ISSN 1226-9999 (print) ISSN 2287-7851 (online)

Korean J. Environ. Biol. 35(3) : 319~328 (2017) https://doi.org/10.11626/KJEB.2017.35.3.319

Palmophyllum crassum, a New Record of an Ancient in Green from Korea

Hyung Woo Lee and Myung Sook Kim*

Department of , Jeju National University, Jeju 63243, Republic of Korea

Abstract - The continuous exploration in deep seawater from Korea makes us lead the discovery of ancient , Palmophyllum, in the Korean coast. The phylogenetic analyses of 18S

rRNA and rbcL genes demonstrate that our specimens are Palmophyllum crassum (Naccari) Rabenhorst, recorded in Japan and clearly distinguished from P. umbracola from New Zealand

and California, USA. Palmophyllum crassum grows in the subtidal region, 8-30 m deep, and has a crustose thallus which is closely adherent to substrates such as non-geniculate crustose coralline algae, sponge, shells, or rocks. P. crassum is composed of numerous spherical cells embedded in the gelatinous matrix. The discovery of this ancient green seaweed implies that the Korean coast is one of the hotspots of algal species diversity and has the suitable marine environment for algal speciation. We suggest the grounds to conserve the Korean coast environmentally as the biodiversity center of marine species by studying the phylogeny of seaweeds.

Key words : 18S rRNA, molecular phylogeny, , Palmophyllum crassum, rbcL

INTRODUCTION that the earliest-diverging Chlorophyta comprises marine with simple morphology by revealing a deep- The green algae distributed in freshwater and seawater, branching clade which is a macroscopic algal group named even terrestrial habitats, are photosynthetic char- as the order including Palmophyllum, Ver- acterized by the presence of chloroplast with two envelope digellas and Palmoclathrus, based on the molecular phylo- membranes, stacked thylakoids, and chlorophyll a and b genetic study. with different morphological organizations such as mona- The palmelloid green algae, Palmophyllum, Palmoclathrus doid, palmelloid, coccoid, coenocytic, and filamentous and Verdigellas, thrive in deep seawater up to >100 m deep, (Pröschold and Leliaert 2007). This green lineage has been where has dimly light marine habitat (Womersley 1971; an essential member in the global ecosystem for a long time Nelson and Ryan 1986; Ballantine and Aponte 1996). These as the ancestor of land evolved by the early diver- seaweeds are characterized by a unique multicellularity, gence of and Chlorophyta, from an ancestral forming firm and well-defined macroscopic thalli, referred green flagellate unicell (Leliaert et al. 2012). Therefore, it to as palmelloid thallus organization (Zechman et al. 2010). was generally recognized that the ancestral Chlorophyta is Despite the lack of evidence of the systematic position unicellular with the presence of flagella and organic body within the green algae, these genera had been classified into scales, but this concept has been a matter of debate (Leliaert the family Palmellaceae of the chlorophycean order Volvo- et al. 2016). Recently, Zechman et al. (2010) challenged cales (Fritsch 1935), the family Palmellaceae of the order (Womersley 1984), or the family Chlorophy-

* ‌Corresponding author: Myung Sook Kim, Tel. 064-754-3523, ceae of the order (Nelson and Ryan 1984). Fax. 064-756-3541, E-mail. [email protected] Recently, since the molecular phylogenetic assessment of

ⓒ2017. Korean Society of Environmental Biology. 320 Hyung Woo Lee and Myung Sook Kim

Table 1. ‌Collection information and GenBank accession number of Palmophyllum crassum (Naccari) Rubenhorst specimens analyzed in this study GenBank accession No. Taxa Collection information Habit (depth/substratum) Voucher 18S rRNA rbcL Gwideok 1-ri, Jeju, Korea; 25-30 m deep / reef with MSK-GA00830 - MF769365 31 May 2011 crustose coralline algae Chujado, Jeju, Korea; 13-15 m deep / reef with MSK-GA00915 MF769362 MF769367 1 Oct. 2011 crustose coralline algae Udo, Jeju, Korea; 18-25 m deep / reef with MSK-GA00912 - MF769366 1 Nov. 2011 crustose coralline algae Udo, Jeju, Korea; 18-25 m deep / reef with MSK131101-10 MF769361 MF769368 Palmophyllum crassum 1 Nov. 2011 crustose coralline algae (Naccari) Rubenhorst Udo, Jeju, Korea; 18-25 m deep / reef with MSK131101-11 - MF769369 1 Nov. 2011 crustose coralline algae Hyeongjeseom, Busan, Korea; 10-15 m deep / reef with MSK160728-02 MF769363 - 28 Jul. 2016 sponge Shimoda, Shizuoka, Japan; 8-15 m deep / reef with MSK170417-01 MF769364 - 17 Apr. 2017 crustose coralline algae Shimoda, Shizuoka, Japan; 8-15 m deep / reef with MSK170417-03 - MF769370 17 Apr. 2017 sponge the -encoded atpB and rbcL genes and the nuclear- analyses. In addition, to compare with the entities from encoded 18S rRNA gene resolved that these palmelloid Korea, we carried out a collection in Japan. The aims of this genera are positioned to the early diverged lineage in Chlo- study are to identify new green algal entities collected from rophyta forming monophyly in order level, the order Palmo- deep subtidal and to resolve the species diversity of Korean phyllales and the family Palmophyllaceae were established green seaweed by reconstructing the green algal phylogeny (Zeckman et al. 2010). Further, this early evolutionary lin- with recognition of ancient lineage. eage was elevated to the class Palmophyllophyceae, based on the chloroplast phylogenomic analyses (Leliaert et al. 2016). MATERIALS AND METHODS The genus Palmophyllum is reported only two species, P. crassum (Naccari) Rabenhorst and P. umbracola Nelson et The samples were collected using SCUBA (from depths Ryan, all over the world (Guiry and Guiry 2017). P. crassum of 8-30 m) in the southern part of Korea including Jeju has been known as the cosmopolitan species, unlike P. Island and the central coast of Japan (Table 1). Underwater umbracola, which is mainly restricted in the Southern images were obtained using a digital camera (Stylus TG-4, hemisphere (Nelson and Ryan 1984). Although it has been Olympus, Japan). Samples were transported to the labora- recorded in Japan (Yoshida et al. 1990), several investiga- tory and photographed by Canon Powershot G7X camera tions of Korean algal flora have not recognized P. crassum (Canon, Japan). After describing the external morphology, distributed in the deep region (Kang et al. 2011; Kang and voucher specimens were made into pressed ones on the her- Kim 2012). barium sheets. Samples for DNA analyses were detached Recently, the molecular methods have been applied to from the thallus and dried via silica-gel. Samples for ana- green seaweeds from Korean coasts to conduct taxonomic tomical investigations were preserved in 5.0% formalin in reexamination or to describe species diversity (Lee and Kim seawater. Sections were done by a bench-top freezing micro­ 2011, 2015). By continuous exploration of deep subtidal tome (NK-101-II; Nippon Optical Works Co., Ltd., Tokyo, regions from Korean coastlines, we obtained crustose and Japan). Photomicrographs were obtained using a BX43 firmly gelatinous green algal specimens unrecognized pre- microscope (Olympus, Tokyo, Japan), with an EOS 600D viously and undertook the molecular and morphological digital camera (Canon, Japan). The digitized images were Palmophyllum crassum from Korea 321

imported into Adobe Photoshop ver. 6.1 software (Adobe RESULTS Systems Inc., San Jose, CA, USA). Voucher specimens were deposited in the herbarium of Jeju National University We analyzed a set of 41 SSU 18S rRNA gene sequences (JNUB), Korea and the National Institute of Biological Re- including the four Palmophyllum crassum (MF769361- sources (NIBR), Incheon, Korea. MF769364) collected from Korea and Japan (Table 1), the DNA was extracted using the DNeasy Mini Kit representative taxa in charophytes, prasinophytes, and core (Qiagen, Hilden, Germany) following the manufacturer’s chlorophytes with Palmophyllophyceae, Palmophyllum instructions. Following DNA extraction, the polymerase umbracola (FJ619275 and FJ619276) and Verdigella peltata chain reaction (PCR) were conducted to amplify the nuclear- (FJ619277 and LT174528). The SSU 18S rRNA data set encoded small subunit (SSU) 18S rRNA gene and the consisted of 1897 characters. Among all sites, 758 (56.1%) ribulose-1, 5-biphospate carboxylase/oxygenage (rbcL). were variable and 506 (26.7%) were parsimoniously infor- Amplification was performed in a 20-μL reaction, using mative. The constructed by ML and BI AccuPower PCR PreMix (Bioneer, Daejeon, Korea) and was congruent, and we exhibited the ML topology including Swift MaxPro thermal cyclers (ESCO, Singapore). The pri­ the bootstrap (BS) value in ML and posterior probability (PP) mer and PCR protocols for SSU 18S rRNA were followed in BI (Fig. 1). --Chlorophy- by Saunders and Moore (2013), and those for rbcL were ceae (UTC) and Streptophyta formed each distinct clade in followed by Heesch et al. (2009). PCR products were pu- the SSU phylogenetic tree, but prasinophytes were polyphy- rified using the AccuPrep Purification Kit (Bioneer) and letic. Our four P. crassum specimens collected from Korea sequenced commercially at Macrogen (Seoul, Korea). and Japan were identical, and they formed an independent Sequence edition and alignment were carried out using clade with Palmophyllum umbracola from USA (FJ619275) Chromas version 1.45 software (Queensland, Australia) and and New Zealand (FJ619276), and Verdigellas peltata from BioEdit software, respectively. Guadeloupe (FJ619277) and USA (LT174528) with strong The uncorrected pair-wise distances were estimated using supports of 100% in BS and 1.0 PP (Fig. 1). Palmophyllum MEGA 5.0 software to assess the genetic variations in both crassum from Korean and Japanese collections showed SSU and rbcL sequences. The program Modeltest version 0.3% genetic divergences with P. umbracola and V. peltata, 3.7 (Posada and Crandall 1998) was used to find the model respectively. P. umbracola from New Zealand (FJ619276), of sequence that best fit the data set by an Akaike the type locality, had 0.6% genetic divergences with P. Information Criterion (AIC). To construct both the SSU and umbracola from California, USA (FJ619275). The Palmo- rbcL phylogenetic tree, maximum likelihood (ML) analyses phyllophyceae clade was separated with other green algal were performed using RAxML software (Stamatakis 2006). taxa distinctly with strong supports (100% in BS, 1.00 in RAxML was performed with all three codons partitioned PP), exhibiting 13.7-26.3% pairwise distances with Strep- and the GTR+Γ +I model. To identify the best tree, 200 tophyta, 12.8-17.4% with prasinophytes, and 16.0-28.0% independent tree inferences using the -# option with default with UTC clade. Especially, this Palmophyllophyceae clade -I (automatically optimized subtree pruning-regrafting re- was located at an earlier diverged position than other green arrangement) and -c (25 distinct rate categories) software seaweed lineages belonged to Ulvophyceae clade, such as options were performed. To generate bootstrap support Capsosiphon fulvelscens (EU099920), Urospora penicilli- values for the phylogeny, 1,000 bootstrap replicates were formis (AB049417) and Ulva prolifera (EU099922), with performed. The Bayesian phylogenetic inferences (BI) were 25.4-26.8% genetic divergences (Fig. 1). generated using MrBayes ver. 3.1.2 software (Ronquist and To understand the phylogenetic relationships within the Huelsenbeck 2003), and obtained the posterior probabilities class Palmophyllophyceae, we obtained six rbcL sequences using a Markov Chain Monte Carlo approach with checking from Korean and Japanese specimens (MF769365-MF by Tracer v1.6 (Rambaut et al. 2014). The ML and BI trees 769370) and aligned a set of 37 rbcL sequences from the were edited with the program FigTree v1.4.0. representative taxa in Ulvophyceae, , Tre- bouxiophyceae and prasinophytes with two Streptophyta 322 Hyung Woo Lee and Myung Sook Kim

Fig. 1. ‌The phylogenetic position of Palmophyllum crassum (Naccari) Rabenhorst from Korea and Japan in the maximum-likelihood (ML) tree of Chlorophyta based on small subunit 18S rRNA sequences. The support values on each branch are from ML bootstrap (>50%, left) and Bayesian posterior probability (>0.50, right). Scale bar represents: substitution/site. species as outgroups ranging over 1272 sites in total. The (AP017927) supported in 99% BS and 1.0 PP, and showed variable position was 605 sites (47.6%) and the parsimony- 0.6% intraspecific divergence with a Japanese specimen informa­tive was 543 sites (42.7%). We conducted both ML (MF769370) from Shimoda, a Pacific coastal region of and BI analyses to construct the rbcL phylogenetic tree Japan. In the ML phylogenetic tree, P. crassum from Korea with BS and PP, and the topology was identical. We showed and Japan were distinguished from P. umbracola from New the rbcL phylogenetic tree derived from ML including BS Zeanad and California, USA, with 1.4-5.3% interspecific and PP supports (Fig. 2). In the rbcL sequence analysis, the divergence, exhibiting closer affinity with P. umbracola class Palmophyllophyceae was also monophyly and early- from New Zealand (EU586182) than one from California, branched in Chlorophyta with powerful supports of 100% USA (EU586180). BS and 1.0 PP. The genus Palmophyllum was clearly dis- tinguished from the genus Verdigellas with 4.0-5.9% inter­ Class Palmophyllophyceae Lelaiaert et al., 2016 generic divergence. The Palmophyllum crassum specimens (국명신칭: 초록방석말강) collected from Korea (MF769365-MF769369) were iden- Order Palmophyllales Zechman et al., 2010 tical with Japanese sequences from the Seto Inland Sea (국명신칭: 초록방석말목) Palmophyllum crassum from Korea 323

Fig. 2. ‌The phylogenetic relationships within the class Palmophyllophyceae in the maximum-likelihood (ML) tree of Chlorophyta based on the plastid encoded rbcL gene sequences. Support values on each branch are from ML bootstrap (>50%, left) and Bayesian posterior probability (>0.50, right). Scale bar represents: substitution/site.

Family Palmophyllaceae Zechman et al., 2010 mophyllum gestroi Piccone 1879; Palmophyllum orbiculare (국명신칭: 초록방석말과). Bornet ex Ardissone 1887; Palmophyllum crassum var. or- Genus Palmophyllum Kützing, 1847 biculare (Bornet ex Ardissone) Feldmann 1937; Palmophyl- (국명신칭: 초록방석말속) lum crassum var. typicum Feldmann 1937; Palmophyllum crassum f. gestroi (Piccone) Giaccone 1965. Palmophyllum crassum (Naccari) Rabenhorst 1868 (국명신칭: 초록방석말) Molecular vouchers: MF769361-MF769364 (SSU 18S Basionym: Palmella crassa Naccari 1828. rRNA); MF769365-MF769370 (rbcL). Synonyms: Palmophyllum flabellatum Kützing 1845; Pal- Specimens examined: MSK-GA00830 (25-30 m deep, 324 Hyung Woo Lee and Myung Sook Kim

Gwideok 1-ri, Jeju, Korea), MSK-GA00915 (13-15 m deep, cells by Kützing (1847). Rabenhorst (1868) transferred from Chujado, Jeju, Korea), MSK131101-09 & 11 (18-25 m Palmella crassa Naccari (1828) to Palmophyllum crassum, deep, Udo, Jeju, Korea), MSK140722-18 & 19 (13-15 m and described that P. crassum (Naccari) Rabenhorst is con- deep, Seopseom, Jeju, Korea), MSK2014-v175 & 176 (15- specific with P. flabellatum Kützing. Currently, P. crassum 20 m deep, Udo, Jeju, Korea), MSK2014-v0216 (28-30 has been accepted as the generic type of Palmophyllum m deep, Dodu, Jeju, Korea), MSK2014-v0234 (14-15 m (Guiry and Guiry 2017). Feldman (1937), which treated P. deep, Yongdam, Jeju, Korea), MSK160330-01 (21-24 m gestroi and P. orbiculare as synonyms of P. crassum, dis- deep, Dodu, Jeju, Korea), MSK160527-s01 (21-24 m deep, cussed varieties of P. crassum dependent on the cell size and Dodu, Jeju, Korea), MSK160627-01 & 02 (10-15 m deep, degree of attachment of the thallus to the substrate. Later, Geomundo, Yeosu, Korea), MSK160728-01, 02 & 03 (10- although Giaccone (1965) proposed species and varieties 15 m deep, Hyeonjeseom, Busan, Korea), MSK170417-01, of Palmophyllum distributed in the Mediterranean Sea as 02 & 03 (8-15 m deep, Shimoda, Shizuoka, Japan), NIBR forms of P. crassum depending on different environment, CL0000112650 (Gwideok 1-ri, Jeju, Korea). Cormaci et al. (2014) assessed that all varieties and forms Habit and anatomy: Plant grows in the subtidal zone, 8- of P. crassum were invalid on the grounds of the Interna- 30 m deep. Thalli are crustose, closely adherent to substrate tional Code of Nomenclature (Melbourne Code, McNeill et and not lobed, which forms and expands irregularly along al. 2012). the substrate plane (Fig. 3a-c). Thalli are usually distrib- In the northwest Pacific region, P. crassum has solely been uted on the shady vertical face of the reef. Thallus patches known in Japanese coastlines (Yoshida et al. 1990). The or sheets attach to non-geniculate crustose coralline algae, entities of P. crassum was firstly reported as P. orbiculare sponge, shells, or rocks. Thallus color is brilliant to dark by Segawa (1936) from Miyake Island, Izu peninsula with green vividly (Fig. 3a-c). Thalli are unstructured and slight­ the description of dark green thallus composed of spherical ly firm gelatinous in texture, and the sizes range from 3- or oblong cells immersed in gelatinous substance, as well as 7×4-8 cm to 10-13×15-18 cm, or largely expanded up to adhering to the substratum such as rocks or calcareous al- about 20×25 cm (Fig. 3a-f). Thalli thickness ranges from gae on the comparison with figures of European specimens. 643.87 μm to 893.92 μm. Thalli are composed of sphe­rical Later, Kajimura (1987) recorded another distribution of this cells, which are unstructured one another (Fig. 3g). In the species from Oki Islands, and Yoshida et al. (1990) listed it transverse section, thalli are dorsiventral (Fig. 3g). From the as P. crassum var. orbiculare. dorsal surface to ventral region, the cell composition and Our molecular and morphological analyses of crustose density are changed. The cell density is greater at margins green seaweed distributed in deep seawater resulted in the and dorsal surface than in central region and ventral side of discovery of an ancient green seaweed, Palmophyllum cras- thalli (Fig. 3g). The dorsal sided cells are overall greenish sum, from the Korean coast. The phylogenetic analysis of and 11.01-14.15 μm in diameter, except the outermost layer rbcL gene supported that the Korean specimens of P. cras- which is composed of little greenish cells, 6.13-7.81 μm in sum is identical to Japanese one (AP017927, Furukawa et diameter (Fig. 3h). In the central and ventral­ regions, cells al. 2017), which are distinguished from the type specimen are colorless, but dark green vacuoles and/or light green of P. umbracola from New Zealand (EU586182, Zechman nucleus are conspicuous (Fig. 3i). These central and ventral et al. 2010). The specimen of P. umbracola from California, cells are 9.92-12.74 μm in diameter. USA (EU586180, Zechman et al. 2010) was not monophyly with the type specimen from New Zealand, exhibiting 5.6% genetic divergence which is presumed that these two spec- DISCUSSION imens can be separated independently. Based on the rbcL analysis, P. crassum known as a cosmopolitan species (Gui- The genus Palmophyllum Kützing was established on the ry and Guiry 2017) should be reassessed from each local basis of the report of P. flabellatum, having gelatinous-carti- specimen in the species level. Originally, P. crassum was laginous, flabellate, and foliose thallus composed of minute established based on specimens from the Mediterranean Sea Palmophyllum crassum from Korea 325

Fig. 3. ‌Palmophyllum crassum (Naccari) Rabenhorst. (a-c) Crustose habits in subtidal coral reef, which are tightly adherent to the substrate. (a) The thallus growing in 25 m deep from Jeju Island, Korea, on 26th May 2016. (b) The thallus growing in 20 m deep from Busan, Korea, on 28th July 2016. (c) The thallus growing in 18 m deep from Shimoda, Japan, on 17th April 2017. (d-f) Voucher specimens of P. crassum. (d-e) MSK131101-10 & 11 collected subtidally in 18-25 m deep from Udo, Jeju, Korea on 1st November 2013. (f) MSK170417-03 collected subtidally in 8-15 m deep from Shimoda, Shizuoka, Japan. (g-i) Cross-section exhibiting coccoid cells embedded in a gelatinous matrix. (g) Transverse view of P. crassum, exhibiting dorsiventral structure. (h) Coccoid cells of dorsal side, entirely filled with greenish cytoplasm. (i) Coccoid cells of middle and ventral portion, showing light green colored nucleus and dark green colored vacuole (arrows), and cup-shaped chloroplast (arrowhead). Scale bars: (a)=3 cm; (b)=5 cm; (c)=6 cm; (d-f)=1 cm; (g)=100 μm; (h)=20 μm; (i)=10 μm. and the Adriatic Sea (Naccari 1828; Hauck 1885; Feldmann rotenoid pigments (Yokohama et al. 1977). These green 1937; Cormaci et al. 2014). Therefore, world-wide exam- seaweeds, referred to as deep-seawater type such as Clado- inations of P. crassum in rbcL analyses will lead to a better phora wrightiana, Codium minus and Umbraulva japonica, understanding of the ancient lineage of green seaweed in contain special chloroplast pigment: siphonixanthin or si- addition to recognition of species diversity. phonein which are efficient photosynthetic pigment under Most green seaweeds distributed in deep water having green illumination (Yokohama et al. 1977; Kageyama and dominant green light have the special composition of ca- Yokohama 1978). Palmophyllum species have not these 326 Hyung Woo Lee and Myung Sook Kim special chloroplast pigments (Yokohama et al. 1977), al- ean coast provides a suitable environment for growing green though they have been found in deep subtidal, growing on seaweeds and further contributed the taxonomic radiation rock faces in the areas of shade, crevices and under rock of green algae. Therefore, we should continuously try to ex- overhangs (Nelson and Ryan 1986). In spite of a lack of plore the green seaweed diversity in the Korean deep water siphonoxanthin or siphonein which functions as a photosyn- and to conserve the oceanic environment of Korea as the thetic pigment in deep seawater, it seems that Palmophyllum biodiversity center of seaweed. species have accommodated itself to dimly environment by maintaining increased chlorophyll b/a ratio (Sartoni et al. 1993). This increased chlorophyll b/a ratio may be regard- ACKNOWLEDGEMENT ed that early branching chlorophytes, like Palmophyllales lineage, have accumulated large quantities of chlorophyll b We thank Dr. Wada at Shimoda Marine Research Center into core antennae of their photosystem to harvest blue-green in University of Tsukuba for helpful cooperation to organize light energy efficiently based on the molecular evolution of sampling in Shimoda, and also all members at the molec- chlorophyllide a oxygenage in green plants (Kunugi et al. ular phylogeny of marine algae laboratory in Jeju National 2016). The ability of Palmophyllum to grow deeply may University. This research was supported by a grant from the play an important role in the diminishment of abiotic stress National Institute of Biological Resources (NIBR), funded and low competition for habitat (Zeckman et al. 2010). by the Ministry of Environment (MOE) of the Republic of In the rbcL analysis, Korean and Japanese specimens Korea (NIBR201701204). of P. crassum had closer affinity with P. umbracola from New Zealand than with Californian P. umbracola specimen supported weakly 57% in BS and 0.76 in PP (Fig. 2), de- REFERENCES spite quite high interspecific divergence, 4.6-5.3%. Recent studies also showed that some red seaweeds from subtidal, Ballantine DL and NE Aponte. 1996. Verdigellas nektongammea Amalthea and Psaromenia, have close intrageneric affinities (Tetrasporales, Chlorophyta), a new deep-water species between Korea and New Zealand (Lee et al. 2016). How- form the Bahamas. 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