transcribed spacer-2 secondary structure and genetic distances. The six species of Glaucocystis were evaluated by compensatory base changes (CBCs) in the secondary structure of the internal transcribed spacer (ITS)-2 of nuclear ribosomal DNA (rDNA) ( Supplementary Figs 6 and 7) and the genetic distances of a plastid gene (Supplementary Fig. 8). Four Glaucocystis species within the crown lineage exhibited CBCs and sufficient genetic distances to be classified as four distinct species (Supplementary Note).

Discussion
Based on the present comparative morphological and molecular examinations of cultured materials from the genus Glaucocystis, six species were clearly delineated (Table 1; Fig. 4). In contrast to previous reports 8,9 , that 45 to 50 years ago had to rely on conventional EM only, ultrastructural diversity of the protoplast periphery was significant within the genus Glaucocystis when examined by UHVEM (Figs 3 and 4; Supplementary Figs 3 and 4). Moreover, ultrastructural diversity was clarified in the arrangement of cellulose filaments of the mother cell under LV FE-SEM (Figs 1 and 2). Based on the differences in these new-generation EM characteristics and LM features of the 13 Glaucocystis strains, we could delineate six morphological species that correspond to six phylogenetic groups (G1-G6) recognised by previous 31 (Table 1; Fig. 1; Supplementary Figs 1 and 5), the in situ ultrastructural features of G. oocystiformis are similar to those of G. geitleri in having periphery type A and gauze fabric-like fibrils ( Supplementary Fig. 5). Among the other four species, G. bhattacharyae and G. incrassata have essentially the same in situ ultrastructures (periphery type C and tightly arranged fibrils), although they were distinguished from one another based on differences in LM characteristics (Table 1; Supplementary Fig. 1). Thus, in addition to new-generation EM observations, LM data and molecular phylogenetic analyses are essential for delineating microalgal species.
The novel strains established here from a single field sample were classified into three species (Table 1;  Supplementary Table 1; Supplementary Fig. 5). Although G. nostochinearum has been considered a cosmopolitan species 24,32,33 , the records may be based on several species, which can be distinguished using the new-generation taxonomic methodology established here (Supplementary Note).
The plasma membrane of five of the six Glaucocystis species had numerous grooves throughout the protoplast surface (Figs 3 and 4; Supplementary Figs 3 and 4) 13,14 . In G. nostochinearum 13 , however, the plasma membrane lacked grooves or invaginations (type B; Supplementary Fig. 4) as in Cyanophora species 10,11 . Since G. nostochinearum belongs to the crown lineage within Glaucocystis ( Supplementary Fig. 5), the lack of grooves or invaginations in G. nostochinearum might have evolved secondarily within Glaucocystis (Fig. 4). Vegetative cells of Cyanophora species are apparently smaller than those of Glaucocystis 10 , and G. nostochinearum exhibits one of the smallest cell sizes within Glaucocystis (Table 1; Fig. 1; Supplementary Fig. 1). In addition, the surface-area-to-volume ratio is smaller (inversely proportional to the cell size) and the transportation of substances across the plasma membrane more limited in larger cells. Thus, the presence of grooves or invaginations at the protoplast periphery in the five Glaucocystis species might contribute to expansion of the surface area of the protoplast and consequently to their large cell size.
The Gloeochaetales are another order of glaucophytes that are characterised by having palmelloid immotile vegetative cells and include two genera, Gloeochaete and Cyanoptyche 20 ; some species have zoospores [34][35][36] . These algae might represent the evolutionarily intermediate stage between the flagellate Cyanophora (Cyanophorales) and the coccoid Glaucocystis (Glaucocystales). Within the Gloeochaetales, cultured strains labelled Cyanoptyche gloeocystis Pascher 37 and Gloeochaete wittrockiana Lagerh 38 are available, but do not number more than three in each species (http://www.ccac.uni-koeln.de/; http://sagdb.uni-goettingen.de/). Although no taxonomic studies have been performed based on EM and/or molecular data, these two taxa are considered cosmopolitan species 34,39 . Therefore, taxonomic studies based on molecular methods and comparative in situ ultrastructural characteristics using various clonal strains, as in Cyanophora 10 and Glaucocystis evaluated here, would be useful for these two species or genera. 3D UHVEM tomography will reveal the peripheral in situ ultrastructures of their  Fig. 1). Phylogeny is based on phylogenetic tree of the concatenated gene sequences ( Supplementary Fig. 5); each species exhibits sufficient genetic distances from other species ( Supplementary  Figs 6-8).
vegetative cells even when enclosed by a non-cellulosic extracellular matrix 35,36 , as in Glaucocystis 13,14 . LV FE-SEM may be applicable in easily inducible, naked zoospores of the Gloeochaetales 35,36 for in situ ultrastructural observation of the protoplast surface, as in Cyanophora 10 . These two types of new-generation EM should be capable of revealing the actual diversity in ultrastructures in the peripheral protoplast in situ, leading to the delineation of more natural species of the gloeochaetalean algae, when combined with molecular phylogenetic results.

Conclusions
In recent taxonomic work on certain microorganisms, there has been a tendency to avoid morphological approaches in favour of molecular ones [15][16][17][18] . However, species delineation based only on molecular data cannot demonstrate how the species live. Even when whole-genome sequence data are available, we can only speculate on the metabolic pathways employed by the organism. Even in bacteria/archaea, species delineation has been carried out on the basis of phenotypic characteristics 40,41 . Next-generation microbial taxonomy, which is just now becoming established, utilises new-generation EM methods (e.g. FE-SEM and UHVEM) to demonstrate detailed in situ ultrastructural features of microscopic organisms in their entirety. Molecular barcoding is only meaningful for lineages within which species have already been delineated and recognised by morphological or phenotypic characteristics. Global and in situ ultrafine microscopy should become the mainstream method used to delineate microbial species, as in the present study on Glaucocystis. balsam xylene, placed on the 60 °C Canada balsam on a glass slide, and then the slide was incubated at 60 °C for a few days. LM observations were carried out as described previously 10 using the permanent slides and living cultured cells.

Glaucocystis nostochinearum
Field-emission scanning electron microscopy. LV FE-SEM was performed as described previously 11 using all 13 Glaucocystis strains, but cells were harvested directly, treated with the critical point dryer JCPD-5 (JEOL) and observed using the UHR FE-SEM SU8220 (Hitachi High-Technologies, Tokyo, Japan).
Transmission electron microscopy and ultra-high-voltage electron microscopy. Since the high-pressure freezing and freeze-substitution fixation method is generally expected to be superior to chemical fixation in preserving the integrity of cellular ultrastructures 12 , this method was performed for TEM and UHVEM as described previously 13 . Ultrathin-section TEM was also performed as described previously 13 for all 13 Glaucocystis strains. In addition, UHVEM and reconstruction of the tomographic images were carried out as described previously 13 in three authentic strains of three Glaucocystis species (Fig. 3).
Molecular phylogenetic analysis and comparative analysis of the secondary structure of ITS-2 in nuclear ribosomal DNA. DNA extraction, polymerase chain reaction (PCR) and direct sequencing of the PCR products were performed as described previously 10,47,48 , using primers designed in a previous study 10,31,49 .
The secondary structure of nuclear rDNA ITS-2 was constructed as described previously 10 . Phylogenetic relationships between Glaucocystis species were examined based on analyses of the concatenated sequences (2,211 base pairs) of the photosystem I P700 chlorophyll a apoprotein A2 (psaB) gene (1,461 base pairs) and the photosystem II P680 chlorophyll a apoprotein D1 (psbA) gene (750 base pairs) from 13 strains of Glaucocystis, representing 10 OTUs (based on identical sequences), and three strains of three other glaucophyte genera as an outgroup (Supplementary Table 1). The sequences were aligned as described previously 10 and subjected to phylogenetic analyses. ML and NJ analyses were performed as described previously 10 , except that one selected model was used: the general time reversible + gamma model with invariant sites for ML.