© 2015 The Japan Mendel Society Cytologia 80(4): 513–524

Taxonomic Study of Pyrobotrys (Spondylomoraceae, Chlorophyceae) Based on Comparative Morphological and Molecular Analyses of

Culture Strains Established Using Novel Methods

Mizuho Sugasawa1, Ryo Matsuzaki1,2, Kaoru Kawafune3, Toshiyuki Takahashi1, Masanobu Kawachi2, Lothar Krienitz4 and Hisayoshi Nozaki1*

1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan

2 Micorobial Culture Collection, Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, 16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan

3 Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 2–12–1 Ookayama, Meguro-ku, Tokyo 152–8550, Japan

4 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Neuglobsow, 16775 Stechlin, Germany

Received May 13, 2015; accepted October 1, 2015

Summary Taxonomic studies of the spondylomoracean genus Pyrobotrys have remained at tradi-tional levels due to the lack of molecular phylogenetic analyses and available culture strains for re-examinations. Here, we established new culture strains of Pyrobotrys using novel culturing methods, and classified them into three species, namely, P. casinoensis, P. elongata, and P. squarrosa, based on comparative microscopic observations and molecular phylogeny. Although P. elongata is charac-terized by its enormous stigma in cells of the most posterior tier in a colony, such a species has not been reported since its original description in 1938. In the present study, the presence of an enormous stigma was confirmed based on light microscopy in three P. elongata strains originating from two localities. Based on transmission electron microscopy, the large stigma of P. elongata was essentially identical to the stigma of other species of Pyrobotrys in having a single layer of stigma globules. Our molecular phylogeny demonstrated that P. elongata was robustly separated from other species of Pyrobotrys.

Key words Molecular phylogeny, Morphology, Pyrobotrys, Pyrobotrys elongata, Spondylomora-ceae, Stigma, Taxonomy, Ultrastructure.

Colonial protists, especially the colonial volvocalean algae, are often studied as models for evolution of multicellularity because they represent successive intermediate organizations between unicellular and complex multicellular organisms (e.g., Arakaki et al. 2013). The Spondylomora-ceae are one of the colonial Volvocales and are characterized as having 4-, 8- or 16-celled cubic colonies without encompassing gelatinous matrices (Ettl 1983, Nozaki 2003). This family includes four morphologically different genera, Spondylomorum Ehrenberg, Chlorcorona Fott, Pyrobotrys Arnoldi and Pascherina P.C. Silva, with approximately 15 currently recognized species (Huber-Pestalozzi 1961, Ettl 1983, Nozaki 1986, 2003). Vegetative cells of this family are quadriflagellate in Spondylomorum whereas in other genera they are biflagellate (Huber-Pestalozzi 1961, Ettl 1983, Nozaki 2003). Although culture strains of Pascherina were recently established and examined by light and electron microscopy and molecular phylogeny (Sugasawa et al. 2015), taxonomic stud-

* Corresponding author, e-mail: nozaki@bs.s.u-tokyo.ac.jpDOI: 10.1508/cytologia.80.513

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ies of other genera of the Spondylomoraceae have remained at traditional levels without molecular phylogenetic analyses and available culture strains for reexaminations.

The genus Pyrobotrys is characterized by having biflagellate cells and by lacking a pyrenoid in the chloroplast, with approximately 10 species (Nozaki 1986). Playfair (1914) described a colo-nial green flagellate as Uva casinoensis Playfair based on material collected from Casino, near the Richmond River, Australia. Similar algae also appear in the literature under the name Pyrobotrys Arnoldi (1916) or Chlamydobotrys Korshikov (1924). Silva (1972) resolved this nomenclatural confusion and used Pyrobotrys. Nozaki (1986) performed a taxonomic study of Pyrobotrys based on 54 strains of Pyrobotrys that were established by anaerobic culture methods using soil samples collected in various localities in Japan. Nozaki (1986) distinguished the strains into four species by differences in vegetative and planozygote morphology. The culture strains investigated were maintained by exchanging the air for nitrogen gas after every inoculation to new medium (Nozaki 1986). Since such anaerobic cultures were difficult to maintain, all of the strains of Pyrobotrys established by Nozaki (1986) no longer exist. Recently, Nakada et al. (2010) analyzed the phyloge-netic position of Pyrobotrys, but they used only two strains of two species. It is thus necessary to increase the number of Pyrobotrys strains for reliable recognition of the species within the genus.

In this paper, we established new strains of Pyrobotrys from Japan and Germany based on new culture methods, and examined their vegetative colonies by light and electron microscopy. Based on the comparative microscopy and molecular phylogeny, the strains were classified into three species including the enigmatic species P. elongata Korshikov that is characterized by having an enormous stigma in the most posterior cells of the colony. Our transmission electron micros-copy (TEM) unambiguously demonstrated the ultrastructural similarity between such a stigma and the stigma of other species of Pyrobotrys.

Materials and methods

CulturesStrains of Pyrobotrys were isolated from soil and water samples (Table 1) according to the pi-

pette-washing method (Pringsheim 1946). The strains of P. casinoensis (Playfair) P.C. Silva (DP11, DP21, and DP31), P. squarrosa Korshikov (YS11 and YS12), and P. elongata (YP316 and YP416) were established from rewetted soil samples; small amounts of dried soil sample and a boiled green pea (Pisum sativum L.) were flooded with distilled water in Petri dishes (90×15 mm) and placed at 20–25°C under 60–200 µmol/m2/s on a 14 : 10 h L : D photoperiod. Colonies appeared in the Petri dishes within 4–10 d. Two or three clones were isolated using the pipette-washing method from each soil sample. The strain of P. elongata (2J-2) was established directly from a water sample (Table 1). Cultures were grown in 18×150-mm screw-cap tubes (Fujimoto Rika, Japan) containing

Table 1. List of Pyrobotrys strains used in this study.

Species Culture designation Origin of strains Date

P. casinoensis DP11, DP21, DP31 Soil samples collected at Brandenburg, Germany (53°08′53.9″N 13°03′13.2″E)

Sep. 29, 2013

P. elongata 2J-2 Water samples collected from a pond in Toneri Park, Adachi-ku, Tokyo, Japan (water temperature 28°C, 35°47′56.75″N 139°46′23.19″E)

Jul. 26, 2013

P. elongata YP316, YP416 Soil samples collected in paddy field at Yokota, Sodegaura-shi, Chiba, Japan (35°23′32.1″N 140°01′36.2″E)

Jul. 21, 2014

P. squarrosa YS11, YS12 Soil samples collected in paddy field at Akiragi, Hagi-shi, Yamaguchi, Japan (34°20′49.6″N 131°25′10.8″E)

Jan. 8, 2013

2015 Taxonomy of Pyrobotrys 515

Table 2. List of accession numbers and strain designationsa in the combined 18S rRNA, rbcL and psaB gene phylogeny (Figs. 3, 4).

Species (strain designation)Accession number

18S rRNA rbcL psaB

Caudivolvoxa; Arenicoliniab

Chlorosarcinopsis arenicola (UTEX 1697) AB218701 AB451192 AB451207Caudivolvoxa; Characiosiphoniab

Characiochloris acuminata (UTEX 2095=NIES-637) AF395435 AB360752 AB451196Characiochloris sasae (NIES-567) AB360741 AB084338 AB084376Characiosiphon rivularis (UTEX 1763) AF395437 EF113419 AB451197Lobocharacium coloradoense (UTEX 2772) AF395436 EF113450 AB451213

Caudivolvoxa; Chlorogoniab

Brachiomonas sp. (MBIC 10757=NBRC 102903) AB183637 AB451191 AB451195Chlamydomonas perpusilla (NIES-1849) AB290339 AB360754 AB451199Chlorogonium capillatum (SAG 12-2e) AJ410442 AB010237 AB451203Chlorogonium complexum (NIES-2296=NBRC 10566) AB477055 AB477057 AB477061Chlorogonium elongatum (NIES-1358) AB278625 AB206329 AB451205Chlorogonium elongatum (UTEX 2571) AJ410444 AB010240 AB451204Chlorogonium euchlorum (SAG 12-2d) AJ410443 AB010229 AB451206Haematococcus lacustris (NIES-144) AB360747 AB084336-7 AB084365Rusalka fusiformis (NIES-123) AB360750 AB010242 AB084370

Caudivolvoxa; Dunalielliniab

Dunaliella minuta (UTEX 1983) M62998 AJ001877 AB084375Gungnir kasakii (CCAP 12/8) AB360742 AB010244 AB084367Gungnir kasakii (NIES-1359) AB360743 AB206331 AB451209Gungnir neglectum (NIES-439) AB360745 AB010243 AB084366Gungnir neglectum (NIES-1869) AB360746 AB360756 AB451210

Caudivolvoxa; Polytominiab

Chlamydomonas applanata (UTEX 225=SAG 11-9) U13984 AB360753 AB451198Chlamydomonas pulsatilla (NIES-122) AB001039 AB003427 AB451200Chlamydomonas pumilio (NIES-1850) AB290340 AB360755 AB451201

Caudivolvoxa; Stephanosphaeriniab

Chlorococcum ellipsoideum (UTEX 972) U70586 EF113431 AB451202Balticola zimbabwiensis (SAG 26.96) AB360748 AB360757 AB451211Hamakko caudatus (NIES-2293) AB451188 AB451193 AB451212Protosiphon botryoides (UTEX 99) U41177 EF113465 AB451214Stephanosphaera pluvialis (UTEX 2409) AB360751 AB360759 AB451215

Caudivolvoxa; Uncertain affinitiesb

Chlamydomonas tetragama (NIES-446) AB007370 AJ001880 AB084357Chlamydomonas sp. (G16) LC093462c LC093463c LC093464c

Pyrobotrys casinoensis (DP11=NIES-3768) LC093465c LC093466c LC093467c

Pyrobotrys elongata (2J-2) LC093468c LC093469c LC093470c

Pyrobotrys elongata (YP416=NIES-3772) LC093471c LC093472c LC093473c

Pyrobotrys squarrosa (NIES-2564) AB542919 AB542927 AB542924Pyrobotrys squarrosa (YS11=NIES-3773) LC093474c LC093475c LC093476c

Pyrobotrys stellata (SAG 10-1c) AB542920 AB542928 AB542925Xenovolvoxa; Moewusiniab

Chlamydomonas moewusii (CC-1419=UTEX 9) U41174 M15842 AB084355Chlamydomonas noctigama (SAG 33.72, NIES-1048) AJ781311 AB101507 AB101513

Xenovolvoxa; Monadiniab

Chlamydomonas kuwadae (NIES-968) AB451190 AB084334 AB084356Xenovolvoxa; Phacotiniab

“Pascherina tetras” (SAG 159-1) AB542921 AB542929 AB542926Pascherina tetras (Isa28=NIES-3749)d LC053638d LC053639d LC053640d

Phacotus lenticularis (NIES-858) X91628 AJ001883 AB084373-4Pteromonas angulosa (SAG 64-3, NIES-861) AF395438 AJ001887 AB084371-2

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10 mL of SVM (Kirk and Kirk 1983) modified by the addition of 200 mg L–1 of sodium acetate and maintained at 25°C with alternating periods of 12-h light and 12-h dark under light conditions of ca. 200 µmol/m2/s provided by cool white fluorescent lamps. P. casinoensis strains DP11, DP21, and DP31 and P. elongata strains YP316 and YP416 were grown under microaerophilic conditions using the AnaeroPouch-MicroAero system (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).

Light microscopyLight microscopy was performed using a BX-53 microscope equipped with Nomarski dif-

ferential interference optics (Olympus, Tokyo, Japan) and images were captured using an Olympus DP71 digital camera (Olympus). To obtain clear images, colonies were attached to polyethylenei-mine-coated coverslips, placed on slides, and sealed with Vaseline or fixed with diluted povidone-iodine (Meiji Seika Pharma, Tokyo, Japan).

Transmission electron microscopyFor TEM, colonies of Pyrobotrys were fixed for 1 h at room temperature with a final concen-

tration of 2% glutaraldehyde obtained by mixing the culture with an equal volume of 4% glutaral-dehyde in 0.025 M sodium cacodylate buffer (pH 7.3). Cells were then rinsed with 0.05 M sodium cacodylate buffer for 30 min at room temperature, after which 2% osmium tetroxide in 0.025 M sodium cacodylate buffer (pH 7.3) was added and incubated for 2 h at room temperature. The fixed

Table 2. List of accession numbers and strain designationsa in the combined 18S rRNA, rbcL and psaB gene phylogeny (Figs. 3, 4).

Species (strain designation)Accession number

18S rRNA rbcL psaB

Chloromonadiniab

Chloromonas radiata (UTEX 966) U57697 AJ001878 AB084345Chloromonas reticulata (UTEX 1970) U70791 AB022534 AB084346-7Chloromonas serbinowii (UTEX 492) U70795 AJ001879 AB084354

Crucicarteriab

Carteria crucifera (NIES-421) D86501 D63431 AB084358Hafniomonas

Hafniomonas montana (NIES-656) AB101518 AB101512 AB101516Oogamochlamydinia

Asterococcus superbus (IAM C-299) AB175836 AB175935 AB504774Radicarteriab

Carteria obtusa (SAG 39.84, NIES-428) AF182818 D89769 AB084361-3Carteria radiosa (UTEX 835, NIES-432) AF182819 D89770 AB084360

ReinhardtiniaChlamydomonas debaryana (SAG 15.72=UTEX 1344) AB542922 D86838 AB044469Chlamydomonas reinhardtii (UTEX 1061, 137C) M32703 J01399 AB044470Eudorina unicocca (NIES-1858, UTEX 737) AB511841 D86829 AB044439Gonium multicoccum (NIES-1708, UTEX 2580) AB511840 D63435 AB044461Paulschulzia pseudovolvox (UTEX 167) U83120 D86837 AB044473Tetrabaena socialis (SAG 12-3, NIES-571) AB278626 D63443 AB044466Volvox carteri (UTEX 1885, NIES-732) X53904 D63446 AB044425Volvox globator (SAG 199.80=UTEX 955) AB542923 D86836 AB044428

a Abbreviations of the culture collections are as following: CC, Chlamydomonas Center culture collection (http://chla-mycollection.org/); CCAP, Culture Collection of Algae and Protozoa (http://www.ccap.ac.uk/); IAM, IAM Culture Collection at the University of Tokyo (it was transferred to NIES); MBIC, Marine Biotechnology Institute Culture Collection (it was transferred to NBRC); NBRC, NITE-Biological Resource Center (http://www.nite.go.jp/en/nbrc/cultures/index.html); NIES, Microbial Culture Collection at the National Institute for Environmental Studies (http://mcc.nies.go.jp/top.jsp); SAG, Sammlung von Algenkulturen at the University of Göttingen (http://www.epsag.uni-goettingen.de/); UTEX, Culture Collection of Algae at the University of Texas at Austin (http://utex.org/). b Phyloge-netic classification based on Nakada et al. (2008). c Sequenced in the present study. d Sugasawa et al. (2015).

Table 2. Continued.

2015 Taxonomy of Pyrobotrys 517

Fig. 1. Light microscopy of newly established Pyrobotrys strains. Scale bars=5 µm. (A–C) P. casinoen-sis strain DP11. (A, B) Two lateral views of colony showing arrangement of cells and stigma (arrowhead). (C) Surface view of cell in the most posterior tier of colony showing stigma (ar-rowhead) positioned in the posterior half of cell. (D–F) P. elongata strain 2J-2. (D, E) Two lateral views of colony showing arrangement of cells and stigma (arrowhead). (F) Surface view of cell in the most posterior tier of colony showing an enormous stigma (arrowhead), positioned in the anterior half of cell. (G–I) P. elongata strain YP416. (G, H) Two lateral views of colony showing arrangement of cells and stigma (arrowhead). (I) Surface view of cell in the most posterior tier of colony showing large stigma (arrowhead). (J–L) P. squarrosa strain YS11. (J, K) Two lateral views of colony showing arrangement of cells and stigma (arrowhead). (L) Lateral view of cell showing two flagella situated on the dorsal side of the cell (arrow).

518 M. Sugasawa et al. Cytologia 80(4)

cells were dehydrated through an ethanol series, replaced by propylene oxide, and embedded in Spurr’s resin (Spurr 1969). Sections were cut using a diamond knife on an Ultracut UCT (Leica, Vienna, Austria), stained with uranyl acetate and lead citrate, and observed with a JEM-1010 elec-tron microscope (JEOL, Tokyo, Japan), as described previously (Takahashi et al. 2014).

Molecular phylogenyThe nucleotide sequences of the 18S ribosomal RNA (rRNA), RuBisCO large subunit (rbcL),

and P700 chlorophyll a apoprotein A2 (psaB) genes were determined based on direct sequencing

Fig. 2. Transmission electron microscopy of three species of Pyrobotrys. Abbreviations: ch, chloro-plast; m, mitochondrion; n, nucleus; s, stigma. (A) Longitudinal section of the most posterior cell of P. elongata strain 2J-2 showing enormous stigma composed of single globule layer. Scale bar=1 µm. (B) Transverse section of enormous stigma in the most posterior cell of P. elongata strain 2J-2, composed of single globule layer. Scale bar=500 nm. (C) Stigma of P. casinoensis strain DP21 composed of single layer of globules. Scale bar=500 nm. (D) Stigma of P. squarrosa strain YS11 composed of single layer of globules. Scale bar=500 nm.

2015 Taxonomy of Pyrobotrys 519

of polymerase chain reaction products, as described previously (Nakada and Nozaki 2007, Hayama et al. 2010). The 18S rRNA, rbcL, and psaB gene sequences were unambiguously aligned with the alignments of Nakada et al. (2010). The final alignment consisted of 58 taxa and 3390 base pairs (excluding third codon positions of rbcL and psaB, see Nakada et al. 2010), representing 15 of 21 major clades of Volvocales and all major clades within the clades Caudivolvoxa and Xenovolvoxa (Nakada et al. 2008) (Table 2). Hafniomonas montana (Geitler) H. Ettl & Moestrup and Carteria

Fig. 3. Bayesian phylogenetic tree of 58 strains of the Volvocales (Table 2) based on combined 18S rRNA, rbcL and psaB gene sequences excluding the third codon positions of rbcL and psaB (3390 base pairs). Hafniomonas montana and Carteria crucifera were treated as an outgroup. The corresponding posterior probabilities (≥0.90) are shown near nodes (top left). The bootstrap values (≥50%) from maximum likelihood (top right), maximum parsimony (bottom left), and neighbor-joining (bottom right) analyses are also shown. Branch lengths and the scale bar rep-resent the expected number of nucleotide substitutions per site. The phylogenetic classifications given by Nakada et al. (2008) are indicated on the right of the tree. Colonial species are shown in gray background.

520 M. Sugasawa et al. Cytologia 80(4)

crucifera Korshikov were treated as an outgroup because these species belong to the most basal lineage within the Volvocales (Nozaki et al. 2003).

The combined data matrix from the 18S rRNA, rbcL, and psaB genes was subjected to Bayesian inference (BI), maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ) analyses. BI was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), as described previously (Nakada and Nozaki 2009). The Markov chain Monte Carlo iteration was stopped at the 500000th generation. The first 25% of generations were discarded as burn-in and the remaining trees were used to calculate a 50% majority-rule consensus tree and determine the posterior probabilities of the individual branches. The evolutionary models of each 18S rRNA (GTR+I+G), and the first (GTR+I+G) and second codon positions (SYM+I+G) of rbcL and psaB, were selected using MrModeltest 2.3 (Nylander 2004) and unlinked in the BI. One thousand rep-licates of bootstrap analyses were performed based on ML using RAxML ver. 7.4.2 (Stamatakis 2006) with the GTR+I+G model. Bootstrap values were also obtained using PAUP* 4.0b10 (Swof-ford 2003) based on 1000 replicates of the MP analysis, with random additions of 10 replicates of a heuristic search using the tree-bisection-reconnection branch-swapping algorithm and NJ analysis (with ML distances deduced from the appropriate substitution model).

To resolve detailed species relationships within Pyrobotrys, phylogenetic analysis using six operational taxonomic units (OTUs) of Pyrobotrys (Table 2) was conducted as described above, ex-cept that the third codon positions of rbcL and psaB were included. The final alignment consisted of 11 OTUs and 4221 base pairs. Chlamydomonas sp. strain G16, C. tetragama (Bohlin) Wille, C. pumilio H. Ettl, C. pulsatilla E. Wollenweber, and C. applanata E.G. Pringsheim were treated as the outgroup because they formed a monophyletic group with the clade composed of Pyrobotrys OTUs in the phylogenetic analysis of the whole volvocalean algae, as described above. In BI phy-logeny, the evolutionary models of each 18S rRNA (GTR+I+G), and the first (F81+G), second (GTR+G), and third (GTR+G) codon positions of rbcL and psaB were selected. For ML phylogeny, the GTR+I+G model was selected.

Fig. 4. Bayesian phylogenetic tree of six OTUs of Pyrobotrys (Table 2) based on combined 18S rRNA, rbcL and psaB gene sequences including the third codon positions of rbcL and psaB (4221 base pairs). Chlamydomonas sp. strain G16, C. tetragama, C. pumilio, C. pulsatilla and C. applanata were treated as an outgroup. The corresponding posterior probabilities (≥0.90) are shown near nodes (top left). The bootstrap values (≥50%) from maximum likelihood (top right), maximum parsimony (bottom left), and neighbor-joining (bottom right) analyses are also shown. Branch lengths and the scale bar represent the expected number of nucleotide substitutions per site.

2015 Taxonomy of Pyrobotrys 521

Results and discussion

CulturesP. elongata strains YP316 and YP416 grew in modified SVM only under microaerophilic

conditions. In contrast, P. casinoensis, P. elongata strain 2J-2, and P. squarrosa grew well in modified SVM, even under aerobic conditions. These results were inconsistent with Nozaki (1986) in that only P. casinoensis cultures grew without exchanging nitrogen gas for air.

Light microscopyBased on the light microscopic characteristics of vegetative colonies (Fig. 1), the strains were

classified into three species (for details, see Taxonomic accounts).

Transmission electron microscopyThe enormous stigma in cells of the most posterior tier of the P. elongata colony is unusual

because the majority of colonial volvocalean algae show gradual decreases in stigma size from anterior to posterior tiers in the vegetative colony (e.g., Ettl 1983, Nozaki 2003). Thus, comparative TEM was performed in P. elongata, P. casinoensis, and P. squarrosa using the present new strains. In P. elongata strain 2J-2, each vegetative cell contained a massive cup-shaped chloroplast and a nucleus inside the chloroplast profiles in the anterior region of the cell (Fig. 2A). The enormous stigma, which had a length of approximately one third that of the cell, consisted of a single layer of globules (Figs. 2A, B). These features were consistent with those of stigmata of P. casinoensis strain DP11 (Fig. 2C) and P. squarrosa strain YS11 (Fig. 2D), with the exception of the size.

Molecular phylogenyTo determine the phylogenetic positions of newly established strains of Pyrobotrys within

the Volvocales, we performed phylogenetic analyses of the 18S rRNA, rbcL, and psaB genes of 58 OTUs including all major groups of Volvocales (Nakada et al. 2010) and the new Pyrobotrys strains (Table 2). Major phylogenetic relationships resolved in the present study were essentially the same as those of Nakada et al. (2010). In the BI phylogenetic tree (Fig. 3), the four OTUs of Pyrobotrys (P. casinoensis strain DP11, P. elongata strain 2J-2, P. elongata strain YP416, and P. squarrosa strain YS11) were resolved as a robust monophyletic group with P. stellata strain SAG10-1c and P. squarrosa strain NIES-2564 (with 1.00 posterior probability in BI and with 96–99% bootstrap values in the ML, MP, and NJ analyses). It is noteworthy that the unicellular alga Chlamydomonas sp. strain G16 was sister to the clade Pyrobotrys with 1.00 posterior probability in BI and with 100% bootstrap values in the ML, MP, and NJ methods.

To resolve more detailed phylogenetic relationships between species of Pyrobotrys, phyloge-netic analysis was performed using six OTUs of Pyrobotrys (Fig. 4) with five OTUs (Chlamydomo-nas sp. strain G16, C. tetragama, C. pumilio, C. pulsatilla and C. applanata) that were designated as an outgroup using 18S rRNA genes and all codon positions of rbcL and psaB genes (4221 base pairs in total). P. stellata strain SAG 10-1c and P. casinoensis strain DP11 formed a monophyletic group with 1.00 posterior probability in BI and with 100% bootstrap values in the ML, MP, and NJ analyses. Two strains of P. elongata formed a robust clade (with 1.00 posterior probability in BI and with 99–100% bootstrap values in the ML, MP, and NJ analyses), but their genetic distance was apparently greater than that between P. stellata strain SAG10-1c and P. casinoensis strain DP11 based on the branch lengths in the tree (Fig. 4). Two P. squarrosa strains were monophyletic (with 1.00 posterior probability in BI and with 100% bootstrap values ML, MP, and NJ calcula-tions). P. squarrosa and P. elongata formed a monophyletic group with 0.99 posterior probability in BI and 85–99% bootstrap values based on other phylogenetic methods.

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Taxonomic accountsPyrobotrys casinoensis (Playfair) P.C. Silva 1972: 204 (Figs. 1A–C).

Basionym: Uva casinoensis Playfair 1914: 108, pl. 2, fig. 3Synonyms: Chlamydobotrys gracilis Korshikov 1924: 55, pl. 2, figs 10–15; Chlorobrachis

gracillima Korshikov 1925: 175, 194, pl. 8, figs 42, 43; Pyrobotrys gracilis (Korshikov) Korshikov 1938: 12.

Colonies were mulberry shaped, consisting of 8 or 16 cells disposed in four alternating tiers of two or four opposed cells each, respectively (Figs. 1A, B). The cells were ovoid to pear-shaped; each had two equal flagella, a stigma (Fig. 1C), and a massive cup-shaped chloroplast without pyre-noid. Cells in the posterior one or two tiers in a colony often protruded and attenuated the posterior ends. Two flagella were situated on the anterior end of the cell. Protoplast was sometimes separated from the cell wall at the posterior end. The sizes of stigmata in the most posterior tier were the same as those of the anterior tier. Colonies were measured 17–44 µm long and cells were 7–16 µm long.

Type locality: Casino, Australia.Distribution: Australia (Playfair 1914), Ukraine (Korshikov 1924, 1925, Schulze 1927,

Strehlow 1929), Poland (Behlau 1935, Bucka et al. 1968), Czech/Slovakia (Ettl 1958), Germany (Schulze 1927, Table 1), United States (Smith 1950, Silva and Papenfuss 1953, Dillard and DaPra 1971), India (Balakrishnan 1966), China (Hu et al. 1980), and Japan (Nozaki 1986).

Strains examined: DP11 (=NIES-3768), DP21 (=NIES-3769), and DP31 (=NIES-3770).Remarks: The present alga agreed well with P. casinoensis characterized by Nozaki (1986) in

having 16-celled vegetative colonies and posterior cells with a protruded posterior end. Although P. casinoensis is characterized by its unique planozygote form (Nozaki 1986), sexual reproduction was not observed in the present culture strains.

Pyrobotrys elongata Korshikov 1938: 13, 20, pl. 3, figs 27–30 (Figs. 1D–I).Synonym: Uva elongata (Korshikov) Fott 1967: 362.Colonies were mulberry shaped, consisting of 8 or 16 cells disposed in four alternating tiers

of two or four opposed cells each, respectively (Figs. 1D, E, G, H). The cells were ovoid to pear-shaped; each had two equal flagella, a stigma, two contractile vacuoles at the base of the flagella and a massive cup-shaped chloroplast without pyrenoid. Cells in the posterior one or two tiers in a colony often protruded and attenuated the posterior ends. Two flagella were situated on the anterior end of the cell. Protoplast was sometimes separated from the cell wall at the posterior end. Cells in the most posterior tier had an enormous stigma, which was nearly one third of the cell length (Figs. 1F, I). Colonies were measured 26–39 µm long and cells were 8–20 µm long.

Type Locality: Kharkiv, Ukraine.Distribution: Ukraine (Korshikov 1938), Japan (Table 1).Strains examined: 2J-2, YP 316 (=NIES-3771), and YP416 (=NIES-3772).Remarks: Although Pyrobotrys elongata is characterized based on its enormous stigma in

cells of the most posterior tier in a colony (Korshikov 1938, Nozaki 1986), such an alga has not been reported since its original description (Korshikov 1938). Here, we established culture strains of P. elongata based on samples collected from two localities of Japan (Table 1). Strains originat-ing from both samples had 16-celled colonies with an enormous stigma in cells of the most posteri-or tier in a colony (Figs. 1D–I). However, morphological differences could be recognized between the two: the stigmata in the most posterior tier of strain 2J-2 (Figs. 1D–F) were more or less larger than those of strains YP316 and YP416 (Figs. 1G–I). The present molecular phylogenetic analysis resolved considerable genetic distance between them, although they formed a monophyletic group (Figs. 3, 4). Thus, until other morphological differences are resolved, these two algae should be identified as P. elongata.

2015 Taxonomy of Pyrobotrys 523

Although P. elongata is characterized based on its enormous stigma in cells of the most poste-rior tier in a colony, other morphological characteristics in vegetative colonies are similar to those of P. casinoensis (Nozaki 1986). The present phylogenetic analysis demonstrated that these two species were robustly separated from each other (Figs. 3, 4), suggestive of the independency of these two species.

Pyrobotrys squarrosa (Korshikov) Korshikov 1938: 12 (Figs. 1J–L).Basionym: Chlamydobotrys squarrosa Korshikov 1928: 236, pl. figs 7–12.Synonyms: Uva squarrosa (Korshikov) Fott 1967: 361; Pyrobotrys squarrosa (Korshikov)

Huber-Pestalozzi in Fott 1967: 361.Colonies were star-shaped, consisting of eight cells disposed in four alternating tiers of two

opposed cells each (Figs. 1J, K). The cells were irregularly pear-shaped with strongly inflated, ventral sides, and a long posterior tail with a somewhat blunted end; each had two equal flagella, a stigma, two contractile vacuoles at the base of flagella and a massive cup-shaped chloroplast with-out pyrenoid. Posterior portions of cells slightly curved backward. Two flagella were situated on the anterior dorsal side, more or less apart from the anterior end of the cell (Fig. 1L). Protoplast was often separated from the cell wall at the posterior end. Colonies were measured 18–31 µm long and cells were 12–20 µm long.

Type Locality: Kharkiv, Ukraine.Distribution: Ukraine (Korshikov 1938), Czech/Slovakia (Fott 1967), Poland (Bucka et al.

1968), and Japan (Nozaki 1986, Table 1).Strains examined: YS11 (=NIES-3773), and YS12 (=NIES-3774).Remarks: The present alga agreed well with P. squarrosa characterized by Nozaki (1986) in

its flagella situated on the dorsal side of the cell, and posterior potions of the cell are curved slightly backward. However, according to the original description of material collected in Ukraine (Korshi-kov 1928), posterior tails of the vegetative cells of P. squarrosa are almost straight. In contrast, the tails of Japanese cultured materials of Nozaki (1986), Nakada et al. (2010), and the present study (Figs. 1J, L) are slightly curved backward. The taxonomic significance of this morphological differ-ence will be resolved by examining other cultured materials originating from Ukraine and/or other countries.

Acknowledgement

This work was supported by Grant-in-Aid for Scientific Research (B) (25304012 to HN) from MEXT/JSPS KAKENHI.

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