A Novel Species in the Genus Heveochlorella

Eur. J. Phycol. (2013), 48(2): 200–209

A novel species in the genus Heveochlorella (Trebouxiophyceae, Chlorophyta) witnesses the evolution from an epiphytic into an endophytic lifestyle in tree-dwelling green algae

SHUAI MA1*, VOLKER A.R. HUSS2*, DEGUAN TAN1, XUEPIAO SUN1, JINGBO CHEN3, YAOYUN XIE3 AND JIAMING ZHANG1

1Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China 2Institute of Molecular Plant Physiology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany 3Hainan Provincial People’s Hospital, Xiuhua Road 19, Haikou, Hainan, China

(Received 20 March 2012; revised 10 November 2012; accepted 19 November 2012)

Roystonea regia is a popular landscape palm famous for its smoothly sculpted white trunk, but sometimes it is spoiled by epiphytic green algae inhabiting the bark. We identified a trebouxiophycean green alga as the single dominant species growing on the trunk of a royal palm in Hainan, China. Light and transmission electron microscopy showed adult cells to be spherical with a diameter of 6.4–13.7 μm, and to contain a single cup-shaped parietal chloroplast. Each chloroplast contained a pyrenoid of 1.4–2.1 μmin diameter. The pyrenoid was penetrated by radially arranged tubular invaginations surrounded by pyrenoglobuli, which is similar to Heveochlorella hainangensis. Phylogenetic analyses inferred from nuclear-encoded 18S rRNA sequences placed strain ITBB A3-8 in the Watanabea clade (Trebouxiophyceae, Chlorophyta), closely related to H. hainangensis. But strain ITBB A3-8 differs sufficiently from H. hainangensis in significant aspects of ultrastructure, physiology, and nuclear and plastid ribosomal RNA and ITS sequences to be treated as a novel species, H. roystonensis Shuai Ma, V. Huss, Xuepiao Sun & Jiaming Zhang, sp. nov. The growth of the epiphytic H. roystonensis seems to be promoted by air pollution in the city, while the endophytic H. hainangensis requires an organic carbon source for optimal growth. Thus in the genus Heveochlorella both an epiphytic and an endophytic lifestyle of tree-dwelling green algae can be observed.

Key words: epiphytic algae, Heveochlorella hainangensis, Heveochlorella roystonensis sp. nov., ITS, pyrenoid, SSU rRNA gene phylogeny, Trebouxiophyceae, taxonomy

Introduction trees (Trémouillaux-Guiller et al., 2002; Zhang et al., Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 Algae inhabit a wide variety of ecosystems including 2008). In Ginkgo biloba, for example, a eukaryotic alga resides inside the cells of this ‘fossil’ plant as a marine, freshwater, terrestrial and even extreme envir- ‘ ’ onments (Huss et al., 2002; La Rocca et al., 2009). precursor form, and is distributed worldwide by Some microalgae have established symbiotic associa- vertical inheritance (Trémouillaux-Guiller & Huss, tions with other organisms, such as Chlorella with 2007). green hydra and Paramecium bursaria (Kovačević The royal palm (Roystonea regia) is a popular orna- et al., 2005, 2007; Kodama et al., 2007), in which mental in tropical countries and was introduced into the algae provide energy and even detoxiﬁcation to the China from India and Sri Lanka. This palm is famous č ć for its massive, symmetrical, smoothly sculpted and hosts (Pardy, 1976; Reisser, 1976; Kova evi et al., ﬁ 2005). Green algae inhabiting trees have been missile-like white trunk, which has an almost arti cial described in increasing numbers in recent years, either appearance and makes a memorable impression on in lichen consortia, or free-living on the surface of millions of tourists in Hainan each year. However, in bamboo or tree trunks (Hanagata et al., 1997; Rindi recent years, some of these palm trees have become et al., 2006; Eliáš et al., 2010; Neustupa et al., 2011). densely colonized by green algae, possibly due to the Some Chlorella-like algae have even been found to be rapid increase of air pollutants by cars, making the endophytic in plants, as shown for ginkgo and rubber trunk green and rough and badly affecting the aes- thetic value of the trees. We took several algal isolates into culture from trunks and sought to characterize and Correspondence to: Jiaming Zhang. E-mail: [email protected]. identify them using morphological and molecular com *These authors contributed equally to the paper. methods. We made an especially close comparison

between the royal palm epiphytes and Heveochlorella amplified using the primers 5′-ACCTGGT hainangensis, previously isolated from rubber trees TGATCCTGCCAGTAG-3′ and 5′-ACCTTGTTACGACT (Zhang et al., 2008). TCTCCTTCCTC-3′ (Zhang et al., 2008), which were designed according to the consensus sequence of the available 18S rRNA sequences of chlorophyte species. The amplifica- Materials and methods tion conditions were as follows: 5 min at 94°C, 30 cycles of 30 Field sampling and isolation of the algae s at 94ºC, 30 s at 58ºC and 2 min at 72ºC, and a final 10 min extension step at 72ºC. The internal transcribed spacer region Field samples were collected from the bark of a royal palm in (ITS), including ITS1, 5.8S rDNA and ITS2, of H. roystonen- ′ ″ ′ ″ Haikou (N 19° 58 57 ,E110°19 37 ), Hainan, China. The sis and H. hainangensis FGG01 was amplified under the same samples were suspended in sterilized distilled water, and conditions, using the primer pair 5′-CTTTG allowed to stand for a few minutes. The supernatant was TACACACCGCCCGTC-3′ and 5′-ATATGCTTAAGTTG sequentially diluted and plated on Tris-Acetate-Phosphate AGCGGGT-3′, which binds to conserved parts of the 3′-region (TAP) medium (Harris, 1989), and incubated at 25°C, under of the 18S rDNA and the 5′-region of the 28S rDNA, respec- 12 h light per day with a light intensity of 100 µmol photons m tively. The chloroplast 16S SSU rRNA sequence was ampli- −2 −1 fl s from cool-white uorescent tubes. The resulting single fied using the primers 5′-AGAG TTYGATCCTGGCTCAGG- cell clones were subcultured on TAP medium until the mor- 3′ and 5′-GTGATCCAYCCY CACCTTCCA G-3′ (Zhang phology of the clones was uniform and no contamination et al., 2008). The amplification conditions were 5 min at 94° detectable. The algal strains have been deposited in the C, 30 cycles of 30 s at 94ºC, 30 s at 55ºC and 1.5 min at 72ºC, Microorganism Collection Center at the Institute of Tropical and a final 10 min extension step at 72ºC. The oligonucleotides Bioscience and Biotechnology, Hainan, and cultures stored at were synthesized by TaKaRa, Dalian, China. PCR products − 80°C in 15% glycerol. Liquid TAP medium was used for were cleaned with a DNA cleaning kit (TaKaRa, Dalian, suspension cultures, which were agitated at 120 rpm on a China) and sequenced by Shangon Biotechnologies ZHWY-2000 orbital shaker (Shanghai Zhicheng, China). (Shanghai, China). The sequences were analysed with Incubation conditions were the same as above. A clonal algal MacVector® 7.1 (Accelrys, Pharmacopeia, USA) and depos- strain ITBB A3-8 was further studied in this paper. ited in the GenBank database under accession numbers JN003600, JN003601, JX290371 and JX290372. Microscopy A small piece of bark with green algae was removed from a Phylogenetic analyses royal palm tree and sliced into thin sections. The sections The nuclear SSU rRNA gene sequences were included in were put on glass slides and observed with a Nikon 80i light large alignments of several hundred trebouxiophycean gene microscope (LM) (Nikon, Tokyo, Japan). Cultured cells from sequences including the most similar sequences as deter- different culture conditions were mounted on glass slides, mined by BLAST searches, and the chloroplast SSU rRNA examined and photographed with a 100× oil immersion lens. sequences were aligned with most available chlorophyte Cell sizes were measured with a micrometre eyepiece. The chloroplast SSU gene sequences. The alignment was carried observations were made either in full exponential phase, or in out manually on a MicroVAX computer using the sequence late stationary phase of growth. editor program distributed by G. Olsen (Olsen et al., 1992) For transmission electron microscopy (TEM), cultured and with reference to secondary structure, determined cells were harvested by centrifugation at 2000 × g for 5 according to the model of Wuyts et al. (2002). Highly vari- min, the pellet was covered by a little egg white, fixed with able regions that could not be aligned unambiguously for a Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 2.5% glutaraldehyde dissolved in 0.2 M phosphate buffered subset of 38 relevant taxa in the 18S rDNA alignment were saline (PBS buffer: 135 mM NaCl, 4.7 mM KCl, 10 mM excluded from the analyses together with PCR primer bind- Na2HPO4, 2mM NaH2PO4, pH 7.2) for 3 h at 4°C, rinsed ing regions and introns, resulting in a total of 1672 positions. three times in PBS buffer for 10 min each time, and post-fixed Phylogenetic trees were inferred by the neighbour joining in 1% osmium tetroxide (aqueous solution) for 2 h at 4°C. (NJ), maximum parsimony (MP) and maximum likelihood The pellet was then rinsed again three times in PBS buffer (ML) methods using PAUP* 4.0b 10 (Swofford, 2002) for NJ and dehydrated by increasing concentrations of acetone and MP, and RAxML for the ML analyses (Stamatakis et al., (50%, 70%, 80%, 90% and 100%, 10 minutes each) at 2008). The tree topology in Fig. 23 was calculated by ML via room temperature. The samples were then embedded in the Web Server http://phylobench.vital-it.ch/raxml-bb/ using Spurr’s resin (Spurr, 1969). Ultrathin sections were prepared the CAT model for rate heterogeneity and choosing ‘Maximum with a A130373 ultramicrotome (Leica, Germany), mounted Likelihood Search’ for the best scoring tree. 1000 bootstrap on carbon-coated formvar slotted grids, sequentially stained resamplings were calculated for NJ under the TrN+I+G model with uranyl acetate and lead citrate, and observed with a selected by Modeltest (Posada & Crandall, 1998; alignment JEOL 1010 (Jeol, Tokyo, Japan). in Supplementary File 1) and for MP choosing ‘New state’ for the treatment of gaps and random addition of sequences with fi each 10 replicates. For ML, 500 bootstraps were calculated DNA extraction, PCR ampli cation and sequencing using the same Web Server as indicated above. The cultured cells were collected by centrifugation at 2000 × g The ITS-sequences were aligned using ClustalOmega for 5 min. The pellets were homogenized with a mortar and (http://www.ebi.ac.uk/Tools/msa/clustalo/) with subsequent pestle. DNA was extracted with a universal DNA isolation kit manual refinement of ITS2 by considering the conserved (TaKaRa, Dalian, China) following the manufacturer’s instruc- secondary structure of this region (Schultz et al., 2005; tions. Nuclear-encoded 18S small subunit (SSU) rDNA was Fig. 25). The phylogenetic analyses for a concatenated S. Ma et al. 202

Figs 1–4. Colonization of the algae on the royal palm (Roystonea regia). 1. Palms with algae growing vigorously on the trunk in a busy street in Haikou, Hainan Province, China. 2. Palms with poor algal growth in the Xinglong Tropical Botanical Garden, CATAS, Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 Wanning County, Hainan. 3. Close-up, showing algae on the bark. 4. Bark section showing algae on the surface.

dataset of 18S+ITS in Fig. 24 (2353 positions) were essen- independent from fungi (Fig. 4), although some alga– tially done as described for the SSU rDNA sequences alone, fungus consortia were also observed. Twelve pure except that GTR+I+G was selected by Modeltest as the best- algal strains were isolated, all of which were morpho- ﬁt model for the NJ bootstrap analysis (alignment in logically identical. Their 18S rDNA was partially Supplementary File 2). sequenced and no variation found; thus all were considered to belong to a single dominant species among Results and discussion the epiphytic algal species dwelling on the palm. One strain (ITBB A3-8) was studied in detail. Isolation of algal strains from royal palms Growth of green algae on the trunk of the royal palm is Morphological observations common in Hainan and seems to be more vigorous in streets with heavy trafﬁc (Fig. 1), compared to the Strain ITBB A3-8 grew best at 28°C in TAP liquid growth in botanical gardens (Fig. 2), suggesting that medium at pH 7.0. Most of the adult cells were sphe- the algae may obtain most of their nutrients from rical, 6.4–13.7 μm in diameter, with a mean diameter the air. Microscopical observation of bark samples of 9.8 μm, and contained a single parietal and cup-- (Fig. 3) revealed that most of the algae were unicel- shaped chloroplast (Fig. 5). Autosporulation was the lular and located on the surface of the bark as clones sole mode of reproduction. Most sporangia contained A novel Heveochlorella species 203

Figs 5–16. Heveochlorella roystonensis, strain ITBB A3-8, LM. 5. Mature vegetative cell with a parietal cup-shaped chloroplast. 6. Sporangia with four or eight autospores. 7. Sporangium with eight autospores. 8. Sporangia with 32 autospores. 9. Sporangium with two autospores, each containing two or three chloroplasts. 10. Sporangium with four autospores of different sizes. 11–13. Dividing Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 chloroplasts. 14. Cell after completion of chloroplast division. 15. Chloroplast dividing from both ends. 16. Unequal division of a chloroplast. Scale bar = 5 μm.

4–16 autospores (Figs 6, 7) but a few contained 32 or chloroplasts then moved to opposite sides of the cell, more (Fig. 8). Sporangia with only two autospores becoming temporarily spindle-shaped (Fig. 14). were rarely observed, probably due to rapid division Sometimes, the longitudinal division was initiated of the autospores; each of the two autospores usually from both ends (Fig. 15), and in a few cases the contained two to three chloroplasts, ready for second- chloroplast divided unequally (Fig. 16). ary division (Fig. 9). The autospores were ovoid, 6–9 TEM micrographs revealed that the cell wall of μm in diameter, with a single ovoid chloroplast. The strain ITBB A3-8 is smooth and double-layered, with- autospores usually divided equally, but in a few cases out any mucilage (Figs 17–19). A cup-shaped parietal they differed in size due to unsynchronized develop- chloroplast accounted for approximately half of the ment and division (Figs 9, 10), as deduced by the volume of mature cells (Fig. 17). A pyrenoid, 1.4–2.1 different numbers and sizes of chloroplasts in the μm in diameter, was located in the centre of the chlor- autospores. The division pattern of the chloroplast oplasts of both the mature cells (Figs 17, 18) and the was similar to that of Heveochlorella hainangensis autospores (Fig. 20). The stroma thylakoids were con- FGG01, an alga isolated from rubber trees (Zhang nected to the surface of the pyrenoid and radiated from et al., 2008), in that it was usually initiated from one it (Figs 21, 22). The pyrenoids were penetrated by end of the chloroplast and progressed longitudinally to radial, tubular invaginations 70–120 nm in diameter, the other end (Figs 11–13). The two daughter with a central rod-like or ﬁbrillar material and many S. Ma et al. 204

surrounding pyrenoglobuli. This kind of pyrenoid hainangensis (EF595524), 96% to ‘Chlorella’ sp. ultrastructure is again similar to that of MBIC10057 (AB058305), and 94% to several Heveochorella hainangensis (Zhang et al., 2008). ‘Chlorella’ species including Heterochlor-ella luteoviridis (Neustupa et al., 2009). Excluding the intron regions Molecular characterization is preferred over using the complete 18S rDNA sequence in blast searches, since group I introns are In order to determine the phylogenetic position of common among green algae and are fast evolving strain ITBB A3-8, its nuclear-encoded 18S rDNA and mobile elements; however, they could not be together with the adjacent ITS, as well as the chlor- excluded from reference strains during the blast oplast-encoded 16S rDNA were sequenced. The search, thereby hiding a high similarity of the 18S sequenced lengths of the 16S rDNA and the ITS exons. The chloroplast 16S rDNA was most similar were 1488 bp and 650 bp, respectively. The ITS of to Heveochlorella hainangensis (95.3%, EF595524), H. hainangensis, which was determined for compar- but many fewer reference sequences were available ison, had a length of 612 bp. The sequenced length of in the database compared with 18S rDNA. the 18S rDNAwas 2500 bp including two introns. The two introns (353 bp and 382 bp) were located at Phylogenetic position of ITBB A3-8 positions 562 and 943 (E. coli numbering), which are well-known positions for group I introns Phylogenetic trees were generated with 18S and 16S (Cannone et al., 2002). The putative secondary struc- rRNA gene sequences and conﬁrmed that strain ITBB tures of the two introns (not shown) were typical for A3-8 belongs to the Trebouxiophyceae. In the 18S group I introns of subgroup IC1 (Cannone et al., rRNA tree, it was included in a clade also containing 2002), as in H. hainangensis, although their primary Heveochlorella hainangensis and ‘Chlorella’ luteo- sequences were divergent. viridis strain MES A5-4 (Fig. 23). The Blast searches in the GenBank database using 605 Heterochlorella luteoviridis strains SAG 211-2a nucleotides of the 18S rRNA sequence (positions 561– (Neustupa et al., 2009), CCAP 211/3, CCAP 211/4, 1165), in which most trebouxiophytes have no intron CCAP 211/10A, CCAP 211/10E and another insertion site, returned highest similarities of 99% to ‘Chlorella’ luteoviridis strain (MBIC 10057) formed ‘Chlorella’ luteoviridis strain MES A5-4 (AB006045), a close sister clade (only one representative strain of followed by 97% similarity to Heveochlorella H. luteoviridis, CCAP 211/10A, was included in the Downloaded by [Dr Volker Huss] at 16:16 30 May 2013

Figs 17–22. Heveochlorella roystonensis, strain ITBB A3-8, TEM. 17, 18. Mature vegetative cell showing the cup-shaped parietal chloroplast, pyrenoid, nucleus and mitochondria. 19. Two-layered cell wall of mature cell. 20. Sporangium containing multiple autospores. 21, 22. Ultrastructure of pyrenoids showing tube-like invaginations arranged radially. C, chloroplast; M, mitochondria; N, nucleus; V, vacuole; CW, cell wall. Scale bars = 1 μm. A novel Heveochlorella species 205

analysis, as all other strains had essentially the same complete 16S sequences of Heterochlorella chloro- sequence). The separation of Heveochlorella and plasts are not yet available, the 16S rDNA phylogeny Heterochlorella was not robustly supported in the did not contribute much to the resolution of the phylogenetic analysis and the lack of a critical stan- Heveochlorella–Heterochlorella lineage (tree not dard to combine or separate two genera (contrast the shown). Instead, we determined the ITS sequences compensatory base changes in ITS2 used a criterion to of ITBB A3-8 and H. hainangensis and compared judge conspecificity: see below) makes it difficult to them with the published ITS sequence of decide whether these two genera should be kept apart Heterochlorella luteoviridis CCAP 211/10A or combined into a single genus Heveochlorella (since (FR865652) to test a sister relationship of H. hainan- Heveochlorella was officially described earlier than gensis and ITBB A3-8, and to provide assurance that Heterochlorella). More evidence and more strains they are distinct species. The phylogenetic tree in Fig. from this lineage are needed to resolve this problem. 24 was based on a concatenated dataset of 18S+ITS However, the separation of the two genera is well sequences, with the trebouxiophytes Watanabea reni- supported by ultrastructural observations, since both formis and Dictyochloropsis reticulata used as out- H. hainangensis (Zhang et al., 2008) and strain ITBB groups. Bootstrap support for a sister relationship of A3-8 (Figs 19, 20) have tube-like thylakoids in the both Heveochlorella species was now excellent for pyrenoid matrix and the pyrenoid is not surrounded by NJ, MP and ML. a starch sheath, whereas Heterochlorella luteoviridis According to Mai & Coleman (1997) and Coleman strain SAG 211-2a has a starch sheath around the (2007), two organisms should be considered as differ- pyrenoid and the pyrenoid matrix is bisected by dou- ent species if the conserved parts of their ITS2 ble-layer thylakoids (Neustupa et al., 2009). sequences differ by at least one compensatory base Unfortunately, the pyrenoid ultrastructure of change (CBC). The ITS2 secondary structure model ‘Chlorella’ luteoviridis MES A5-4, the closest relative in Fig. 25 for both Heveochlorella species shows that of strain ITBB A3-8, could not be determined, as this besides a considerable sequence variation (67.6% strain was not available from any public alga collec- overall similarity for ITS2) there is one CBC at the tion, nor was any information about this strain known, conserved base of helix I, one hemi compensatory including its habitat. The same applies to ‘Chlorella’ base change (hCBC) in helix II, one CBC and one sp. MBIC 10057 which was also not available from hCBC in the conserved part of helix III, and at least public alga collections. The recently described genus one hCBC in the conserved part of helix IV. From this and species Kalinella bambusicola (Neustupa et al., and all the other evidence given above, we conclude 2009) was the next most closely related to the that ITBB A3-8 is a novel species of Heveochlorella, Heveochlorella/Heterochlorella lineage in our phy- which we name H. roystonensis. logeny, although clearly divergent in its 18S rDNA. Its pyrenoid is surrounded by separate starch grains Heveochlorella roystonensis Shuai Ma, V. Huss, and bisected by many parallel thylakoids (Neustupa Xuepiao Sun & Jiaming Zhang, sp. nov. et al., 2009). A morphological comparison of the described taxa most closely related to ITBB A3-8, Figs 5–22 including pyrenoid ultrastructure as a useful taxo- Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 DESCRIPTIO: Algae microscopicae virides solitariae ter- nomic marker (Ikeda & Takeda, 1995), is given in ricolae. Cellulae sphaericae, 6.4–13.7 μm diametro. Table 1. Paries cellulae laevis bistratus. Chromatophora in cel- The chloroplast 16S rDNA sequences of strain lulis iuvenalibus fusiformia, in cellulis adultis parie- ITBB A3-8 and Heveochlorella hainangensis clus- talia patelliformiaque, unoquoque pyrenoide unica tered together with 100% bootstrap support, but as 1.4–2.1 μm diametro praedito; pyrenoides a

Table 1. Morphological comparison between Heveochlorella roystonensis ITBB A3-8 and three closely related strains, Heveochlorella hainangensis FGG01, Kalinella bambusicola CAUP H7901 and Heterochlorella luteoviridis SAG 211-2a.

H. roystonensis H. hainangensis K. bambusicola H. luteoviridis Characteristics ITBB A3-8 FGGO1 CAUP H7901 SAG 211-2a

Autospore shape spherical spherical or triangular spherical spherical or triangular Cell size (μm) 6.4–13.7 3.8–8.7 4.6–10.5 3.8–6.4 Cell wall layers 2 2 2 2 Pyrenoids per chloroplast 1 multiple 1 1 Pyrenoid diameter (μm) 1.4–2.1 0.3–0.7 1.7–3.3 1.1–2.3 Pyrenoid inserts (thylakoids/invaginations) tube-like invaginations tube-like invaginations multiple bands single band Directions of pyrenoid inserts multiple multiple multiple single Starch sheath absent absent present present References this research Zhang et al. (2008) Neustupa et al. (2009) Neustupa et al. (2009) S. Ma et al. 206

DQ530055 Elliptochloris bilobata SAG 245.80 79/65/93 AM167525 Coccomyxa glaronensis CCALA 306 X89012 Choricystis minor SAG 251-1 92/64/69 AJ581912 Botryococcus braunii Titicaca AJ311639 Raphidonema spiculiforme CCAP 470/2 AB080302 Picochlorum maculatum CCAP 251/3 AF124336 Marvania geminata SAG 12.88 63/73/79 X13688 Chlorella vulgaris SAG 211-11b 94/71/59 FM205836 Micractinium pusillum CCAP 248/5 Y17470 Closteriopsis acicularis SAG 11.86 X56105 Parachlorella kessleri SAG 211-11g -/60/77 X56101 Auxenochlorella protothecoides SAG 211-7a X74003 Prototheca wickerhamii SAG 263-11 94/78/84 X74000 „Chlorella“ mirabilis Andreyeva 748-I X63520 Pseudochlorella pringsheimii SAG 211-1a AJ416105 „Chlorella“ sphaerica SAG 11.88 AJ416106 Prasiola crispa SAG 43.96 85/96/95 AJ416107 Stichococcus bacillaris SAG 379-1b Z21551 Trebouxia impressa UTEX 892 Z28971 Myrmecia biatorellae UTEX 907 FJ829884 Phyllosiphon arisari AM260450 Symbiont of Psoroglaena epiphylla L-1016 -/73/82 X63505 Chloroidium saccharophilum SAG 211-9a FM946011 Chloroidium engadinensis SAG 812-1 86/93/90 FM946012 Chloroidium ellipsoideum SAG 3.95 FM946019 Chloroidium angusto-ellipsoideum SAG 2115 EU346910 Kalinella bambusicola CAUP H7901 FR865652 Heterochlorella luteoviridis CCAP 211/10A AB058305 „Chlorella“ sp. MBIC10057 JN003601 Heveochlorella roystonensis ITBB A3-8 AB006045 „Chlorella luteoviridis“ MES A5-4 96/88/95 58/56/65 EF595524 Heveochlorella hainangensis FGG01 X73991 Watanabea reniformis SAG 211-9b EU090195 Watanabea sp. MSC1 AJ439401 Viridiella fridericiana SAG 10.92 83/52/- GU017650 Dictyochloropsis reticulata SAG 53.87 M84320 Dunaliella salina M32703 Chlamydomonas reinhardtii CC-400

Fig. 23. Phylogenetic tree deduced from nuclear 18S rDNA sequences of Heveochlorella roystonensis and related trebouxiophycean green algae. The tree was rooted with two chlorophycean sequences as outgroups. Tree topology is based on a maximum likelihood (ML) analysis, which was mostly consistent with trees calculated by maximum parsimony (MP) and neighbour joining (NJ). The numbers at nodes indicate bootstrap support for NJ/MP/ML (see Methods). Statistical support of more than 90% for all three methods is indicated by a thick line. Bootstrap values lower than 50% are not shown. Branch lengths reﬂect the evolutionary distances indicated by the scale. GenBank accession numbers precede the taxon names.

JN003601, JX290371 Heveochlorella roystonensis ITBB A3-8 85/100/100

100/100/100 EF595524, JX290372 Heveochlorella hainangensis FGG01

FR865652 Heterochlorella luteoviridis CCAP 211/10A

FM958480 Watanabea reniformis SAG 211-9b

100/100/100 GU017650 Dictyochloropsis reticulata SAG 53.87 0.1 substitutions/site Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 Fig. 24. Phylogenetic tree based on concatenated 18S+ITS sequence data of Heveochlorella and Heterochlorella with two more remotely related trebouxiophycean green algae as outgroups. A maximum likelihood analysis was used to infer the tree topology. The bootstrap support for NJ/MP/ML is indicated at the nodes (see Methods). Branch lengths reﬂect the evolutionary distances indicated by the scale. GenBank accession numbers precede the taxon names.

invaginationibus peripheralibus tubiformibus plusmi- surrounding the invaginations or scattered in the pyr- nusve radialibus penetrato; pyrenoglobuli invagina- enoid matrix. Reproduction by 4–16 spherical to ellip- tiones cingentes vel per matricem pyrenoidis soidal autospores. dispersi. Propagatio per 4–16 autosporas globosas HOLOTYPE: Deposited as ITBB A3-8 in the vel ellipsoideas. Microorganism Collection Center of the Institute of DESCRIPTION: Green solitary microalgae, terrestrial. Tropical Bioscience and Biotechnology (ITBB), Cells spherical, 6.4–13.7 μm in diameter. Cell walls CATAS, Hainan, China and cryopreserved there in a smooth and double-layered. Chloroplasts spindle metabolically inert state at −80°C. shaped in young cells, parietal and cup-shaped in ETYMOLOGY: Derived from Roystonea regia, the royal adult cells, with a single pyrenoid of 1.4–2.1 μmin palm. diameter, which is penetrated by more or less radial, tubelike, peripheral invaginations; pyrenoglobuli TYPE LOCALITY: Hainan Province, China. A novel Heveochlorella species 207

III III U C U U G C G C C C G C G A U A U G C G C U G U G G C G C G A G A A U A U U G U G G C G C G C G C U U U U U U U U A G C G C A U G C CBC C G C G U A U G hCBC C G A C G C G G C G C G C G C G U G U UU U U A A U C G C G U IV A U C A G C A C G C C G C C G G G C G C C A G C U A G G G C G U A G C U U U C C G C G G IV U A A U C C G C U A C G C U C G C A U U G A G G hCBC C A U A A A G G A C GC GAGU CUGCUG C A hCBC A C II G A A C C C C C CGCU C GA CGGU C U G C U G C A ITS2 A A C U C C GC GAGU CUGCUG Heveochlorella hainangensis C A II A FGG01 A A C G LSU U CGUU CU GACGGU C U UCGAC CUG U A AG U GG CU G GA C U C C U G G U A A G G U C C C C 5.8S UU G C C C C G G A A A U U U C U G U G U G A G C G C U C C CBC G C C A C G A C C C U G G C U C U C I U U CU

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Fig. 25. Secondary structure model of the internal transcribed spacer 2 (ITS2) of Heveochlorella hainangensis FGG01. Helices I, II, IV and the conserved part of helix III are shown for H. roystonensis ITBB A3-8 for comparison. Hemi-compensatory base changes (hCBCs) and full compensatory base changes (CBCs) are indicated for each helix.

DISTRIBUTION: Royal palms at the type locality, not However, the lack of distinctive morphological fea- known from elsewhere. tures and the asexual reproduction of these algae caused considerable problems in taxonomic descrip- ﬁ General discussion tions and classi cation (Kessler & Huss, 1992). In a biochemical and phylogenetic study, Chlorella spe- The genus Chlorella was originally described as coc- cies were found to be distributed in two classes of coid green algae surrounded by a smooth cell wall and green algae, the Trebouxiophyceae and the reproducing exclusively by asexual autosporulation Chlorophyceae (Huss et al., 1999). From the 50 spe- (Shihira & Krauss, 1965; Fott & Nováková, 1969). cies previously assigned to Chlorella, only three spe- About 50 species were once assigned to this genus cies now remained there: C. vulgaris, C. lobophora with traditional morphological methods (Fott & and C. sorokiniana (Krienitz et al., 2004). Other Nováková, 1969), including Chlorella luteoviridis. Chlorella-like species have been reclassiﬁed into S. Ma et al. 208

distinctive genera, such as Auxenochlorella (Kalina & might be regarded as an adaptation to its supposed Punčochářová, 1987), Parachlorella (Krienitz et al., endophytic lifestyle (Zhang et al., 2008). In the genus 2004), Chloroidium and Pseudochlorella (Darienko Heveochlorella we may therefore witness the evolu- et al., 2010), Heveochlorella (Zhang et al., 2008) tion from an epiphytic into an endophytic lifestyle of and Heterochlorella (Neustupa et al., 2009). tree-dwelling green algae. However, there are still a number of Chlorella-like species not officially reclassified. Acknowledgements Heveochlorella roystonensis generally resembles H. hainangensis in morphology and ultrastructure, This research was supported by the Chinese International Science and Technology Cooperation having a similar cell wall and chloroplast division Program (2010DFA62040), Natural Science pattern, and a pyrenoid with tube-like radial invagina- Foundation of Hainan Province, China (No. 309051) tions. But their differences are also significant: H. and National Nonprofit Institute Research Grant of roystonensis grows well without sugar supplementa- CATAS-ITBB (ITBBZX2008-4-3). tion and has a single large pyrenoid, while H. hainangensis has several small pyrenoids and requires sugar Supplementary information supplementation for optimal growth (Zhang et al., 2008). In turn, the pyrenoid structure of both strains The following supplementary material is available for differs from that in their phylogenetically close rela- this article, accessible via the Supplementary Content ’ tive Heterochlorella luteoviridis SAG 211-2a, whose tab on the article s online page at fi pyrenoid is surrounded by a starch sheath and Supplementary Files 1 and 2. Nexus les of the 18S whose pyrenoid matrix is bisected by double-layer rDNA and 18S+ITS alignments that were used to thylakoids (Neustupa et al., 2009), allowing their calculate the phylogenetic trees in Figs 23 and 24, morphological distinction on the basis of pyrenoid respectively. The results of Modeltest evaluations for fi ultrastructure. Therefore, judging by the molecular the best- t models of DNA substitution (TrN+I+G phylogeny, the ultrastructure of the pyrenoid is a and GTR+I+G, respectively) are included at the end fi more important feature for classification than the of the les. mere number and size of pyrenoids. Both Heveochlorella roystonensis and H. hainan- References gensis belong to the former Chlorella luteoviridis ABOAL,M.&WERNER, O. (2011). Morphology, fine structure, life lineage within the Watanabea clade, together with cycle and phylogenetic analysis of Phyllosiphon arisari, a sipho- Kalinella and Chloroidium. Examples of symbiotic nous parasitic green alga. European Journal of Phycology, 46: associations of species belonging to the Watanabea 181–192. clade are numerous: strain HHG of Chlorella sacchar- CANNONE, J.J., SUBRAMANIAN, S., SCHNARE, M.N., COLLETT, J.R., D’SOUZA, L.M., DU, Y., FENG, B., LIN, N., MADABUSI, L.V., ophila (= Chloroidium saccharophilum; Darienko MULLER, K.M., PANDE, N., SHANG, Z., YU,N.&GUTELL, R.R. et al., 2010) was isolated as an endosymbiont from (2002). The comparative RNAweb (CRW) site: an online database the foraminiferan Heterostegina depressa (Lee et al., of comparative sequence and structure information for ribosomal, 1982; Huss et al., 1987), strain CAUP H7901 of intron, and other RNAs. BMC Bioinformatics, 3:2. COLEMAN, A.W. (2007). Pan-eukaryote ITS2 homologies revealed

Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 Kalinella bambusicola is an epiphyte of the bamboo by RNA secondary structure. Nucleic Acids Research, 35: 3322– genus Gigantochloa (Neustupa et al., 2009), and 3329. Phyllosiphon arisari is a parasite of several species DARIENKO, T., GUSTAVS, L., MUDIMU, O., MENENDEZ, C.R., SCHUMANN, of Araceae (Aboal & Werner, 2011). Furthermore, R., KARSTEN, U., FRIEDL,T.&PRÖSCHOLD, T. (2010). Chloroidium, a common terrestrial coccoid green alga previously assigned to Heveochlorella hainangensis was isolated as an endo- Chlorella (Trebouxiophyceae, Chlorophyta). European Journal of phyte of rubber trees (Zhang et al., 2008). Our strains Phycology, 45:79–95. of Heveochlorella roystonensis, however, were iso- ELIÁŠ, M., NĚMCOVÁ,Y.,ŠKALOUD, P., NEUSTUPA, J., KAUFNEROVÁ,V. lated as epiphytes from the surface of the bark of & ŠEJNOHOVÁ, L. (2010). Hylodesmus singaporensis gen. et sp. nov., a new autosporic subaerial green alga (Scenedesmaceae, royal palms. Its growth seems to be promoted by air Chlorophyta) from Singapore. International Journal of pollution in the city and the abundance of green algae Systematic and Evolutionary Microbiology, 60: 1224–1235. on tree bark has been reported elsewhere to be posi- FOTT,B.&NOVÁKOVÁ, M. (1969). A monograph on the genus tively correlated with airborne nitrogen pollution Chlorella. The fresh water species. In: Studies in phycology (FOTT, B., editor), 10–74. Academia, Prague. (Poikolainen et al., 1998), as has lichen growth GOMBERT, S., ASTA,J.&SEAWARD, M.R.D. (2003). Correlation (Gombert et al., 2003). One of the differences between between the nitrogen concentration of two epiphytic lichens and the two Heveochlorella species is that the epiphytic H. the trafﬁc density in an urban area. Environmental Pollution, 123: roystonensis grows well autotrophically in media 281–290. HANAGATA, N., KARUBE,I.&CHIHARA, M. (1997). Bark-inhabiting without carbon supplements, while the growth of the green algae in Japan (3) Chlorella trebouxioides and Ch. angusto endophytic H. hainangensis is considerably enhanced ellipsoidea, sp. nov. (Chlorelloideae, Chlorellaceae, by supplying a carbon source to the medium, which Chlorococcales). Journal of Japanese Botany, 72:36–43. A novel Heveochlorella species 209

HARRIS, E.H. (1989). The Chlamydomonas sourcebook. Academic OLSEN, G.J., OVERBEEK, R., LARSEN, N., MARSH, T.L., MCCAUGHEY,M. Press, San Diego. J., MACIUKENAS, M.A., KUAN, W.-M., MACKE, T.J., XING,Y.& HUSS, V.A.R., SCHWARZWALDER,E.&KESSLER, E. (1987). WOESE, C.R. (1992). The ribosomal database project. Nucleic Deoxyribonucleic acid reassociation in the taxonomy of the Acids Research, 20: 2199–2200. genus Chlorella. II. Chlorella saccharophila. Archives of PARDY, R.L. (1976). The morphology of green hydra endosymbionts Microbiology, 147: 221–224. as influenced by host strain and host environment. Journal of Cell HUSS, V.A.R., FRANK, C., HARTMANN, E.C., HIRMER, M., KLOBOUCEK, Science, 20: 655–669. A., SEIDEL, B.M., WENZELER,P.&KESSLER, E. (1999). Biochemical POIKOLAINEN, J., LIPPO, H., HONGISTO, M., KUBIN, E., MIKKOLA,K.& taxonomy and molecular phylogeny of the genus Chlorella sensu LINDGREN, M. (1998). On the abundance of epiphytic green algae in lato (Chlorophyta). Journal of Phycology, 35: 587–598. relation to the nitrogen concentrations of biomonitors and nitrogen HUSS, V.A.R., CINIGLIA, C., CENNAMO, P., COZZOLINO, S., PINTO,G.& deposition in Finland. Environmental Pollution, 102:85–92. POLLIO, A. (2002). Phylogenetic relationships and taxonomic POSADA,D.&CRANDALL, K.A. (1998). Modeltest: testing the model position of Chlorella-like isolates from low pH environments of DNA substitution. Bioinformatics, 14: 817–818. (pH < 3.0). BMC Evolutionary Biology, 2: 13. REISSER, W. (1976). Die stoffwechselphysiologischen Beziehungen IKEDA,T.&TAKEDA, H. (1995). Species-specific differences of zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der pyrenoids in Chlorella (Chlorophyta). Journal of Phycology, 31: Paramecium bursaria-Symbiose II. Symbiose-spezifische 813–818. Merkmale der Stoffwechselphysiologie und der Cytologie des KALINA,T.&PUNČOCHÁŘOVÁ, M. (1987). Taxonomy of the subfam- Symbioseverbandes und ihre Regulation. Archives of ily Scotiellocystoideae Fott 1976 (Chlorellaceae, Chlorophyceae). Microbiology, 111: 161–170. Algological Studies, 45: 473–521. RINDI, F., LOPEZ-BAUTISTA, J.M., SHERWOOD, A.R. & GUIRY, M.D. KESSLER,E.&HUSS, V.A.R. (1992). Comparative physiology and (2006). Morphology and phylogenetic position of biochemistry and taxonomic assignment of the Chlorella Spongiochrysis hawaiiensis gen. et sp. nov., the first known ter- (Chlorophyceae) strains of the Culture Collection of the restrial member of the order Cladophorales (Ulvophyceae, University of Texas at Austin. Journal of Phycology, 28: 550–553. Chlorophyta). International Journal of Systematic and KODAMA, Y., NAKAHARA,M.&FUJISHIMA, M. (2007). Symbiotic alga Evolutionary Microbiology, 56: 913–922. Chlorella vulgaris of the ciliate Paramecium bursaria shows SCHULTZ, J., MAISEL, S., GERLACH, D., MÜLLER,T.&WOLF,M. temporary resistance to host lysosomal enzymes during the early (2005). A common core of secondary structure of the internal infection process. Protoplasma, 230:61–67. transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA, 11: KOVAČEVIĆ, G., KALAFATIĆ,M.&LJUBEŠIĆ, N. (2005). Endosymbiotic 361–364. alga from green hydra under the influence of cinoxacin. Folia SHIHIRA,I.&KRAUSS, R.W. (1965). Chlorella: physiology and Microbiologia (Praha), 50: 205–208. taxonomy of forty-one isolates. University of Maryland KOVAČEVIĆ,G.,KALAFATIĆ,M.&LJUBEŠIĆ, N. (2007). New observations (College Park), Maryland. on green hydra symbiosis. Folia Biologica (Krakow), 55:77–79. SPURR, A.R. (1969). A low-viscosity epoxy resin embedding med- KRIENITZ, L., HEGEWALD, E.H., HEPPERLE, D., HUSS, V.A.R., ROHR,T. ium for electron microscopy. Journal of Ultrastructure Research, &WOLF, M. (2004). Phylogenetic relationship of Chlorella and 26:31–43. Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae). STAMATAKIS, A., HOOVER,P.&ROUGEMONT, J. (2008). A rapid boot- Phycologia, 43: 529–542. strap algorithm for the RAxML Web Servers. Systematic Biology, LA ROCCA, N., ANDREOLI, C., GIACOMETTI, G., RASCIO,N.&MORO,I. 57: 758–771. (2009). Responses of the Antarctic microalga Koliella antarctica SWOFFORD, D.L. (2002). PAUP*. Phylogenetic analysis using parsi- (Trebouxiophyceae, Chlorophyta) to cadmium contamination. mony (*and other methods). Version 4.0b 10. Sinauer Associates, Photosynthetica, 47: 471–479. Sunderland, MA. LEE, J.J., REIDY,J.&KESSLER, E. (1982). Symbiotic Chlorella spe- TRÉMOUILLAUX-GUILLER,J.&HUSS, V.A.R. (2007). A cryptic intra- cies from larger Foraminifera. Botanica Marina, 25: 171–176. cellular green alga in Ginkgo biloba: ribosomal DNA markers MAI, J.C. & COLEMAN, A.W. (1997). The internal transcribed spacer reveal worldwide distribution. Planta, 226: 553–557. 2 exhibits a common secondary structure in green algae TRÉMOUILLAUX-GUILLER, J., ROHR, T., ROHR,R.&HUSS, V.A.R. and flowering plants. Journal of Molecular Evolution, 44: (2002). Discovery of an endophytic alga in Ginkgo biloba. 258–271. American Journal of Botany, 89: 727–733. Downloaded by [Dr Volker Huss] at 16:16 30 May 2013 NEUSTUPA, J., NĚMCOVÁ, Y., ELIÁŠ,M.&ŠKALOUD, P. (2009). WUYTS, J., VAN DE PEER,Y.&DE WACHTER, R. (2002). The European Kalinella bambusicola gen. et sp. nov. (Trebouxiophyceae, database on small subunit ribosomal RNA. Nucleic Acids Chlorophyta), a novel coccoid Chlorella-like subaerial alga from Research, 30: 183–185. Southeast Asia. Phycological Research, 57: 159–169. ZHANG, J., HUSS, V.A.R., SUN, X., CHANG,K.&PANG, D. (2008). NEUSTUPA, J., ELIÁŠ, M., ŠKALOUD, P., NĚMCOVÁ,Y.&ŠEJNOHOVÁ,L. Morphology and phylogenetic position of a trebouxiophycean (2011). Xylochloris irregularis gen. et sp. nov. green alga (Chlorophyta) growing on the rubber tree, Hevea bra- (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid siliensis, with the description of a new genus and species. green alga. Phycologia, 50:57–66. European Journal of Phycology, 43: 185–193.