http://dx.doi.org/10.4490/algae.2013.28.2.149 Open Access
A novel species Symphyocladia glabra sp. nov. (Rhodomelaceae, Rhodophyta) from Korea based on morphological and molecular analyses
Jeong Chan Kang
1and Myung Sook Kim
1,*
1Department of Biology and Research Institute for Basic Sciences, Jeju National University, Jeju 690-756, Korea
Six species of the genus Symphyocladia are currently recognized worldwide, all of which are reported to grow on the Korean coast. We described a new species Symphyocladia glabra sp. nov. based on morphological and molecular evi- dence. The new species is characterized by mostly an erect and broad thallus, the parallel arrangement of numerous apical cells, completely fused congenital cells, a corticated basal portion of faint midrib with six to eight pericentral cells, rhizoids cutting off from pericentral cells, and absent vegetative trichoblasts. The new species has morphological similar- ity to S. marchantioides and S. jejuinsula. However, S. glabra sp. nov. is distinguished from S. marchantioides by the cor- ticated lower portion of the thallus and the absence of vegetative trichoblasts, and S. jejuinsula by number of pericentral cells and the fact that the thallus does not taper upward. The phylogeny of rbcL sequences indicated that S. glabra sp. nov.
is definitely a separate entity within the genus Symphyocladia.
Key Words: morphology; rbcL; Rhodophyta; Symphyocladia glabra sp. nov.; taxonomy
INTRODUCTION
Six species of Symphyocladia are currently recognized including the type species S. marchantioides (Harvey) Falkenberg, and S. linearis (Okamura) Falkenberg, S. la- tiuscula (Harvey) Yamada, S. pumila (Yendo) Uwai and Masuda, S. lithophila M. S. Kim, and S. jejuinsula S. Y.
Kim, W. Nelson and M. S. Kim. The genus Symphyocladia, a member of the Rhodomelaceae, was established by Falkenberg based on S. marchantioides, which was first reported by Harvey as Amansia marchantioides from New Zealand (Harvey 1855, Schmitz and Falkenberg 1897).
Falkenberg (1901) established the tribe Pterosiphonieae and included Symphyocladia in this tribe. At this time, he transferred Placophora linearis Okamura (1895) (see De Toni 1895) from Japan and Dictyomenia gracilis Martens (1866) from China to the genus Symphyocladia. Yamada
(1941) identified that S. gracilis (Martens) Falkenberg was the same species as Rytiphlaea latiuscula Harvey (1857) from Japan, and he made the combination S. latiuscula (Harvey) Yamada. Okamura (1923) added S. pennata from Japan; however, Uwai and Masuda (1999) reported that S. pennata is conspecific with Pterosiphonia pumila from Japan, which was first described by Yendo (1920) and they made the combination, S. pumila (Yendo) S. Uwai and M.
Masuda. Recently, two more species (S. lithophila and S.
jejuinsula) were added from the Korean coast (Kim et al.
2010, 2012b).
The morphological characteristics (e.g., habit of thal- lus, degree of cortication and congenital fusion, number of pericentral cells, occurrence of vegetative trichoblasts, and tetrasporangial stichidia) have been used to separate
Received April 6, 2013, Accepted May 9, 2013
*
Corresponding Author E-mail: myungskim@jejunu.ac.krTel: +82-64-754-3523, Fax: +82-64-756-3541 This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://cre- ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
assigned an identification number, and photographs were taken with a D-80 camera (Nikon, Tokyo, Japan) on white paper. A part of the thalli was detached, cleaned in filtered seawater under the microscope to remove other organisms, and desiccated with silica-gel for DNA extrac- tion, while the remaining major part of the thalli was used to make pressed specimens or preserved in 5% formalin / seawater for further morphological analysis. Sections were made using a freezing microtome (NK-101-II; Nip- pon Optical Works Co. Ltd., Tokyo, Japan). Sections on slides were stained with 1% aqueous aniline blue acidi- fied with a drop of 1% HCl and mounted in 30% Karo corn syrup. Photomicrographs were taken using QIMAGING 1394 camera (QImaging, Surrey, BC, Canada) attached to a BX50 microscope (Olympus, Tokyo, Japan). Voucher specimens were deposited in the herbariums of Jeju Na- tional University (JNUB), Jeju and of National Institute of Biological Resources (NIBR), Incheon, Korea.
The total DNA of samples was extracted from the sili- ca-gel dried specimens using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The extracted DNA was stored at -20°C and used to amplify the rbcL gene. Polymerase chain reaction (PCR) amplification was performed using rbcL primers designed by Kim et al. (2010). For samples the failed am- plification, we redesigned the primers (e.g., rbcLJNF1 [5′- TGG TGA ATC ATC TAC AGC TA-3′] / rbcLJNR1 [5′-CAA TAC GCA TCC ATT TAC AA-3′] and rbcLJNF2 [5′-CAT AGA GCT GGT AAT TCA AC-3′] / rbcLJNR2 [TTC TAC AAA GTC AGC TGT ATC-3′] combination for the front and back part of the rbcL region, respectively). PCR amplification was performed in a total volume of 20 µL with AccuPower PCR PreMix (Bioneer, Daejeon, Korea). PCR was conducted with an initial denaturation at 95°C for 5 min, followed by 35 cycles of amplification (denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 2 min), with a final extension at 72°C for 7 min. The PCR prod- ucts were purified using the AccuPrep PCR Purification Kit (Bioneer) and then sequenced commercially (Macro- gen, Seoul, Korea). Both electropherogram outputs from each sample were edited using Chromas version 1.45 (Technelysium Pty Ltd., Helensvale, QLD, Australia). To- tal rbcL sequence was organized using the multiple-se- quence editing program BioEdit (Hall 1999) and aligned visually. None of the alignments posed a problem, as no gaps were observed.
To confirm the taxonomic position of Symphyocladia sp., maximum likelihood (ML) analysis was performed using RAxML software (Stamatakis 2006) and the GTR + Г evolutionary model. Fifteen rbcL sequences of Symphyo- the symphyocladian species (Choi and Lee 1995, Uwai
and Masuda 1999, Kim et al. 2010, 2012b). DNA sequenc- es also have been widely used to determine the taxonomy and phylogeny of red algae, and they allow us to answer questions when identifying species without clear-cut morphological diagnostic criteria (Gavio and Fredericq 2002, Kim et al. 2004, 2012a, Conklin et al. 2009). In par- ticular, the chloroplast-encoded rbcL gene is considered more phylogenetically informative in the genus or spe- cies level than the other markers for the red algae (Kim et al. 2010). For the symphyocladian species, rbcL sequenc- es are the most useful marker in taxonomic study because the verified sequences through previous studies (Kim et al. 2010, 2012b) are available from GenBank.
It is reported that all of the symphyocladian species inhabit the Korean coast (Guiry and Guiry 2013). We col- lected species of Symphyocladia, including unidentified species, from several localities on the Korean coast. The unidentified specimens exhibited habits similar to those of S. marchantioides and S. jejuinsula in terms of thin, flat and flabellate thalli, and numerous apical cells in a roundish parallel shape. However, they were corticated on the lower part of thalli and had larger blades than S.
marchantioides, and were not tapered upward in contrast with S. jejuinsula. Therefore, the aims of this study are as follows: 1) to determine whether the unidentified speci- mens are different species from S. marchantioides and S.
jejuinsula; if so, 2) to confirm their taxonomic position in the genus Symphyocladia; and 3) to investigate the phylo- genetic relationships based on the rbcL gene. To achieve these aims, we collected individuals of unidentified spe- cies in three phases of the life cycle including tetrasporo- phytes and male and female gametophytes, and analyzed their morphological and molecular characteristics.
MATERIALS AND METHODS
Specimens were collected from the intertidal zone for S. latiuscula and S. marchantioides, and the subtidal zone for the other species by SCUBA. We used 40 sym- phyocladian samples from the Korean coast, two from Japan, and 21 rbcL sequence data of Symphyocladia and Pterosiphonia from previous studies. Field observations and collections of Symphyocladia sp. were made from eight sites on the eastern and southern coasts of Korea.
The specimen collection data and the GenBank acces- sion numbers of the rbcL data are listed in Table 1. Field- collected samples were put into an icebox with seawater and transported to the laboratory. Every individual was
Ta bl e 1.
Collection information of samples used in this study Taxa Collection data Habitat (depth / substratum) Voucher GenBank accession No.References Symphyocladia glabra sp. nov.Biyangdo, Jeju Korea; Apr 8, 201210 m depth / bed rock120408-26 (⊕)KF048951This study Dueokdo, Wando, Korea; Jul 27, 20125-15 m depth / bed rock120727-14KF048963This study Dueokdo, Wando, Korea; Jul 27, 20125-15 m depth / bed rock120727-15KF048964This study Geomundo, Yeosu, Korea; Jul 25, 201210 m depth / bed rock120725-41KF048962This study Gangneung, Korea; Oct 27, 20123-8 m depth / bed rock121027-27KF048968This study Jocheon, Jeju, Korea; Feb 14, 20128 m depth / ropeS307 (♂)KF048952This study Jocheon, Jeju, Korea; Feb 14, 20128 m depth / ropeS308 (♀)KF048953This study Jocheon, Jeju, Korea; Feb 14, 20128 m depth / bed rockS309 (⊕)KF048965This study Jongdal, Jeju, Korea; Feb 4, 201210 m depth / ropeS310 (⊕)KF048954This study Jongdal, Jeju, Korea; Feb 3, 20136 m depth / A. longiramulosa130203-11 (⊕)KF048966This study Jongdal, Jeju, Korea; Feb 3, 20136 m depth / A. longiramulosa130203-12 (♂)KF048967This study Jongdal, Jeju, Korea; Feb 3, 20136 m depth / A. longiramulosa130203-13 (⊕)KF048970This study Jongdal, Jeju, Korea; Feb 3, 20136 m depth / A. longiramulosaNIBRAL 0000136687 (⊕)KF048971This study Namhaedo, Namhae, Korea; May 19, 20125 m depth / bed rock120519-38 (⊕)KF048961This study Namae, Gangneung, Korea; Oct 27, 20125-10 m depth / bed rock121027-65KF048969This study S. jejuinsula S. Y. Kim, W. Nelson & M. S. KimChujado, Jeju, Korea; Jul 4, 2008 -T14JF967647Kim et al. (2012b) Dueokdo, Wanddo, Korea; Jul 27, 20123-7 m depth / bed rock120727-10KF048958This study Dueokdo, Wando, Korea; Jul 27, 20123-7 m depth / bed rock120727-11KF048959This study Gijang, Busan, Korea; Mar 9, 2005 -M4-2JF967649Kim et al. (2012b) Hado, Jeju, Korea; May 15, 20125 m depth / bed rock120515-2KF048950This study Jocheon, Jeju, Korea; May 25, 2009-S 199JF967651Kim et al. (2012b) Songjeong, Busan, Korea; Dec 20, 20123-8 m depth / bed rock121220-61KF048960This study S. latiuscula (Harvey) YamadaAnin, Gangneung, Korea; May 19, 2012Low intertidal / bed rock120519-70KF048941This study Taean, Korea; May 10, 2006 -S 67GQ867072Kim et al. (2010) Chujado, Jeju, Korea; May 10, 2006 -S 136GQ867070Kim et al. (2010) Gijang, Busan, Korea; Jul 21, 2012Low intertidal / bed rock120721-33KF048940This study Gangneung, Korea; Oct 27, 201215-18 m depth / bed rock121027-31KF048943This study Gangneung, Korea; Oct 27, 201215-18 m depth / bed rock121027-32KF048944This study Namae, Gangneung, Korea; Oct 27, 20125-10 m depth / bed rock121027-72KF048945This study Namae, Gangneung, Korea; Oct 27, 20125-10 m depth / bed rock121027-73KF048946This study Wando, Korea; Jun 9, 2012Low intertidal / bed rock120609-38KF048942This study Hakodate, Japan; Feb 24, 2012Low intertidal / bed rock120224-28KF048938This study Hakodate, Japan; Feb 25, 2012Low intertidal / bed rock120225-26KF048939This studyTa bl e 1.
Continued Taxa Collection data Habitat (depth / substratum)Voucher GenBank accession No. References S. linearis (Okamura) FalkenbergAnin, Gangneung, Korea; Feb 9, 2008 -S 203GU731227Kim et al. (2010) Biyangdo, Jeju, Korea; Apr 8, 20125 m depth / bed rock120408-39KF048947This study Gangneung, Korea; Oct 27, 20123-8 m depth / bed rock121027-24KF048949This study Gwanganri, Busan, Korea; Nov 3, 20123-5 m depth / bed rock121103-3KF048956This study Songjeong, Busan, Korea; Nov 3, 20125-10 m depth / bed rock121103-77KF048957This study Uljin, Korea; Apr 28, 20126 m depth / rope120428-13 (⊕)KF048948This study Uljin, Korea; Apr 28, 20126 m depth / rope120428-15KF048955This study S. lithophila M. S. KimDadaepo, Busan, Korea; Feb 4, 2008Low intertidal / bed rockS132GQ867075Kim et al. (2010) Dadaepo, Busan, Korea; Feb 21, 2008Low intertidal / bed rockS145GQ867076Kim et al. (2010) Gampo, Gyeongju, Korea; Mar 16, 2003Low intertidal / bed rockS146GQ867078Kim et al. (2010) Gijang, Busan, Korea; Dec 14, 2008Low intertidal / bed rockS133GQ867077Kim et al. (2010) S. marchantioides (Harvey) FalkenbergSagye, Jeju, Korea; Jan 10, 2013Low intertidal / bed rock130110-7KF048979This study Cultured at KU-MACC, Japan -S144GQ867074Kim et al. (2010) Black Reef, New Zealand; Mar 11, 2011 -ASL128JF967662Kim et al. (2012b) Wairarapa, New Zealand; Jan 5, 2010 -ASK006GU731229Kim et al. (2010) Waitara, New Zealand; Jan 20, 2011 -ASL79JF967661Kim et al. (2012b) S. pumila (Yendo) S. Uwai & M. MasudaBiyangdo, Jeju, Korea; Feb 17, 201310 m depth / G. angusta130217-21 (⊕)KF048976This study Biyangdo, Jeju, Korea; Feb 17, 201310 m depth / G. angusta130217-22KF048977This study Biyangdo, Jeju, Korea; Feb 17, 201310 m depth / G. angusta130217-23KF048978This study Seogwipo, Korea; Dec 16, 2009 -S204GU731228Kim et al. (2010) Eoyoung, Jeju, Korea; May 7, 2012Low intertidal120507-1(⊕)KF048972This study Jigwido, Jeju, Korea; Dec 17, 2009- / G. angustaS249-1 (⊕)KF048974This study Jigwido, Jeju, Korea; Dec 17, 2009- / G. angustaS249-2 (♀)KF048975This study Sinchon, Jeju, Korea; Sep 11, 201212 m depth / G. angusta120911-1KF048973This study Cultured at KU-MACC, Japan -S143GQ867073Kim et al. (2010) Pterosiphonia ardreana Maggs & HommersandLourido, A Coruna, Spain; Jul 14, 2008 -21372JX857427Kim et al. (2012a) P. arenosa M. S. Kim & B. KimGimnyeong, Jeju, Korea; Jan 21, 2012 -12pte01JX857414Kim et al. (2012a) P. cloiophylla (C. Agardh) FalkenbergKommetjie, South Africa; Feb 18, 2006 -P 2024GQ867081Kim et al. (2010) P. complanata (Clemente) FalkenbergFinisère, France; Jun 24, 2006 -P 732GQ867079Kim et al. (2010) P. pennata (C. Agardh) SauvageauA Coruña, Spain; Jun 6, 2008 -20940JX857426Kim et al. (2012a) A. longiramulosa, Acanthopeltis longiramulosa; G. angusta, Grateloupia angusta.arranged in a roundish parallel shape; pericentral cells 6-8; rare vegetative trichoblasts; procarps producing on fugacious trichoblast along the margins of the blade with a four-celled carpogonial branch; ostiolate globu- lar cystocarp with a polysiphonous stalk; spermatangia bearing on trichoblast along the upper margins of the blade; corn-shaped antheridia adaxially curved with 1-2 monosiphonous stalk cells; tetrasporangia spherical and tetrahedrally divided, formed one per segment in straight rows in laterally fused stichidia on the margins of the blade, surrounded by two lateral and one basal cover cell.
Holotype. JN130203-11 (tetrasporophyte), collected from 6 m depth of Jongdal, Jeju province, Korea (33°29′
53′′ N, 126°54′50′′ E) on Feb 3, 2013, and deposited in the JNUB (the Herbarium of Department of Biology, Jeju National University, Korea).
Isotypes. JNUB (JN130203-12-13) and KB (NIBRAL00 00136687-8).
Distributions. Collected from Jeju Island, and the southern (Wando to Namhaedo) and eastern (Gan- gneung) coasts of Korea.
Etymology. The specific epithet (glabra) was chosen to represent the morphological difference from the type species, S. marchantioides, as lack of hair.
Korean name. 민털보라색우무.
Habitat. Symphyocladia glabra was collected at 3-30 m depth of the subtidal zone and they were growing on bed rock, abandoned rope or other red algae, such as Ac- anthopeltis longiramulosa, Gelidium elegans. Reproduc- tive fronds were mostly discovered between winter and spring season.
Other specimens examined. Jocheon, Jeju (JN 110520, tetrasporophyte, May 20, 2011; S 307, male gameto- phyte, Feb 14, 2012; S 308, female gametophyte, Feb 14, 2012; S309, tetrasporophyte, Feb 14, 2012); Gwideok, Jeju (JN110531, tetrasporophyte, May 31, 2011); Udo, Jeju (JN130203-7, female gametophyte, Feb 3, 2013); Jongdal, Jeju (S310, tetrasporophyte, Feb 4, 2012); Biyangdo, Jeju (JN120408-26, tetrasporophyte, Apr 8, 2012); Namhaedo, Namhae (JN120519-38, tetrasporophyte, May 19, 2012);
Geomundo, Yeosu (JN120725-41, vegetative thallus, Jul 25, 2012); Dueokdo, Wando (JN120727-14-15, vegetative thallus, Jul 25, 2012); Gyeongpo, Gan gneung (JN121027- 27, vegetative thallus, Oct 27, 2012); Namae, Gan gneung (JN121027-65, vegetative thallus, Oct 27, 2012).
Morphology. Fronds are mostly erect. Erect thalli are thin, flat, and broadly membranous; 3-10 cm high; 0.5-1.2 cm wide; and 200 µm thin in the middle part of the blade, tapering at the base and wider toward the upper part (Fig.
1A). Growth occurs from numerous apical cells, which are cladia sp. were aligned with 43 of the other specimens of
Symphyocladia, and five species of Pterosiphonia were used as an out group. We used 200 independent tree in- ferences using the -# option with default -I (automati- cally optimized subtree pruning and regrafting [SPR] re- arrangement) and -c (25 distinct rate categories) options of the program to identify the best tree. To generate boot- strap values for the phylogenetic tree, we used the same program with the same settings for 1,000 replications.
RESULTS
Symphyocladia glabra J. C. Kang et M. S. Kim sp.
nov. (Figs 1 & 2)
Description. Thalli plerumque erecti, large plana et membranacea, rubro ad nigrantis purpurascentibus brunneis, attachiatus ad substratum, a pluribus singu- lae rhizoids a ventral pars implicati decumbentes palmes, rhizoids interibit de pricentral cellulis et terminatur in multicellulare haptera; erectis thalli 3-10 cm altus, 0.5- 1.2 cm in diameter et attenuatae ad basim, et latiores ad partem superiorem, divisa alternate vel opposite pinnatis modo, habere crenulate margine, corticated in basali pars deficimus midnervo lateralibus ramis omnino congenita traditio miscet parentali axe; numerosa apicalibus cellulis disposita surrotundus parallela figura; pericentral cellulis 6-8; vegetabilis trichoblasts rara, procarps sunt produca- tur in fugaces trichoblasts per laminae marginem cum quattuor-cellularibus carpogonial ramus, ostiolata cysto- carp globosam cum polysiphonous culmus; spermatangia ortus in trichoblasts per margines superiores ense; anther- idia adaxially curvus segeti figuram 1-2 monosiphonous culmus cellulis; tetrasporangiis sphaericum et tetrahe- drally divisa, formatur unum per segmentum simplices ordines in lateraliter excoquuntur stichidia in marginibus laminis, 90 µm diameter, ab duabus circumventa later- alem et unum basali cover cellulis.
Thallus mostly erect, broadly flat and membranous, reddish to dark-purplish brown, attached to the substra- tum by numerous unicellular rhizoids from the ventral part of entangled decumbent branch, rhizoids cutting off from the pericentral cell and terminating in multicellular haptera; erect thallus growing 3-10 cm high, 0.5-1.2 cm in diameter and tapering at the base and wider toward the upper part, divided alternate or oppositely pinnate manner, crenulate margin, corticated in the basal part of the faint midrib; lateral branches completely congeni- tal fused with the parental axis; numerous apical cells
cells (Fig. 1B). In the surface view, mature pericentral cells are arranged in a nodular fan shape, about 30-40 µm in diameter and 200-300 µm long (Fig. 1C). In the transverse section, the single central cell is surrounded by six to eight pericentral cells (Fig. 1D & E). The main branch has a faint arranged in a roundish parallel shape. The apical cells are
divided obliquely at first, cutting off one segment trans- versely downward and forming pericentral cells in an al- ternate sequence. The apical cells and pericentral cells of the thalli become congenitally fused with the row of axial
A
C
D
B
E
G
F
H I
Fig. 1.
Vegetative structure of Symphyocladia glabra sp. nov. (A) Holotype specimen from Jongdal (JN130203-11), Jeju Island. (B) Apical cells showing roundish parallel arrangement. (C) Surface view of middle part of thallus showing congenitally fused polysiphonous filaments. (D-F) Cross section views of the parts of thallus showing a central cell (cc) surrounded by 6-8 pericentral cells (arrows), and cortication (arrowheads) on the basal part, the below apical (D), the middle (E), and the basal part (F). (G) Thallus margin showing undulate shape and lack of vegetative trichoblast. (H) Decumbent branches attached by numerous unicellular rhizoides forming haptera (arrowheads). (I) Rhizoid cutting off from the pericentral cell of decumbent branch. Scale bars represent: A, 3 cm; B & E, 50 µm; C & H, 100 µm; D, 1,000 µm; F, 500 µm; G, 30 µm; I, 300 µm.cladian specimens including 15 S. glabra, and five ptero- siphonian species as an outgroup. Of all sites, 208 (18.1%) were variable and 176 (15.3%) were phylogenetically in- formative. The 15 specimens of S. glabra from eight sites in Korea formed an independent monophyletic clade with 0-0.3% divergence that separated from the other clades. There was a similar divergence between S. glabra and the other Symphyocladia species by 4.3-4.8%, except S. lithophila by 5.2-5.4%, which had the highest differ- ence among the Symphyocladia clades.
In the phylogenetic tree (Fig. 3), the genus Symphyo- cladia was clearly separated from the Pterosiphonia clade and supported monophyly by the 100% ML bootstrap value. Each species of Symphyocladia including S. glabra, also formed independent clades with strong bootstrap values.
DISCUSSION
We intensively collected symphyocladian species along the Korean coast and found a new species named Sym- phyocladia glabra sp. nov. This species is morphologically similar to S. marchantioides in characters of the flabel- late and pinnate outline, the faint midrib and roundish crenulate margin of the blade, the complete congenital fusion of the axis with laterals, the roundish parallel ar- rangement of the numerous apical cells, the number of pericentral cells, and the production of tetrasporangial stichidia. However, they differ in thallus size, the lack of vegetative trichoblasts, and the cortication on the lower portion of the midrib (Kim et al. 2012b). Moreover, S. gla- bra also closely resembles to S. jejuinsula having the fea- tures of the mostly erect thalli, the membranous flabellate erect branches, the cortication on the basal part of the thalli, and the lack of vegetative trichoblasts. Conversely, S. jejuinsula has more slender and tapering branches to- ward the apical part, 10 segments of congenitally fused branchlets, and eight to ten pericentral cells (Kim et al.
2012b). While comparing new species with the other spe- cies of Symphyocladia, we found the obvious morpho- logical characters separating them from the others (Table 2). The development of reproductive structures is essen- tially the same as in other symphyocladian species, such as S. pumila (Uwai and Masuda 1999), and S. latiuscula (Matsuyama and Masaki 1975, Choi and Lee 1991). The recognition of this new species is strongly supported by not only morphological but also molecular evidence. Kim et al. (2012b) mentioned that the divergence of the rbcL sequence among the species of Symphyocladia ranges midrib, which is corticated in the basal part (Fig. 1F).
Branches are divided alternate or oppositely in a pinnate manner in a plane and become a crenulate margin (Fig.
1A & G). The erect thalli are occurred from the entangled decumbent branch, which adheres to the substratum by numerous protruding rhizoids from the under-surface (Fig. 1H). Rhizoids are unicellular, 30 µm in diameter, cut- ting off from the pericentral cell of the decumbent branch and forming multicellular haptera on their terminal part (Fig. 1H & I). No vegetative trichoblasts are observed. The color is bright red when alive but becomes light brown when fixed by formalin solution. The texture is membra- nous and adheres well on a paper.
The procarps are initiated with forming trichoblast ini- tial and the fertile pericentral cell derived from the api- cal cell. The apical cell cuts off the fertile pericentral cell obliquely and becomes the trichoblast initial itself. The trichoblast initial is divided transversely and is alternately branched three times, and it consequently forms a repro- ductive trichoblast that is bent adaxially (Fig. 2B-E). At the same time, the fertile pericentral cell is more developed, and it forms the basal sterile initial and the four-celled carpogonial branch (Fig. 2E & F). The procarps are cov- ered prior to fertilization by a pericarp derived from the periaxial cells of the suprabasal reproductive trichoblasts (Fig. 2E & F). Before maturation of the carpogonium, the reproductive trichoblasts are decayed. The mature ostio- late cystocarps are globular, 400-500 µm in diameter and have polysiphonous stalks (Fig. 2A).
Spermatangial branchlets are produced on the protu- berances along the upper margins of the blades (Fig. 2G).
The spermatangial branchlets are initiated with the mod- ification of apical cells to unbranched and monosipho- nous trichoblasts by several transverse cell divisions. The monosiphonous trichoblasts are born spirally in an alter- nate manner on every segment of the marginal protuber- ance, and they become polysiphonous by longitudinal di- vision (except basal 1-2 cells). The mature spermatangial branchlets are corn-shaped, 300-350 µm long, 70-90 µm in diameter, and curved adaxially (Fig. 2G-J).
The tetrasporangia are spherical, divided tetrahedrally, and produced one per segment in straight rows on the laterally fused stichidia at the margins of the blades. The fertile branchlets swell on both sides with tetrasporangial maturity. The mature tetrasporangia are 90 µm in diame- ter with two cover cells. The tetrasporangia are connected with a stalk cell by a pit-connection (Fig. 2K & L). No veg- etative trichoblasts are observed.
Molecular analysis. We aligned 1,150 nucleotide base pairs of the rbcL gene from 58 individuals of symphyo-
Fig. 2.
Reproductive structure of Symphyocladia glabra sp. nov. (A) Female reproductive organ producing on the upper margin of blade. (B- F) Development of female reproductive organ (bs, basal sterile cell; cb, carpogonial branch; fp, fertile pericentral cell; ls, lateral sterile cell; tb, trichoblast; tbi, trichoblast initial; tr, tricogyne). (G) Spermatangial branchlets producing along the upper margin of blade. (H & I) Development of male reproductive organ. (J) Cross-section view of mature spermatangial branchlet showing a central cell (cc) and pericentral cells (arrows) surrounded by numerous spermatangia (st). (K) Tetrasporangial stichidia producing on the upper margin of blade. (L) Development of tetrasporangia, which are connected with a stalk cell by a pit-connection (arrows). Scale bars represent: A, 500 µm; B-D, H-J & L, 20 µm; E & F, 50 µm; G, 100 µm; K, 300 µm.A B C D
E
G
F
H
I
L J
K
tioides, S. jejuinsula, and S. glabra) have been identified to one species as S. marchantioides. Okamura (1912) il- lustrated four specimens of S. marchantioides, one large and three small. The largest specimen has morphological features of the many linear prostrate branches and the erect thallus tapering upward that are similar to those of S. jejuinsula. His description is also consistent with the description of S. jejuinsula by Kim et al. (2012b) in fea- ture of thallus habit and size, and cortication of midrib.
between 2.6-5.6%. From rbcL data in this study, the new species had more than 4% of divergence and formed a monophyletic clade strongly supported by the 100% ML bootstrap value. The results of our molecular analysis suggest that the chloroplast-encoded rbcL gene is very useful marker in delimitating morphologically similar taxonomic groups in the genus Symphyocladia.
It is possible that in the Northwestern Pacific region, three similar symphyocladian species (i.e., S. marchan-
Fig. 3.
Maximum likelihood phylogenetic tree for the genus Symphyocladia and relatives derived from plastid-encoded rbcL sequence data. The bootstrap values (1,000 replicates) are shown above branches.Ta bl e 2.
Comparison of distinguishing morphological characters of Symphyocladia glabra sp. nov. and other Symphyocladia species Species charactersS. glabra sp. nov.S. marchantioides (Harvey) Falkenberg S. jejuinsula S. Y. Kim, W. Nelson & M. S. Kim S. pumila (Yendo) S. Uwai & M. Masuda S. lithophila M. S. KimS. linearis (Okamura) FalkenbergS. latiuscula (Harvey) Yamada Height 3-10 cm 2-4 cm 3-10 cm 1-2 cm 1-2 cm 10-30 cm 5-15 cm Width 0.5-1.2 cm 3-5 mm 1-3 mm 900 μm300 μm - 1.0-1.5 mm Habit Membranous / flabellate Membranous Alternate- distichous Alternate- distichous Alternate- distichous Linear Flabellate Substratum Epilithic / epiphyticEpilithic / epiphytic Epilithic Epiphytic Epilithic Epilithic Epilithic / epiphytic Cortication Present (in basal part)Absent Present (in lower part)Absent Absent Present (in lower part)Present (thoroughly) Vegetative trichoblast Absent Present Absent Absent Absent Absent Present Congenital fusion Complete Complete More than 10 Complete 5-77-85-7 Pericentral cells 6-86-88-107-98-107-86-7 Tetrasporangial stichidiumPresent Present Absent Stichidia-like Absent Absent Present Type locality Jongdal, Jeju, KoreaHawke’s Bay, New Zealand Jocheon, Jeju, Korea Misaki, Japan Gijang, Korea Japan Hakodate, Japan References This study Falkenberg (1901), Kim et al. (2012a)Kim et al. (2012b)Uwai and Masuda (1999)Kim et al. (2010) Choi and Lee (1995)Choi and Lee (1991, 1995)
ACKNOWLEDGEMENTS
This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR No. 2013-02-001).
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In Korea, Lee (2008) described S. marchantioides in his pictorial book on macroalgae. We observed the speci- mens identified as S. marchantioides (Lee 2008) depos- ited in the JNUB, which were voucher specimens of the plate on page 321. As a result, we confirmed that the two specimens collected from Biyangdo (sample No. 2006- 138) and Jeju Harbor (sample No. 2007-72) had the same characteristics as S. glabra in terms of thallus habit, lack of vegetative trichoblasts, thallus size, and cortication on the lower portion of the faint midrib.
In the genus Symphyocladia, the following diagnostic characters for classification at the species level have been used: habit of thallus, degree of cortication and congeni- tal fusion, number of pericentral cells, and occurrence of vegetative trichoblasts and tetrasporangial stichidia (Choi and Lee 1995, Kim et al. 2010, 2012b). The other four species are easily distinguishable by their morpho- logical characteristics from S. marchantioides, S. jejuin- sula, and S. glabra as follows: 1) S. linearis by its linear and large thalli, seven to eight pericentral cells, and proxi- mal cortication; 2) S. latiuscula by its thick and entirely corticated thalli (except on the apical part of the blade), abundant vegetative trichoblasts, and six to seven peri- central cells; 3) S. pumila by its small ecorticate thalli with alternate-distichous branches at two-segment intervals, seven to nine pericentral cells, seven to eleven segments congenitally fused between lateral branches and parental axes, and stichidia-like tetrasporangial branchlets; and 4) S. lithophila by its small ecorticated thalli with alternate- distichous branches on every second segment of the axes, eight to ten pericentral cells, congenital fusions in five to seven segments, and epilithic habitation (Table 2).
In conclusion, we intensively collected symphyocladi- an species along the Korean coast and revealed (after add- ing a new species) that, in total, seven species inhabit in the Korean coast. Based on morphological and molecular analysis, we identified one novel species, Symphyocladia glabra sp. nov. that has been known as S. marchantioides.
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