Research Article

Algae 2013, 28(2): 185-192 http://dx.doi.org/10.4490/algae.2013.28.2.185 Open Access

Cold-tolerant strain of pluvialis (, ) from Blomstrandhalvøya (Svalbard)

Tatyana A. Klochkova1, Min Seok Kwak2, Jong Won Han2, Taizo Motomura3, Chikako Nagasato3 and Gwang Hoon Kim2,* 1Kamchatka State Technical University (KamchatGTU), Petropavlovsk-Kamchatsky, Klyuchevskaya 35, 683003, Russia 2Department of Biology, Kongju National University, Kongju 314-701, Korea 3Muroran Marine Station, Field Science Centre for Northern Biosphere, Hokkaido University, Muroran 051-0003, Japan

A new cold-adapted Arctic strain of from Blomstrandhalvøya Island (Svalbard) is described. This strain is predominantly always in non-motile palmelloid stage. Transmission electron microscopy showed the pres- ence of very thick cell wall and abundant lipid vesicles in the palmelloids, including red and green cells. The external morphology of the non-motile palmelloid and motile bi-flagellated cells of our strain is similar toH. pluvialis; however it differs from H. pluvialis in physiology. Our strain is adapted to live and produce astaxanthin in the low temperature (4- 10°C), whilst the usual growth temperature for H. pluvialis is between 20-27°C. Phylogenetic analysis based on 18S rRNA gene data showed that our strain nested within the Haematococcus group, forming a sister relationship to H. lacustris and H. pluvialis, which are considered synonymous. Therefore, we identified our Arctic strain asH . pluvialis.

Key Words: astaxanthin; Blomstrandhalvøya; cold resistance; Haematococcus pluvialis; Svalbard; transmission electron microscopy

INTRODUCTION

The genus Haematococcus (, Volvo- induced stress conditions, such as nutrient deprivation, cales) is cosmopolitan, reported from all continents ex- increased salinity, high irradiance, and exposure to high cept Antarctica (Guiry and Guiry 2013). Seven species of temperature (30-33°C), as well as combinations of these Haematococcus have been currently accepted taxonomi- stresses (summarized in Collins et al. 2011). In natural cally and among them, H. pluvialis Flotow is the best environment, accumulation of the astaxanthin is an ad- studied. This ubiquitous microalga occurs primarily in aptation to habitats that exhibit strong radiation, in addi- principally ephemeral small fresh water pools in temper- tion to the formation of cysts having rigid cell walls (e.g., ate climate, where it passes through the life cycle in as few Hagen et al. 1994, 2002, Montsant et al. 2001). as 4 days (e.g., Droop 1954, Czygan 1970, Thompson and Astaxanthin is generally known to be used as a food Wujek 1989). It is well known for the ability to synthesize coloring agent, natural feed additive for the poultry in- and accumulate esters of the red ketocarotenoid astax- dustry and for aquaculture, especially as a feed supple- anthin (3,39-dihydroxy-b,b-carotene-4,49-dione) for up ment in the culture of salmon, trout and shrimp, and for to 4% of the total cellular dry weight under laboratory- medicinal and cosmetic application due to its powerful

This is an Open Access article distributed under the terms of the Received April 15, 2013, Accepted May 18, 2013 Creative Commons Attribution Non-Commercial License (http://cre- Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted * non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] provided the original work is properly cited. Tel: +82-41-850-8504, Fax: +82-41-850-8479

Copyright © The Korean Society of Phycology 185 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860 Algae 2013, 28(2): 185-192

antioxidant capacity. Most of the astaxanthin used for with an argon-krypton laser using a 488-nm excitation aquaculture is synthetically derived; however, growing line and AOBS filter-free system collecting emitted light demand exists for commercial production of astaxan- between 498 and 700 nm. A series of optical sections of thin from natural sources. Therefore, numerous studies chloroplast was captured and used for 3D reconstruction regarding Haematococcus focused on the astaxanthin- of morphology. The autofluorescence of the chlorophyll producing properties of H. pluvialis, using strains mainly was exploited for visualization of chloroplast structure. from culture collections (i.e., The Culture Collection of Al- gae at the University of Texas at Austin [UTEX], National Electron microscopic observations Institute for Environmental Studies, Japan [NIES], Cul- ture Collection of Algae and Protozoa [CCAP], The Global Three fixation methods were applied to fix cells in Bioresource Center [ATCC], Culture Collection of Algae vegetative (green) and aplanospore (red) stages, includ- at Goettingen [SAG], Algae Culture Collection at Norsk ing chemical fixation, cryofixation, and high-pressure Institutt for Vannforskning [NIVA]) and grown under a freezing. The chemical fixation and embedding methods wide range of culture conditions (González et al. 2009). were performed as described by Klochkova et al. (2006), The usual growth temperature for H. pluvialis is between and the cryofixation and embedding methods were per- 20-27°C. formed as described by Terauchi et al. (2012). For high We recently found a new cold-adapted Arctic strain pressure freezing and freeze substitution, a pellet of of Haematococcus on Blomstrandhalvøya Island (Sval- cells was placed between two copper planchets and rap- bard) (Kim et al. 2011). Our strain, tentatively named as idly frozen in a high-pressure freezing instrument (EM Haematococcus sp. (Kim et al. 2011), exhibits growth be- PACT2; Leica, Solms, Germany) at a pressure of approxi- tween 4-15°C, whereas the average summer temperature mately 2,000 atm and at the temperature of liquid nitro- of Svalbard reaches 4-6°C, and January averages at -12°C gen. The samples were then placed in liquid nitrogen, and to -16°C. The purpose of the present study was to provide the top of the copper tube was peeled away for further more morphological and physiological details for this pe- processing. The following cryofixation and embedding culiar Arctic strain of Haematococcus and to address its methods were performed as described by Terauchi et al. phylogenetic relationships to other species in the genus. (2012). Resin-embedded samples were sectioned with a diamond knife, and thin sections stained with 4% uranyl acetate for 30 min and lead citrate (Reynolds 1963) for 10 MATERIALS AND METHODS min were viewed and photographed on a Hitachi H-300 transmission electron microscope (TEM) (Hitachi, Tokyo, Sample collection and culture Japan).

Specimen of Haematococcus pluvialis was collected on Molecular phylogenetic analysis Blomstrandhalvøya Island (78°59′ N, 12°03′ E) in a small freshwater basin in the rock fed with melted snow by the The DNA of H. pluvialis was extracted using an Invi- authors G. H. K., T. A. K., and J. W. H. (Kim et al. 2011). sorb Spin Plant Mini Kit (Invitek, Berlin-Buch, Germany) The unialgal strain was isolated as described by Kim et according to the manufacturer’s protocol. Extracted DNA al. (2011) and grown in a modified liquid ATCC Medium was stored at -20°C and used for the amplification of 18S 625 (Klochkova et al. 2006), Bold’s basal medium (Bischoff rRNA. The 18S rRNA was amplified and sequenced using and Bold 1963), and conventional BG11 medium in 90 × the following primers: G01 (5′-CACCTGGTTGATCCTGC- 60-mm plastic Petri dishes and 150-mL glass flasks at CAG-3′), G14 (5′-CCTTGGCAGACGCTTTCGCAG-3′), G04 4-15°C, 30-35 µmol photons m-2 s-1 provided by cool- (5′-CAGAGGTGAAATTCTTGGAT-3′), and G07 (5′-GCTT- white fluorescent lighting and 12 : 12 h light : dark regime. GATCCTTCTGCAGGTTCACCTAC-3′) (Saunders et al. Micrographs were taken with Olympus DP50 digital cam- 1995). Polymerase chain reaction and sequencing were era (Olympus, Tokyo, Japan) affixed to an Olympus BX50 performed as detailed in Klochkova et al. (2006, 2008). All microscope using Viewfinder Lite and Studio Lite com- sequences of the forward and reverse strands were deter- puter programs. mined and the electropherograms were edited using the For investigation of chloroplast morphology, cells were program Chromas v.1.45 (McCarthy 1998). Nucleotide observed under an Olympus Fluoview laser scanning sequences were aligned using Se-Al v2.0a11 (Rambaut confocal microscope. The microscope was equipped 2002).

http://dx.doi.org/10.4490/algae.2013.28.2.185 186 Klochkova et al. Haematococcus pluvialis from Svalbard

Pleurastruminsigne Z28972 100 87 Characium vacuolatum M63001

94 Protosiphon botryoides U41177

100 Chlorococcum ellipsoideum U70586

100 Haematococcus zimbabwiensis U70797 sp. AB360751 98 Haematococcus lacustris AB360747 72 Haematococcuspluvialis (Svalbard) KC986379 78 14 100 Haematococcus sp. FJ877140

Haematococcus pluvialis AF159369

100 Dunaliella parva M6299 98 Dunaliella salina DQ009779

18S rRNA 98 Chloromonas subdivisa AF517080 100 Chlamydomonas monadina AY220559

88 Chlamydomonas pulsatilla AF514404

Pteromonasprotracta X91627 99 Phacotus lenticularis X91628

Carteria radiosa AF182819

Scenedesmus obliquus EF564131 0.01 Fig. 1. Maximum likelihood tree estimated from 18S rRNA sequence data. Numbers above the branches indicate bootstrap values.

Phylogeny of 18S rRNA was reconstructed using maxi- (AB360747; isolate NIES-144, collected from Sapporo, mum likelihood (ML). ML analyses were performed with Hokkaido) and Haematococcus sp. (FJ877140; origin un- RAxML v.7.2.8 (Stamatakis 2006) using the GTRGAMMA known). The clade included H. pluvialis (AF159369; iso- model. We used 300 independent tree inferences, apply- late SAG 34-1b, collected from former Czechoslovakia), ing options of automatically optimized surface plasmon although it had less affinity to our strain. SpeciesH. lacus- resonance (SPR) rearrangement and 25 distinct rate cat- tris has been synonymized with H. pluvialis. If they are egories in the program to identify the best tree. Statistical conspecific, our strain positioning between them should support for each branch was obtained by 1,000 bootstrap also be attributed to H. pluvialis until more information replications with the same substitution model. The se- is available on various strains of the H. pluvialis-lacustris quence determined in this study has been deposited in complex. GenBank under accession number KC986379. Morphology

RESULTS Our strain is predominantly always in non-motile palmelloid stage. Both green- and red-colored spheri- Molecular phylogeny cal non-motile palmelloid cells (Fig. 2A, B & G) are 19.2- 44.8 µm in diameter (28.2 ± 1.9 µm on average) and have Our strain nested within the Haematococcus group a thick cell wall (secondary wall) reaching up to 930 nm (Fig. 1), forming a sister relationship to H. lacustris thick (Fig. 3A). Some cells are ellipsoidal. The centrally

187 http://e-algae.kr Algae 2013, 28(2): 185-192

A B C D

E F G

Fig. 2. Light microscopy of Haematococcus pluvialis cells. (A & B) Through-focus images of the same green palmelloid cell cultured at 15°C, showing chloroplast with several pyrenoids occupying almost the whole cell’s periphery (arrows). (C & D) Morphology of large parietal chloroplast seen with confocal microscopy. (C) Reconstruction of the chloroplast shape by overlaying serial sections. (D) Section taken through the cell’s center, showing lack of chlorophyll autofluorescence in the center and its restricted localization to the cells’ periphery. (E) Motile bi-flagellated cell in 20°C. (F) Red pigmentation due to astaxanthin accumulation appears towards the center of palmelloid cells in 6°C (arrowheads). (G) Crimson- colored astaxanthin-containing palmelloid cell developed in 6°C. Scale bars represent: A-G, 10 μm.

positioned nucleus was up to 9.5 µm in size, with a large F) of 200-1,000 nm in diameter. The cytoplasm was het- nucleolus (data not shown), and a small Golgi body posi- erogeneous and segregated (Fig. 3E); however this might tioned next to the nucleus was observed (Fig. 3G). Large have been an artifact of the fixation. chloroplast with numerous starch grains occupies the Motile bi-flagellated cells Fig( . 2E) were rarely seen whole cell’s periphery (Figs 2C, D, 3B & C). Chloroplast of in field-collected samples (4 bi-flagellated cells among young cells contains 2-4 pyrenoids and large old cells have several hundred non-motile astaxanthin-containing red up to 8-12 pyrenoids. No lamellae enter the pyrenoid and cells). Also, they are rarely seen in the laboratory culture. the starch sheath is composed of numerous grains (Fig. Motile spores have a cup-shaped chloroplast with 1-2 3B & D). The thylakoids are stacked in a rather random pyrenoids and one large vacuole positioned in the cell manner (Fig. 3B-D). Numerous small mitochondria were center or several smaller scattered vacuoles. Cytoplasmic mostly located beneath the cell membrane (Fig. 3A) and threads extending from the protoplast to the wall are ex- some mitochondria were seen in the cytoplasm (Fig. 3C). tremely fine. The cells retain motility for 1 day after re- Lipid vesicles packed almost the whole cytoplasmic lease, and thereafter they settle on the substrate and be- volume in the cells. Massive lipid accumulation occurred gin to develop palmelloid morphology. regardless of the pigment accumulation, since green- colored palmelloids (Fig. 2A & B) also contained numer- Growth temperature ous lipid vesicles (Fig. 3C). In the green palmelloids lipid vesicles were less electron-dense and 200-400 nm in di- When this strain was first brought from the field, it ameter, whereas red palmelloids (i.e., astaxanthin-con- could live only at 4-6°C; however >80% of the cells were taining) had more electron-dense lipid vesicles (Fig. 3E & always red-colored. The cells show slow growth at 4-6°C.

http://dx.doi.org/10.4490/algae.2013.28.2.185 188 Klochkova et al. Haematococcus pluvialis from Svalbard

A B

C D

E F G

Fig. 3. Electron microscopy of Haematococcus pluvialis palmelloid cells. (A) Thick cell wall of the palmelloid cell, showing trilaminar sheath (tls, arrow) and smooth secondary wall (sw). Arrowheads point to mitochondria located beneath the cell membrane. (B) Arrangement of the thylakoids in the chloroplast. (C) Numerous lipid vesicles (asterisks) scattered in the cytoplasm of green palmelloid cell. (D) Pyrenoid within a starch-containing chloroplast. (E) Electron-dense astaxanthin-containing lipid vesicles (asterisks) scattered in the heterogeneous segregated cytoplasm (arrowheads) of the red palmelloids. (F) Enlarged lipid vesicle. Arrows point to a membrane surrounding the vesicle. (G) Vertical section through the Golgi body (g). Arrows point to the adjacent Golgi vesicles. ecm, extracellular matrix; is, interspace; m, mitochondrion; p, pyrenoid; s, starch; t, thylakoids. Scale bars represent: A, C & E-G, 200 nm; B & D, 500 nm.

189 http://e-algae.kr Algae 2013, 28(2): 185-192

After a period of 860 days in culture at this temperature, backs in its vegetative culture to increase the growth rate we counted only 230,000-866,000 cells in a total volume and culture productivity. of 20 mL of BG11 medium, which were progeny from 1 Knowledge of the morphology and life cycle of H. cell. pluvialis becomes increasingly important due to the in- Over the subsequent period of laboratory culture for terest in this organism as a biotechnological source of 1.5 years, this isolate was gradually acclimated to grow at astaxanthin. However, studies on its morphogenesis and 15°C. When cultured at 15°C under 30-35 µmol photons ultrastructure proceed slowly in comparison to other m-2 s-1, palmelloids are mostly green-colored. This strain microalgae. The palmelloid cells have so thick cell wall reproduces efficiently by means of motile flagellated that chemical fixation and embedding prior to ultrathin spores after 1-2 days-long exposure to 20°C; however it sectioning and electron microscopy becomes almost im- is not able to live at this temperature for more than 2-3 possible (Hagen et al. 2002). The cells of our strain were days. Astaxanthin is accumulated by stressing cells grown notoriously difficult to fix for TEM using chemical fixa- at 15°C under 30-35 mol photons m-2 s-1 with low tem- tion method and cryofixation. These treatments resulted perature between 4-10°C under 30-35 µmol photons m-2 in turgor breakage and complete disintegration of the s-1. Under cold stress, efficient astaxanthin production cellular contents, and formation of massive ice crystals occurs even in the low light (approximately 1.7-9 µmol rupturing the organelles, respectively. Only high-pressure photons m-2 s-1). Red globular regions appear towards the freezing with the following cryofixation was partially suc- center of palmelloid cells (Fig. 2F), and after several days cessful to preserve some cell structures more or less in- of the same treatment cells appear completely red (Fig. tact. Our TEM results were conclusive with Hagen et al. 2G). (2002), showing presence of thick and smooth secondary wall, which is believed to be mainly composed of granu- lose non-fibrillar mannan I, a trilaminar sheath, and a DISCUSSION layer of extracellular matrix in the palmelloid cells. The cytoplasm of the green palmelloids was packed with lipid The cold-tolerant strain of Haematococcus pluvialis vesicles, implying that abundant lipid accumulation oc- discussed in the present study was collected only from curs prior to the accumulation of the astaxanthin inside one site on Blomstrandhalvøya Island, which was a small the vesicles. rock pool located at a distance of several meters from Phylogenetic relationships within Haematococcus have the sea, but we did not find it in other surveyed areas been poorly resolved and only four members of the genus on this island and in the vicinity of Ny-Ǻlesund (Kim et have been sequenced to date, including H. droebakensis al. 2008, 2011). Droop (1961) also noted that H. pluvialis Wollenweber, H. lacustris (Girod-Chantrans) Roststafin- typically inhabited rock pools, often, although not nec- ski, H. pluvialis, and H. zimbabwiensis Pockock, result- essarily, within a few feet of the sea. We previously sug- ing in 1, 11, 211, and 6 nucleotide sequences of different gested anthropogenic introduction of some microalgae products for each species deposited in the GenBank, to this Arctic area (Kim et al. 2011). It is noteworthy that respectively (National Center for Biotechnology Infor- no Haematococcus microalgae were recorded from this mation 2013). Also, nucleotide sequences of different region before and in general H. pluvialis inhabits temper- products for 3 unidentifiedHaematococcus sp. have been ate climate. When defining area of species distribution deposited in the GenBank (National Center for Biotech- one should consider that it is limited by the temperature nology Information 2013). Although H. pluvialis is the for maximum and / or optimum reproduction and not by most often used for sequencing, no comprehensive study the temperatures for survival. Our strain of H. pluvialis was undertaken to resolve relationships among different from Blomstrandhalvøya reproduces efficiently at 15- strains registered in the database. The name H. pluvialis 20°C, while the average summer temperature of Svalbard might have been commonly attached to any red-colored reaches 4-6°C. If introduction supposedly took place, it palmelloid Haematococcus, while different similar look- is not a recent event and should have occurred at least ing isolates might potentially be numerous new cryptic a number of years ago, since the cells had to go through species. For instance, species H. lacustris is also synony- several generations of their life cycle to adapt to the harsh mized with H. pluvialis (e.g., Hagen et al. 2002, Pentecost Arctic environment. The unfavorable environment might 2002), however they have a principal morphological dif- have influenced the specifically low growth rate of this ference such as H. lacustris is predominantly always in strain. Further work is required to overcome the draw- motile flagellated stage (Pocock 1960), whereasH. plu-

http://dx.doi.org/10.4490/algae.2013.28.2.185 190 Klochkova et al. Haematococcus pluvialis from Svalbard

vialis is predominantly always in non-motile palmelloid Droop, M. R. 1961. Haematococcus pluvialis and its allies. III: stage. Until more comprehensive phylogenetic analysis Organic nutrition. Rev. Algol. N. S. 5:247-259. on different strains of H. pluvialis from around the world González, M. A., Cifuentes, A. S. & Gómez, P. I. 2009. Growth is complete, we suggest attributing our strain to this spe- and total carotenoid content in four Chilean strains of cies based on current phylogenetic result. Morphologi- Haematococcus pluvialis Flotow, under laboratory con- cally, it is not very different from H. pluvialis to support its ditions. Gayana Bot. 66:58-70. separation as a distinct species; however it significantly Guiry, M. D. & Guiry, G. M. 2013. AlgaeBase. World-wide differs in physiology. Unlike other described strains of H. electronic publication, National University of Ireland, pluvialis, our strain is adapted to live and produce astax- Galway. Available from: http://www.algaebase.org. Ac- anthin in the low temperature (4-10°C). Most commonly cessed Mar 27, 2013. cultured species of algae are able to grow at temperatures Hagen, C., Braune, W. & Björn, L. O. 1994. Functional as- between 16 and 27°C and the optimal growth tempera- pects of secondary carotenoids in Haematococcus la- tures are usually in the range of 18-20°C (Chen et al. 2009). custris (Volvocales). III. Action as a “sunshade”. J. Phycol. As evident from the numerous references on H. pluvialis, 30:241-248. its usual growth temperature for commercial cultivation Hagen, C., Siegmund, S. & Braune, W. 2002. Ultrastructural is between 20-27°C. Our psychrophilic strain is therefore and chemical changes in the cell wall of Haematococcus an exception and can potentially be used for astaxanthin pluvialis (Volvocales, Chlorophyta) during aplanospore exploitation in colder climates. formation. Eur. J. Phycol. 37:217-226. Kim, G. H., Klochkova, T. A., Han, J. W., Kang, S. -H., Choi, H. G., Chung, K. W. & Kim, S. J. 2011. Freshwater and ter- ACKNOWLEDGEMENTS restrial algae from Ny-Ålesund and Blomstrandhalvøya Island (Svalbard). Arctic 64:25-31. We express our sincere thanks to Dr. G. C. Zuccarello Kim, G. H., Klochkova, T. A. & Kang, S. -H. 2008. Notes on for his help with the phylogenetic analysis of Haemato- freshwater and terrestrial algae from Ny-Ålesund, Sval- coccus. This research was a part of the project entitled bard (high Arctic sea area). J. Environ. Biol. 29:485-491. ‘Long-term change of structure and function in marine Klochkova, T. A., Cho, G. -Y., Boo, S. M., Chung, K. W., Kim, S. ecosystems of Korea’ funded by the Ministry of Oceans J. & Kim, G. H. 2008. Interactions between marine facul- and Fisheries, Korea. tative epiphyte Chlamydomonas sp. (Chlamydomonad- ales, Chlorophyta) and ceramiaceaen algae (Rhodophy- ta). J. Environ. Biol. 29:427-435. REFERENCES Klochkova, T. A., Kang, S. -H., Cho, G. Y., Pueschel, C. M., West, J. A. & Kim, G. H. 2006. Biology of a terrestrial Bischoff, H. W. & Bold, H. C. 1963. Phycological studies. IV. green alga, Chlorococcum sp. (Chlorococcales, Chlo- Some soil algae from enchanted rock and related algal rophyta), collected from the Miruksazi stupa in Korea. species. Univ. Texas Publ. No. 6318:1-95. Phycologia 45:349-358. Chen, P., Min, M., Chen, Y., Wang, L., Li, Y., Chen, Q., Wang, C., McCarthy, C. 1996-1998. Chromas. Version 1.45 (32-bits). Wan, Y., Wang, X., Cheng, Y., Deng, S., Hennessy, K., Lin, Nathan, Queensland: School of Health Science, Griffith X., Liu, Y., Wang, Y., Martinez, B. & Ruan, R. 2009. Review University. Available from: http://technelysium.com. of the biological and engineering aspects of algae to fu- au/chromas.html. Accessed Dec 7, 2012. els approach. Int. J. Agric. Biol. Eng. 2:1-30. Montsant, A., Zarka, A. & Boussiba, S. 2001. Presence of a Collins, A. M., Jones, H. D. T., Han, D., Hu, Q., Beechem, T. nonhydrolyzable biopolymer in cell wall of vegetative E. & Timlin, J. A. 2011. Carotenoid distribution in living cells and astaxanthin-rich cysts of Haematococcus plu- cells of Haematococcus pluvialis (Chlorophyceae). PLoS vialis (Chlorophyceae). Mar. Biotechnol. 3:515-521. ONE 6:e24302. National Center for Biotechnology Information. 2013. Gen- Czygan, F. C. 1970. Blood-rain and blood-snow: nitrogen- Bank. Available from: http://www.ncbi.nlm.nih.gov. Ac- deficient cells ofHaematococcus pluvialis and Chlam- cessed Mar 27, 2013. ydomonas nivalis. Arch. Mikrobiol. 74:69-76. Pentecost, A. 2002. Order Volvocales. In John, D. M., Whitton, Droop, M. R. 1954. Conditions governing haematochrome B. A. & Brook, A. J. (Eds.) The Freshwater Algal Flora of formation and loss in the alga Haematococcus pluvialis the British Isles: An Identification Guide to Freshwater Flotow. Arch. Mikrobiol. 20:391-397. and Terrestrial Algae. Cambridge University Press, Cam-

191 http://e-algae.kr Algae 2013, 28(2): 185-192

bridge, pp. 303-327. Acad. Sci. U. S. A. 92:244-248. Pocock, M. A. 1960. Haematococcus in southern Africa. Trans. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood- R. Soc. S. Afr. 36:5-55. based phylogenetic analyses with thousands of taxa and Rambaut, A. 2002. Se-Al: sequence alignment editor, version mixed models. Bioinformatics 22:2688-2690. 2.0a11. Available from: http://tree.bio.ed.ac.uk/soft- Terauchi, M., Nagasato, C., Kajimura, N., Mineyuki, Y., Oku- ware/seal. Accessed Mar 27, 2013. da, K., Katsaros, C. & Motomura, T. 2012. Ultrastructural Reynolds, E. S. 1963. The use of lead citrate at high pH as an study of plasmodesmata in the brown alga Dictyota di- electron-opaque stain in electron microscopy. J. Cell chotoma (Dictyotales, Phaeophyceae). Planta 236:1013- Biol. 17:208-212. 1026. Saunders, G. W., Potter, D., Paskind, M. P. & Andersen, R. A. Thompson, R. H. & Wujek, D. E. 1989. Haematococcus caro- 1995. Cladistic analyses of combined traditional and cellus sp. nov. (Haematococcaceae, Chlorophyta) from molecular data sets reveal an algal lineage. Proc. Natl. the United States. Phycologia 28:268-270.

http://dx.doi.org/10.4490/algae.2013.28.2.185 192