Rhodophyta) Based on Carposporophyte Development and Molecular Data

Botanica Marina 2017; 60(5): 515–532

Jeong Chan Kang, Mi Yeon Yang and Myung Sook Kim* Neoharaldiophyllum, a new genus of Delesseriaceae (Rhodophyta) based on carposporophyte development and molecular data

DOI 10.1515/bot-2017-0003 Received 9 January, 2017; accepted 20 April, 2017; online first 22 Introduction June, 2017 Zinova (1981) established the genus Haraldiophyllum Abstract: The new genus Neoharaldiophyllum J.C. Kang et based on three species of Nitophyllum (i.e. Nitophyllum M.S. Kim belonging to the tribe Myriogrammeae, subfam- bonnemaisonii Greville [=Myriogramme bonnemaisonii ily Phycodryoideae of the Delesseriaceae, is described. Kylin], N. mirabile Kylin and N. versicolor Harvey). The This new genus consists of four species, namely Neoharal- three species had been mentioned by Kylin (1934, 1956) as diophyllum udoense (M.S. Kim et J.C. Kang) gen. et comb. having the same pattern of procarp development but differ nov. from Jeju Island Korea as the type species, Neoharal- from those of the generitype species Nitophyllum punc- diophyllum nottii (R.E. Norris et M.J. Wynne) J.C. Kang et tatum (Stackhouse) Greville. Kylin (1925, 1934) described M.S. Kim comb. nov. from the Hood Canal, Mason County, that the procarp in the three species has two sterile-cell Washington, USA, Neoharaldiophyllum mirabile (Kylin) groups, whereas that of N. punctatum has a single sterile- J.C. Kang et M.S. Kim comb nov. from Canoe Island, San cell group. He also noted that the two pericentral cells cut Juan County, Washington, USA, and Neoharaldiophyl- off from the fertile central cell in the procarp are borne lum erosum (Harvey) J.C. Kang et M.S. Kim comb. nov. perpendicular to the longitudinal axis of the frond in the from Garden Island, Western Australia. The morphologi- three species, while they are arranged in parallel with cal traits of the new genus are very similar to the genera the axis in N. punctatum. These two major differences Haraldiophyllum and Myriogramme in terms of the veg- between the three species and N. punctatum became the etative and reproductive structures. There are differences primary basis for establishing the genus Haraldiophyl- among the three genera in the developmental patterns lum by Zinova (1981). At that time, she designated Haral- of the carposporophyte: the primary gonimoblast cells diophyllum bonnemaisonii (Kylin) Zinova from Atlantic of Neoharaldiophyllum are prostrate on the floor cells of Europe as generitype species. Among the three species, the cavity of the cystocarp and fuse together secondarily, N. versicolor is currently regarded as a taxonomic synonym whereas in the two other genera they remain free without of Drachiella heterocarpa (Chauvin ex Duby) Maggs et a secondary incorporation with the floor cells; the car- Hommersand (Wynne 2014, Guiry and Guiry 2016). In the posporangia of Myriogramme are borne in short chains same study, Zinova (1981) established another new genus, terminating the gonimoblast filaments, while those in Hideophyllum, based on Myriogramme yezoensis Yamada the other two genera are borne as solitary structures. The et Tokida from Hokkaido, Japan. molecular phylogenies based on rbcL and LSU sequences The morphological traits of Haraldiophyllum are strongly support the significance of the developmental known to be very similar to those of Myriogramme Kylin patterns of the carposporophyte and support the separa- and Hideophyllum Zinova in terms of habit, thallus struc- tion of Neoharaldiophyllum from Haraldiophyllum in the ture, morphology of spermatangial and tetrasporan- tribe Myriogrammeae. gial sori, and composition of procarp (i.e. with anterior cover-cell group and two sterile-cell groups). However, Keywords: cystocarp structure; Delesseriaceae; molecular Haraldiophyllum differs from the two other genera in that phylogeny; Neoharaldiophyllum gen. nov.; taxonomy. it has single terminally borne carposporangia, while the other genera produce short chains of carposporangia *Corresponding author: Myung Sook Kim, Department of Biology, on the gonimoblast filaments (Zinova 1981, Maggs and Jeju National University, Jeju 690-756, Korea; and Research Institute Hommersand 1993, Hommersand and Fredericq 1997a, for Basic Sciences, Jeju National University, Jeju 690-756, Korea, e-mail: [email protected] Lin et al. 2007, Wynne 2014). Another important charac- Jeong Chan Kang and Mi Yeon Yang: Department of Biology, Jeju ter of Haraldiophyllum could be observed in the cystocarp, National University, Jeju 690-756, Korea that is, the incorporation of the fusion cell with the floor 516 J.C. Kang et al.: Neoharaldiophyllum gen. nov. cells in the cavity of the cystocarp (Zinova 1981, Maggs and desiccant for DNA extraction, and the samples for mor- Hommersand 1993, Millar 1994, Millar and Huisman 1996, phological observation were preserved in 5% formalin/ Hommersand and Fredericq 1997a, Lin et al. 2007, Wynne seawater. Voucher specimens were deposited in the her- 2014). barium of Jeju National University (JNUB, Jeju, Korea) While only seven species of Haraldiophyllum have and the National Institute of Biological Resources (NIBR, been recognized to date, they are scattered across Atlantic Incheon, Korea). Europe, the northern Pacific America, the north-western Sections for morphological examination were cut Pacific region and the southern hemisphere (Wynne 2014, by hand using razor blades or with bench-top freezing Guiry and Guiry 2016). In the north-western Pacific region, microtome (MFS no. 222; Nippon Optical Works, Tokyo, Nam and Kim (1996) initially recorded H. bonnemaisonii Japan). Materials were stained with Wittmann’s (1965) based on specimens from the southern coast of Korea, aceto-iron-hematoxylin-chloral hydrate and mounted and the second species, namely Haraldiophyllum udoense in 50% Hoyer’s mounting medium, or stained with 1% [“udoensis”] from Jeju Island, was described by Kim and aniline blue acidified with 1% HCl and mounted in 40% Kang (2011). As H. udoense was described based on tet- corn syrup solution (Lin et al. 2007). Images were cap- rasporic plants, the morphological features of female and tured using a G7x digital camera (Canon, Tokyo, Japan) male reproductive structures are still unknown. attached to an SZ2-ILST dissecting microscope (Olympus, To investigate their distribution and confirm the game- Tokyo, Japan); anatomical images were captured with a tophytic reproductive structures, we collected the species BX 43 microscope (Olympus, Tokyo, Japan) equipped with along the coast of the Korean peninsula, and gametan- an EOS 600 D digital camera (Canon, Tokyo, Japan). gial plants were successfully collected from several sites Total genomic DNA was extracted from specimens from the western and southern coasts of Korea including with a DNeasy Plant Mini kit (Qiagen, Hilden, Germany), the type locality Jeju Island, during 2012–2016. When we following manufacturer instructions. Amplification for observed the cross-section of the cystocarp, we were con- the nuclear LSU, the plastid ribulose-1, 5-bisphosphate fused because the shape of their carposporophyte is very carboxylase large subunit gene (rbcL) and the mitochon- different from those of ordinary Delesseriacean species drial cytochrome c oxidase 1 barcode region (COI-5P) was including the type species, H. bonnemaisonii. To investi- conducted using primers T01N, G, X, 28F, T05, T15 (LSU; gate the taxonomic and phylogenetic significance of the Freshwater et al. 1999, Harper and Saunders 2001), F7, shape of the carposporophyte, we analyzed the morphol- R898, F762, R1442 (rbcL; Gavio and Fredericq 2002, Kim ogy as well as two gene sequences (rbcL and LSU) and et al. 2010), and GWSFn, COIR686 (COI; Le Gall and Saun- then compared the results with previous studies of closely ders 2010, Sherwood et al. 2010). related taxonomic groups. To certify the genetic homo- Amplification reactions were set up and run as geneity among the various populations of H. udoense, described in Saunders and Moore (2013). The PCR prod- we also compared the rbcL gene sequences from our ucts were purified with an AccuPrep PCR Purification Kit specimens including the holotype. In the present study, (Bioneer, Daejeon, Korea) and sequenced commercially we describe in detail the morphological characters from by Macrogen (Seoul, Korea). Electropherogram outputs of male and female reproductive structures to supplement each sample were edited using the Chromas Lite 2.01 soft- the previous study by Kim and Kang (2011), especially ware (Technelysium, Helensvale, Queensland, Australia) focussing on the developmental pattern of the carpospor- and PHYDIT version 3.1 software (Chun 2001). Sequences ophyte, and we establish the new genus Neoharaldiophyl- were assembled in BioEdit 7.0.5 (Hall 1999) and aligned lum based on H. udoense from Korea. with Clustal W (Sievers et al. 2011). The LSU alignment was adjusted by eye, and ambiguous regions and introns were removed prior to analyses. The alignments of rbcL and combined LSU + rbcL Materials and methods sequence data were constructed to explore the relationships of the Neoharaldiophyllum with related taxa. This Specimens were collected from the subtidal on the coasts alignment included 12 newly generated sequences (two of of South Korea by SCUBA diving and from the intertidal on LSU and 10 of rbcL). Phylogenetic analyses were performed the coasts of New Zealand and Australia (Table 1). Field- using RAxML (Stamatakis 2006) using the GTR + Γ + I collected samples were kept fresh in a cool box with an model. To identify the best tree, we constructed 200 inde- ice pack and then transported to the laboratory. A clean pendent tree inferences using the –# option with default fragment of each specimen was quickly dried in silica gel –I (automatically optimized Subtree Pruning-Regrafting J.C. Kang et al.: Neoharaldiophyllum gen. nov. 517

Table 1: Two species in the genera Neoharaldiophyllum and Haraldiophyllum sampled for molecular analysis in this study with collection information and GenBank accession numbers.

Species Collection information/Generation No. of voucher GenBank accession no.

rbcL COI LSU

Neoharaldiophyllum udoense (M.S. Strait of Udo, Jeju Island, Korea, coll. J.C. JN120705-35 KY497915 Kim et J.C. Kang) J.C. Kang et M.S. Kang, 5 Jul. 2012/ ⊕ Kim gen. et comb. nov Strait of Udo, Jeju Island, Korea, coll. J.C. JN120705-36 KY497927 KY497921 Kang, 5 Jul. 2012/ Sinjindo Island, Taean, Korea, coll. M.S. Kim JN120809-17 KY497928 and M.Y. Yang, 9 Aug. 2012/- Gwanganri, Busan, Korea, coll. J.C. Kang and JN121103-10 KY497934 M.Y. Yang, 3 Nov. 2012/ Gwanganri, Busan, Korea, coll. J.C. Kang and JN121103-11 KY497926 KY497920 M.Y. Yang, 3 Nov. 2012/ Songjeong beach, Songjeongri, Busan, Korea, JN121103-81 KY497925 coll. J.C. Kang and M.Y. Yang, 3 Nov. 2012/ ⊕ Songjeong beach, Songjeongri, Busan, Korea, JN121103-82 KY497933 coll. J.C. Kang and M.Y. Yang, 3 Nov. 2012/ ⊕ Songjeong beach, Songjeongri, Busan, Korea, JN121103-83 KY497916 coll. J.C. Kang and M.Y. Yang, 3 Nov. 2012/ ⊕ Gwanganri, Busan, Korea, coll. J.C. Kang and JN121220-01 KY497924 KY497919 M.Y. Yang, 20 Dec. 2012/ Gwanganri, Busan, Korea, coll. J.C. Kang and JN121220-03 KY497914 M.Y. Yang, 20 Dec. 2012/ Strait of Udo, Jeju Island, Korea, coll. J.C. JN130607-1 KY497923 KY497918 Kang, 7 Jun. 2013/ ⊕ Sinheungri shore, Jangheung, Korea, coll. J.C. JN140418-03 KY497929 Kang, 18 May 2014/ ⊕ Sinheungri shore, Sinheungri, Jangheung, JN140418-06 KY497930 Korea, coll. J.C. Kang, 18 May 2014/ Sinheungri shore, Sinheungri, Jangheung, JN140418-07 KY497931 KY497912 Korea, coll. J.C. Kang, 18 May 2014/ ⊕ Strait of Udo, Jeju Island, Korea, coll. J.C. JN150509-R2 KY497932 KY497913 Kang, 9 Apr. 2015/ Sinjindo Island, Taean, Korea, coll. J.C. Kang, JN160224-1 KY497922 KY497917 24 Feb. 2016/ ⊕ Haraldiophyllum crispatum (Hooker Short beach, Sandy Bay, Tasmania, Australia, JN151105-14 KY497939 f. et Harvey) S.-M. Lin et W. Nelson coll. J.C. Kang, 5 Nov. 2015/ Short beach, Sandy Bay, Tasmania, Australia, JN151105-9 KY497938 coll. J.C. Kang, 5 Nov. 2015/ ⊕ Short beach, Sandy Bay, Tasmania, Australia, JN151105-12 KY497935 coll. J.C. Kang, 5 Nov. 2015/ Short beach, Sandy Bay, Tasmania, Australia, JN151105-20 KY497936 coll. J.C. Kang, 5 Nov. 2015/ Short beach, Sandy Bay, Tasmania, Australia, JN151105-23 KY497937 coll. J.C. Kang, 5 Nov. 2015/ Southern Pirates Bay, Eaglehawk Neck, JN151108-84 KY497940 Tasmania, Australia, coll. J.C. Kang, 8 Nov. 2015/ Southern Pirates Bay, Eaglehawk Neck, JN151108-85 KY497941 Tasmania, Australia, coll. J.C. Kang, 8 Nov. 2015/

rearrangement) and –c (25 distinct rate categories) soft- substitution model and RAxML program setting. A total of ware options. Statistical support for each branch was eight COI-5P sequences generated for DNA barcoding were obtained from 1000 bootstrap replications using the same submitted to GenBank. 518 J.C. Kang et al.: Neoharaldiophyllum gen. nov.

Results Type species

Neoharaldiophyllum udoense (M.S. Kim et J.C. Kang) J.C. Neoharaldiophyllum J.C. Kang et M.S. Kim Kang et M.S. Kim comb. nov. gen. nov.

Plant epilithic or epiphytic, erect and lobed, attached by Neoharaldiophyllum udoense (M.S. Kim et small disc often with several stolons; thalli flat and membranous with a cylindrical stipe; blades broad linear to J.C. Kang) J.C. Kang et M.S. Kim comb. nov. obovate often deeply cleft or subdichotomously divided, (Figures 1–54) margins undulate and entire or denticulate with multi- Basionym cellular spines, midrib and microscopic veins absent but basal subdichotomous or flabellate nerves present; upper Haraldiophyllum udoense M.S. Kim et J.C. Kang, ALGAE 26: blade monostromatic and lower blade polystromatic; 212, figs. 1–3 (as “udoensis”) (2011). in cross-sections of polystromatic portion, similar-sized isometric rectangular cells arranged in horizontal tiers in vertical rows; cortical cells polygonal with numerous Holotype small discoid chloroplasts. Growth by means of diffuse marginal meristems or a transversely (often obliquely) JNUB (MSK30601HU, tetrasporophyte, 14 Jun. 2009). dividing apical cell, with intercalary cell divisions. Game- tophyte dioecious. Cystocarps scattered on both surfaces of blade; procarp initiated by a pair of pericentral cells Type locality cut off from a fertile central cell perpendicular to the axis of the growing thallus, the anterior pericentral cell Haumokdong, Udo, Jeju Island, Korea (33°30´21˝ N, becomes a cover-cell group and the posterior one func- 126°56´09˝ E). tions as a supporting cell; mature procarp consisting of a supporting cell, a four-celled carpogonial branch, a single-celled first sterile-cell group, a one- to two-celled Distribution second sterile-cell group and an anterior cover-cell group; a fusion cell derived by cell fusion of a supporting cell, Southern and western coasts of Korea including Jeju both sterile-cell groups, and an auxiliary cell during early Island (This study, Kim and Kang 2011). development of cystocarp; the primary gonimoblast filaments extend by cell divisions along the floor cells of the cystocarp cavity and incorporate together at the point of Specimens examined contact; singly borne terminal carposporangia formed on highly branched secondary gonimoblast filaments, which Jeju Island, Korea: Udo (JN120705-34~36, 05 Jul. 2012, are produced from the primary gonimoblast filaments; female, tetrasporic, on rhodolith, subtidal; JN130607-1, cystocarp hemispherical with a single ostiole. Spermatan- 07 Jun. 2013, female, tetrasporic, on rhodolith, subtidal; gial sori formed in irregular shapes on both sides of upper JN150509-4, R2, 09 May 2015, female, tetrasporic, on blade. Tetrasporangial sori scattered on both surfaces of rhodolith, subtidal), Seongsan (JN100222-2, 22 Feb. the blade, small round to linear in shape; tetrasporan- 2010, male, on rope, subtidal); Busan, Korea: Gwan- gia spherical and tetrahedrally divided, arranged in two ganri (JN121103-10~13, 03 Nov. 2012, male, female, tet- layers, which are clearly separated by a central cell layer. rasporic, on metal piers, subtidal; JN121220-1~4, male, female, tetrasporic), Songjeongri (JN121103-80~83, Etymology 03 Nov. 2012, female, tetrasporic, on stone or bedrock, subtidal), Namchunli (NIBRAL0000126677, 01 Mar. 1965, The generic epithet (Neoharaldiophyllum) was chosen with tetrasporic, on rocks, subtidal, coll. J.W. Kang, identified the combination of “new” (neo), “Harald” (the first name as Myriogramme crozieri (Hooker f. et Harvey) Kylin); of Harald Kylin) and “phyllo” (leaf). Harald Kylin was the Taean, Korea: Sinjindo (JN160224-1~5, 24 Feb. 2016, first to observe the main characteristic of this genus and male, female, tetrasporic, on rope, subtidal), Padori mentioned the likely necessity of the new genus. (NIBRAL0000102466~8, 26 Dec. 2007, vegetative, coll. J.C. Kang et al.: Neoharaldiophyllum gen. nov. 519

E.H. Bae and K.H. Lee, identified as Myriogramme crozieri young cells near the meristematic portion, such as at the (Hooker f. et Harvey) Kylin); Haenam, Korea: Sinheungri thallus tip or upper margin, and are soon dissected into (JN140418-02~08, 18 Apr. 2014, female, tetrasporic, in small discoid granules (Figures 20–23). drift); Yeosu, Korea: Geomundo (JN160627-1~2, 27 Jun. The apical organization of the young thalli is pre- 2016, vegetative, on bedrock, subtidal); Wando, Korea: sented in Figures 25–27. The growth is initiated by trans- Cheongsando (JN160629-1~2, 29 Jun. 2016, vegetative, on verse (Figures 24 and 25), often oblique (Figure 26) bedrock, subtidal). division of an apical cell to form a primary cell row. OTHER TAXA EXAMINED: Haraldiophyllum crispa- The cells in the primary cell row are laterally divided tum: Wellington, New Zealand (Evans Bay: JN130826- to produce the second-order cell rows on both sides. 01~03, 26 Aug. 2013, female, tetrasporic, in drift; Muritai: The second-order cell rows are abaxially elongated by JN130914-15, 14 Sep. 2013, male, in drift; Chaffers marina: continued cell division and always extend to the blade JN140207-1, male, female, tetrasporic, in drift); Tasmania, margins. Most of the third- and higher-order cell rows Australia (Short beach, Sandy Bay: JN151105-1~27, 05 Nov. are cut off upwardly from the previous cell row and are 2015, male, female, tetrasporic, in drift; Pirates Bay, Eagle- abaxially elongated. The intercalary cell divisions often hawk Neck: 151108-84~85, 08 Nov. 2015, male, female, in occur in primary and higher-order cell rows. The traces drift). of oblique divisions are easily observed in any of these cell rows (Figure 24, red arrowheads). Some cells bud off thick- walled cells, which are linked to adjacent cells Habit and vegetative morphology by means of secondary pit connections (Figure 24, black arrows). The nature of the apical organization is main- Plants are erect and epilithic, up to 40 cm high, greenish tained in the mature thallus tip, including transverse red when alive and turning pink to red-brown upon drying. and oblique division of an apical cell (Figures 27 and 28). They consist of a disc-like or several fibrous holdfasts, a The blade margins are entire or denticulate with small variable length of subdichotomously dividing stipe, and spines (Figure 29 and 30). several membranous and broad linear or obovate blades, which are often deeply cleft with undulate margins and an acute to obtuse apex (Figures 1–10). Reproductive morphology Thalli are attached usually by means of a discoid holdfast and creeping stolons on the rock or rope (Figure 11), Gametophytes are dioecious. Procarps are scattered while the fibrous holdfasts are strongly developed when on both sides of thallus surface near the meristems or growing on rhodoliths (Figure 12). The stipe is cylindri- growing region (Figure 31). The fertile central cells cut off cal at the lower end and compressed toward the base of an anterior polygonal vegetative pericental cell and a pos- the blade, 0.5–1.2 mm thick (Figures 11–14). New blades terior roundish fertile pericentral cell, which correspond often arise from the stipe and creeping stolons (Figure 11). to a cover-cell group and a supporting cell, respectively In cross-sections of the stipe, 10–20 layers of irregularly (Figure 32). The supporting cell cuts off a first sterile-cell shaped cortical cells surround the central 8–15 layers of group initial (Figure 33) and then the carpogonial branch similarly sized and shaped cells, which are arranged in an initial, which forms a carpogonial branch by sequential elongated concentric circle (Figure 13). division (Figure 34–36). When the carpogonial branch The blades are at the ends of the compressed branches becomes three-celled, the supporting cell cuts off a basal extending from the stipe, which is cuneate to decurrent second sterile-cell group initial, which is usually once at the base (Figure 14). At the base of the blade, the eva- divided before fertilization (Figures 35–37). The mature nescent midrib-like or the fanwise radiating thickened procarp consists of a one- to two-celled cover-cell group, nerves from the end of the stipe were usually observed a supporting cell, a four-celled carpogonial branch with (Figure 14). The cells composing the nerves are uniformly a terminal trichogyne, a single-celled first sterile-cell isometric and arranged in horizontal tiers and vertical group, and usually a two-celled second sterile-cell group rows in cross-section view (Figures 15 and 16). The blade (Figure 36 and 37). This process of procarp development is thin and monostromatic except for the lower portion, occurs on both sides of the thallus from a common fertile basal nerves and reproductive structures (Figures 15–19). central cell (Figure 38): however, only one procarp is suc- There is no midrib or microscopic veins on the blade cessfully developed into a cystocarp, and the opposite one except the basal nerves. The cortical cells of the blade are remains as a trace while the opposite fertile sterile cell polygonal with parietal chloroplasts, which are lobed in could be easily observed under the floor cell layer of the 520 J.C. Kang et al.: Neoharaldiophyllum gen. nov.

Figures 1–10: Neoharaldiophyllum udoense (M.S. Kim et J.C. Kang) J.C. Kang et M.S. Kim comb. nov. from Korea. Thallus habit. (1) In situ habit from the subtidal of Udo, Jeju Island. (2–10) Pressed specimens on herbarium sheets from Udo, Jeju Island (2, JN1505098-R2, female; 3, JN120705-36, female; 4, 130607-1, tetrasporic), Busan (5, JN121103-11, female; 6, JN121220-01, male; 7, 121103-80, tetrasporic), Haenam (8, JN140418-06, female; 9, JN140418-07, tetrasporic), and Taean (10, JN160224-1, tetrasporic). Scale bars: 3, 5, 7 = 2 cm; 2, 4, 6, 8–10 = 5 cm. cystocarp during the maturation (Figures 40–43 and 45). was not observed. The auxiliary cell cuts off a gonimoblast Presumably after fertilization, the supporting cell cuts off initial, which forms chained gonimoblast cells (Figures 41 an auxiliary cell, and the carpogonium cuts off distal and and 42). In this stage, the auxiliary cell and support- proximal connecting cells (Figures 39 and 40). The fusion ing cell are not fused. As the number of gonimoblast between the auxiliary cell and proximal connecting cell cells increases, the auxiliary cell, supporting cell, both J.C. Kang et al.: Neoharaldiophyllum gen. nov. 521

Figures 11–30: Neoharaldiophyllum udoense (M.S. Kim et J.C. Kang) J.C. Kang et M.S. Kim comb. nov. Vegetative structures. (11–12) Holdfast and stipe from rope (11) and rhodolith (12). (13) Cross-section of stipe. (14) Basal portion of blade with the evanescent fanwise radiating thickened nerves. (15–19) Cross-sections of blade through base (15), nerves (16), lower (17), middle (18) and upper (19) portions of blade. (20–23) Cortical cells and chloroplasts from the near meristematic (20), upper (21), middle (22) and lower (23) parts of blade. (24) Diagram of apical organization of young blade with the order of cell-rows (numbers) and cells produced by intercalary divisions (i), thick-walled cells (black arrows) budded off from cortical cells, and linked by secondary pit connections to adjacent cells, and traces of oblique divisions (red arrowheads). (25–26) Tips of young thalli growing by means of transverse (25) and oblique (26) divisions of an apical cell. (27–28) Tips of mature blades with transversely (27) and obliquely (28) dividing apical cell. (29–30) Marginal crenulations. Scale bars: 12, 14 = 5 mm; 11 = 1 mm; 15–16 = 500 μm; 13, 17 = 200 μm; 18, 29 = 100 μm; 19, 25–28, 30 = 50 μm; 21–23 = 30 μm; 20 = 10 μm. 522 J.C. Kang et al.: Neoharaldiophyllum gen. nov. sterile-cell groups and inner gonimoblast cells are fused Molecular analysis and form a fusion cell (Figure 43). The primary gonimoblast filaments are extending along the floor of the cystocarp, We performed phylogenetic analyses of species in the while becoming incorporated with adjacent floor cells family Delesseriaceae using rbcL and LSU + rbcL sequences. and cut off secondary gonimoblast cells (Figures 43–45). The rbcL alignment included 1446 sites and 550 parsimony- In the cross-section of a mature cystocarp, the primary informative sites (38%). Combining the LSU and rbcL align- gonimoblast filaments appear to be forming roots while ment resulted in 3839 sites and 997 parsimony-informative they extend horizontally over the floor cells (Figures 44 sites (26%). Phylogenetic trees together with bootstrap value and 45). The fertile central cell does not participate in the for maximum likelihood (ML) are shown in Figures 55 and fusion cell and remains intact (Figures 41–45). The mature 56 that resulted in two subfamilies, Phycodryoideae and carposporangia are pyriform, 50–63 μm long and 25–37 Nitophylloideae, with good support. The subfamily Phy- μm wide, and are formed singly on each terminal end of codryoideae was divided into four tribes: Schizoserideae, the secondary gonimoblast filaments (Figures 44 and 46). Cryptopleureae, Myriogrammeae, and Phycodryeae. Newly The mature cystocarp is dome-shaped, 450–520 μm high generated rbcL sequences of Neoharaldiophyllum udoense and 1200–1500 μm in diameter, and has a pericarp with are identical to a specimen cited by Kim and Kang (2011). 5–6 cell layers and a non-protruding ostiole (Figures 44 Neoharaldiophyllum udoense is sister to Haraldiophyllum and 47). sp. from Chile and Haraldiophyllum mirabile from the USA Spermatangial sori are scattered on both surfaces of with 4.1–4.7% sequence divergence in rbcL gene. They are the blade and appear as white or pale spots. The sori are separated from a clade including Haraldiophyllum bonne- at first small round to elliptical shapes and elongated by maisonii and Haraldiophyllum crispatum with 100% sup- the coalescence of adjacent sori (Figure 48). In surface porting value. Our rbcL sequences from New Zealand and view of the sorus margin, 8–12 spermatangial mother cells Tasmania are identical to H. crispatum from GenBank are borne on a polygonal fertile central cell (Figure 49). (DQ916305) with no sequence variation. Neoharaldiophyl- The spermatangial sori are initiated in the monostromatic lum udoense has sequence divergences with H. crispatum portions of the blade. Spermatangial sori are initiated by and H. bonnemaisonii in rbcL of 5.4% and 6.4%, respec- two periclinal divisions of multiple cells in the unicellu- tively. Eight COI-5P sequences of N. udoense are totally lar portion of the blade to form three cell layers. At this identical (data not shown). stage, the innermost cell and two outermost cells correspond to the fertile central cell and spermatangial mother cell initials, respectively. The initials become horizontally divided several times, becoming spermatangial mother Discussion cells, which cut off one to two clavate spermatangia (Figures 50 and 51). When the genus Myriogramme Kylin (as well as the Myri- Tetrasporangial sori are scattered randomly over ogramme-group) was established, Kylin (1924) described both blade surfaces, except at the base of the blade and the genus based on heterogeneous taxa: the type species, along the margins, and are round to elliptical in shape M. livida (Hooker f. et Harvey) Kylin, for the cystocarp up to 800 μm wide (Figure 49). As blades continue to (illustration in Hooker 1847: pl. 179, figs. 3–4) and Myrio- grow, the sori coalesce with neighbors and elongated gramme minuta Kylin (now = Drachiella minuta (Kylin) (Figures 4 and 7). The cortical cells first divide pericli- Maggs et Hommersand: assigned to the tribe Schizos- nally to both sides, and the innermost cell becomes a erideae Hommersand et Fredericq at the present time) central cell while the two outermost cells are cortical for vegetative, tetrasporangial sori and procarp. Sub- cells. The central cell cuts off laterally a tetrasporan- sequently, the concept of Kylin’s Myriogramme corre- gial initial; then the cortical cells divide periclinally to sponded to the tribe Schizoserideae rather than to the produce subcortical cells and become five-cell layers. As Myriogrammeae Hommersand et Fredericq (Hommersand the sori grow, both cortical and subcortical cells divide and Fredericq 1997a,b). The major difference between the several times to become small cover cells. Each central two tribes Schizoserideae and Myriogrammeae could be cell can cut off the tetrasporangia towards both sides of observed in the structure of the procarp: the cover cell the thallus. The two layers of tetrasporangia are clearly group is absent in the former tribe while present in the separated by a central cell layer. Mature tetrasporangia latter (Hommersand and Fredericq 1997a,b). are spherical, 50–70 μm in diameter and tetrahedrally Because there was no information about the procarp divided (Figures 53 and 54). structure for the type species of Myriogramme until J.C. Kang et al.: Neoharaldiophyllum gen. nov. 523

Hommersand and Fredericq (1997a), there had been borne terminal carposporangia rather than the short- taxonomic confusion between Myriogramme and Nito- chained carposporangia on each distal end of gonimo- phyllum Greville (Kylin 1924, 1925, 1934, 1956, Yamada blast filaments (Figure 46). 1935, Mikami 1972, Zinova 1981). Kylin (1934) noticed that The vegetative and reproductive anatomies in Neo- some of the taxa that had been transferred from several haraldiophyllum and authentic Haraldiophyllum (Lin genera to Myriogramme by him in the previous study et al. 2007) are almost identical in terms of their blade (Kylin 1924) have a cover-cell group and single terminal structure, cell arrangement in cross-section, nerves, carposporangia, and he transferred again two species, M. shape of chloroplasts, composition and direction of bonnemaisonii Kylin and M. versicolor (Harvey) Kylin, to procarp formation, determination of sterile-cell groups Nitophyllum. Kylin (1934) also put a special emphasis on (fused with an auxiliary cell and a supporting cell in the differences between the type species of Nitophyllum, early stage of cystocarp development), and arrangement N. punctatum (Stackhouse) Greville, and the three species of carposporangia. However, significant differences of Nitophyllum, adding the species Nitophyllum mirabile, between these two genera are found in the carposporo- which he had described in a previous study (Kylin 1925) phyte and in the tetrasporangial sori. In Neoharaldio- of the other two species, namely N. bonnemaisonii and N. phyllum, a fusion cell does not fuse with floor cells of versicolor: the two pericentral cells cut off from a fertile the cystocarp cavity, the primary gonimoblast cells fuse central cell are arranged parallel to the axis of the growing secondarily with many floor cells of the cystocarp cavity thallus in N. punctatum, while those of the three species (Figures 43–46), and tetrasporangia are in two sym- are perpendicular. However, he assigned the three species metrical layers in cross-sections of tetrasporangial sori to Nitophyllum because he regarded the components of (Figure 54). In contrast, Haraldiophyllum has a fusion the procarp and the features of the carposporangia to be cell incorporated with a few of the floor cells of the similar to those features in Nitophyllum. cystocarp, free primary gonimoblast filaments without After Zinova (1981) established the genera Haraldio- secondary fusion and an asymmetric arrangement of phyllum and Hideophyllum, Hommersand and Fredericq tetrasporangia. (1997a) confirmed that the procarp from authentic mate- The differences between Neoharaldiophyllum and rial of M. livida is the same as that of Haraldiophyllum Haraldiophyllum that we described in the previous par- bonnemaisonii, Haraldiophyllum mirabile and Hideophyl- agraph are not newly observed characters; they were lum yezoense; however, the carposporangia are in chains already described by Kylin (1925, 1934). When Nito- unlike in Haraldiophyllum. Subsequently, they erected the phyllum mirabile (=Haraldiophyllum mirabile) was first tribe Myriogrammeae and included Myriogramme, Goni- described by Kylin (1925), he emphasized that the gonimocolax Kylin, Platyclinia J. Agardh, genera which had moblast filaments in the mature cystocarp are connected been assigned to Kylin’s Myriogramme-group (Kylin 1924, with many cells on the bottom of the cystocarp, unlike 1956), and added Haraldiophyllum and Hideophyllum to in ordinary Delesseriacean species. For these reasons, this tribe. The detailed observations on the type species he mentioned the possibility of a new genus for N. mira- of Haraldiophyllum, H. bonnemaisonii, were conducted by bile. In the later study (Kylin 1934), he again emphasized Maggs and Hommersand (1993) and Lin et al. (2007). the differences of the carposporophyte and arrangement In our study, all specimens of Neoharaldiophyllum of tetrasporangia between Nitophyllum mirabile and udoense (M.S. Kim et J.C. Kang) J.C. Kang et M.S. Kim Nitophyllum bonnemaisonii Greville (=Haraldiophyllum comb. nov. that are depicted in Figures 2–11 were con- bonnemaisonii), presenting illustrations of a sectioned firmed to be identical with the holotype of H. udoense cystocarp and a tetrasporangial sorus (Kylin 1934: M.S. Kim et J.C. Kang by means of rbcL sequence data figs. 2A–B). More than 70 years later, Lin et al. (2007) (Table 1). The habits of N. udoense from Jeju, Busan and mentioned again the differences among the species of Jindo of Korea (Figures 3, 5, 7 and 11) closely resemble Haraldiophyllum. Myriogramme livida (Hommersand and Fredericq 1997a: In the phylogenetic trees based on rbcL and LSU figs. 1–2) rather than the type species of Haraldiophyl- sequences (Figures 55 and 56), the tribe Myriogram- lum, H. bonnemaisonii (Lin et al. 2007: figs. 1–2) as well as meae, which uniquely among the subfamily Phycodry- other Haraldiophyllum species (Norris and Wynne 1969: oideae has the cover-cell group, forms a monophyletic figs. 3–4; Millar 1994: fig.1; Millar and Huisman 1996: clade as well as three other tribes. The clade of the tribe figs. 1–3; Lin et al. 2007: figs. 28–36). However, N. udoense Myriogrammeae is first divided by a feature of carpospo- should be closer to Haraldiophyllum than Myriogramme rangial arrangement, namely chained (Myriogramme) from an anatomical perspective in terms of the singly and terminal (Haraldiophyllum and Neoharaldiophyllum) 524 J.C. Kang et al.: Neoharaldiophyllum gen. nov. J.C. Kang et al.: Neoharaldiophyllum gen. nov. 525

Figures 31–47: Neoharaldiophyllum udoense (M.S. Kim et J.C. Kang) J.C. Kang et M.S. Kim comb. nov. Female reproductive structures. (31) Procarps (white arrowheads). (32) Young procarp with a vegetative pericentral cell (vpc) and a fertile pericentral cell (fpc); white arrow indicates direction to apex. (33) Young procarp with a cover-cell group (co), supporting cell (sc) and a first sterile group (st1). (34–35) Three- celled carpogonial branch (cb1, cb2, cbi). Note the second sterile group (st2) cut off from a supporting cell at this stage. (36–37) Below-surface and surface views of same point on mature procarp with a supporting cell (sc), a carpogonial branch (cb1, cb2, cb3) with a carpogonium (cp), a terminal trichogyne (tr), a one-celled first sterile-cell group (st1), one to two celled second sterile-cell group (st2), and a cover-cell group (co). (38) Cross-section through a pair of procarps formed from a common fertile central cell (fcc) on both sides of blade. (39) Early post-fertilization stage with two connecting cells (con) cut off from a carpogonium (cp), and an auxiliary cell (au) from a supporting cell (sc). (40–42) Young carposporophytes showing a fertile central cell (fcc), opposite a fertile pericentral cell (fpc), a supporting cell (sc), an auxiliary cell, a cover cell group (co), two sterile cell groups (st1, st2), a gonimoblast initial (goi) and several gonimoblast cells (gon). (43) Young cystocarp with a fusion cell (fu) and primary gonimoblast cells (gon1), which incorporate gametophytic cells (arrowheads) while extending onto floor of cystocarp cavity, and secondary gonimoblast cells (gon2). (44–46) Cross-sections through mature cystocarp. Note terminal carposporangia (ca) on secondary gonimoblast filaments, primary gonimoblast cells incorporated with floor cells of cystocarp (arrowheads, fc), and fertile central cell remaining intact (fcc). (47) Surface view of fully mature, hemispherical cystocarp. Scale bars: 47 = 2 mm; 31, 44 = 200 μm; 43, 45 = 100 μm; 40–42, 46 = 50 μm; 38–39 = 30 μm; 32–37 = 20 μm.

Figures 48–54: Neoharaldiophyllum udoense (M.S. Kim et J.C. Kang) J.C. Kang et M.S. Kim comb. nov. (48–51) Male reproductive structures. (48–49) Surface views of blades bearing spermatangial sori (ss) with spermatangial mother cells (sm), spermatangia (st), and sperm (sp). (50–51) Cross-sections through young (50) and mature (51) spermatangial sorus showing fertile central cells (cc) and spermatangial mother cell initial (smi), spermatangial mother cell (sm), and spermatangia (st). (52–54) Reproductive structures of tetrasporophyte. (52) Surface view of tetrasporangial sori (tss). (53–54) Cross-sections of young (53) and mature (54) tetrasporangial sorus showing tetrasporangial initials (ti) connected with inner fertile central cells (cc) by pit-connections (arrowheads), cortical cells (ct), subcortical cells (sct), and two layers of tetrasporangia. Scale bars: 48, 52 = 2 mm; 54 = 100 μm; 49–51, 53 = 50 μm. 526 J.C. Kang et al.: Neoharaldiophyllum gen. nov.

Figure 55: Maximum likelihood phylogenetic tree for the subfamily Phycodryoideae derived from plastid-encoded rbcL sequence data. Bootstrap values (1000 replicates) are shown above branches. Scale bar represents substitutions per site. carposporangia. The clade having terminal carpospo- secondary incorporation with floor cells) and Neoharal- rangia is divided again with 100% bootstrap support by diophyllum-type (i.e. gonimoblast filaments secondarily the shape of the carposporophyte, such as Haraldiophyl- incorporated with many floor cells of the cystocarp). The lum-type (i.e. gonimoblast filaments remain free without clade with Neoharaldiophyllum-type carposporophytes J.C. Kang et al.: Neoharaldiophyllum gen. nov. 527

Figure 56: Maximum likelihood phylogenetic tree for the subfamily Phycodryoideae derived from combined rbcL and LSU sequence data with bootstrap values (1000 replicates) above branches. Scale bar represents substitutions per site. To right of tree, diagrams of female reproductive structures for the clades Schizoserideae and Myriogrammeae. (A) Comparatively small fusion cell (fu: black cell), and secondarily fused primary gonimoblast filaments (gon: gray cells) with many floor cells of cystocarp in the genus Neoharaldiophyllum. (B) Free primary gonimoblast filaments (gon: gray cells) without secondary fusion, and a large fusion cell (fu: black cell) incorporated with a few floor cells of cystocarp in the genera Haraldiophyllum and Myriogramme. (C) Single terminal carposporangia in the genera Neoharaldiophyllum and Haraldiophyllum. (D) Chains of carposporangia in the genus Myriogramme and the tribe Schizoserideae. (E) Procarp with a cover cell group (co) and two sterile cell groups (gray cells) in the tribe Myriogrammeae. (F) Procarp with two sterile cell groups (gray cells) without any cover cell group in the tribe Schizoserideae. 528 J.C. Kang et al.: Neoharaldiophyllum gen. nov. includes H. mirabile, N. udoense, and Haraldiophyllum Types sp. from Chile (AF254188, rbcL; AF259430, LSU), which species were said by Lin et al. (2007) to have carposporo- Dennis J. Russell (Wynne 1203); fig. 1; 18 December 1967; phytes with a similar development to H. mirabile. 10 m depth. WTU 238051 (Norris and Wynne 1969: 143, After Zinova’s (1981) study, several Nitophyllum and Wynne 2014, Guiry and Guiry 2016). Myriogramme species, such as N. nottii R.E. Norris et M.J. Wynne, N. sinuosum Lucas, M. erosa (Harvey) Kylin Nomenclatural synonym and M. crispata (Hooker f. et Harvey) Kylin [=M. denticu- lata (Harvey) Kylin] were transferred to Haraldiophyllum Haraldiophyllum nottii (R.E. Norris et M.J. Wynne) M.J. (Wynne 1983, Millar 1990, Millar and Huisman 1996, Lin Wynne 1983: 444. et al. 2007). Later, two more species of Haraldiophyllum, H. infossum A.J.K. Millar and H. udoense, were described (Millar 1994, Kim and Kang 2011). Among those species, Type locality four, including H. mirabile, H. notii, H. erosum and H. udoense, were described as forming a Neoharaldiophyl- Hood Canal, 1 mile S. of Eldon, Mason County, Washing- lum-type carposporophyte. ton, USA (Norris and Wynne 1969: 143, Wynne 2014, Guiry Several recent taxonomic studies of molecular phylog- and Guiry 2016). eny with attention paid to female morphology indicated that the developmental patterns of carposporophytes are Distribution a proper character for defining boundaries at the generic level in the orders Gigartinales and Halymeniales, as well North America (British Columbia, Washington) (Guiry and as Ceramiales (Lin et al. 2002, 2012, Díaz-Tapia et al. 2013, Guiry 2016). Gargiulo et al. 2013). In our phylogenetic trees (Figures 55 and 56), the original concept of the Myriogramme-group by Kylin (1924) is first divided by procarp shape (i.e. with vs. Neoharaldiophyllum mirabile (Kylin) J.C. without a cover-cell group in the procarp) at the tribal level, Kang et M.S. Kim comb. nov. namely Schizoserideae and Myriogrammeae, followed by the arrangement of carposporangia (i.e. terminal vs. in Basionym chains) at the generic level or above. Also, it is reasonable to assume that the fate of primary gonimoblast filaments Nitophyllum mirabile Kylin, Lunds Univ. Årsskrift, N. F., (either fusing with many floor cells or remaining free) is one Andra Avd. 2, 21(9): 64, figs. 42–43, 1925. of the important characters for delimitation at the generic level. Isosyntypes By this collective evidence, including the morphology of the carposporophyte and phylogenetic relation- UC279580, female and tetrasporic, coll. H. Kylin, 24 June ships from rbcL and LSU, we propose to recognise the new 1924 (Hommersand and Fredericq 1997a). genus Neoharaldiophyllum based on N. udoense comb. nov. In addition, we transfer three more species of Haral- diophyllum with Neoharaldiophyllum-type carposporo- Nomenclatural synonym phytes, including H. erosum, H. mirabile and H. nottii, to the new genus Neoharaldiophyllum. Haraldiophyllum mirabile (Kylin) A.D. Zinova 1981: 13.

Type locality Neoharaldiophyllum nottii (R.E. Norris et M.J. Wynne) J.C. Kang et M.S. Kim comb. nov. Canoe Island, San Juan County, Washington, USA.

Basionym Distribution Nitophyllum nottii R.E. Norris et M.J. Wynne, Syesis 1: 141, figs. 1–8, 20, 22. 1969 [“1968”] (Wynne 2014, Guiry and North America (Alaska, British Columbia, Washington) Guiry 2016). (Guiry and Guiry 2016). J.C. Kang et al.: Neoharaldiophyllum gen. nov. 529 Kylin Gonimocolax A supporting cell, a A supporting cell, branch, carpogonial groups sterile two Parasitic In chains Unknown Secondarily fused with with fused Secondarily of floor cells many cystocarp Unknown australis Gonimocolax Kylin (Skottsberg) Kylin (1924), Wynne (1924), Wynne Kylin (2014)

J. Agardh Platyclinia A cover-cell group, group, A cover-cell a supporting cell, branch, carpogonial groups sterile two Independent In chains Unknown Wholly fused with with fused Wholly of floor cells of many cystocarp Asymmetrical in two in two Asymmetrical layers stipitata Platyclinia J.Agardh Womersley (2003), Womersley (2014) Wynne

Tokida) Tokida) Zinova Hideophyllum A cover-cell group, group, A cover-cell a supporting cell, two branch, carpogonial sterile groups Independent In chains Unknown Free without secondary secondary without Free of floor cells with fusion cystocarp Uncertain yezoense Hideophyllum et (Yamada A.D.Zinova Mikami (1972) Mikami

Harvey) Kylin Myriogramme A cover-cell group, group, A cover-cell a supporting cell, two branch, carpogonial sterile groups Independent In chains A supporting cell, an A supporting cell, inner cell, auxiliary a gonimoblast, primary a few cell, fertile central cystocarp of floor cells Free without secondary secondary without Free of floor cells with fusion cystocarp Uncertain Myriogramme livida Myriogramme et f. (Hooker Kylin Hommersand and and Hommersand (1997a) Fredericq

Zinova Haraldiophyllum A cover-cell group, supporting group, A cover-cell two branch, a carpogonial cell, sterile groups Independent Single, terminal A supporting cell, an auxiliary an auxiliary A supporting cell, inner groups, sterile-cell cell, a fertile gonimoblast, primary of floor cells a few cell, central cystocarp Free without secondary fusion fusion secondary without Free cystocarp of floor cells with Asymmetrical in two layers in two Asymmetrical Haraldiophyllum Haraldiophyllum bonnemaisonii (Kylin) A.D.Zinova Kylin (1934), Zinova (1981), Zinova (1934), Kylin Hommersand and Maggs (1994), Lin et al. (1993), Millar (2007)

M.S. Kim comb. nov. M.S. Kim comb. Neoharaldiophyllum gen. nov. Neoharaldiophyllum A cover-cell group, supporting group, A cover-cell two branch, a carpogonial cell, sterile groups Independent Single, terminal A supporting cell, an auxiliary an auxiliary A supporting cell, inner group, sterile-cell cell, gonimoblasts primary Secondarily fused with many many with fused Secondarily cystocarp of floor cells Symmetrical in two layers in two Symmetrical Neoharaldiophyllum udoense Neoharaldiophyllum J.C. J.C. Kang) (M.S. Kim et et Kang This study, Kylin (1925, 1934), (1925, Kylin study, This (1969), Wynne and Norris (1996), Huisman and Millar (2003) Womersley

Comparison of diagnostic morphological characters among genera assigned to the tribe Myriogrammeae. the tribe to assigned among genera characters morphological diagnostic of 2: Comparison Table Genus Composition of of Composition procarp Host Carposporangia Participating Participating for components fusion of formation cell Primary Primary gonimoblast filaments Arrangement of of Arrangement tetrasporangia Type species Type References 530 J.C. Kang et al.: Neoharaldiophyllum gen. nov.

Neoharaldiophyllum erosum (Harvey) J.C. Neoharaldiophyllum and Haraldiophyllum have many Kang et M.S. Kim comb. nov. morphological features in common, except for the final development of the primary gonimoblast filaments (sec- Basionym ondarily fused with floor cells of cystocarp vs. remaining free without secondary incorporation), size of fusion cell Nitophyllum erosum Harvey, Phycologia Australica, vol. 2: (comparatively small vs. large), fusion cell formation (not pl. XCIV. 1859. incorporated with any floor cells vs. incorporated with a few floor cells) and arrangement of tetrasporangia (clearly separated into two layers vs. irregularly arranged; Table 2). Lectotype In this study, only three species of Haraldiophyllum (i.e. Haraldiophyllum bonnemaisonii, Haraldiophyllum crispa- Clifton; Herb. Harvey, TCD (Womersley 2003, Guiry and tum and H. infossum) with a Haraldiophyllum-type carpo- Guiry 2016). sporophyte (Millar 1994: figs. 6 and 15; Lin et al. 2007: figs. 27 and 60) remain in the genus Haraldiophyllum. On the Korean coast, four species assigned to the tribe Nomenclatural synonyms Myriogrammeae, including Myriogramme livida, H. bonnemaisonii, Haraldiophyllum udoense and Hideophyllum Aglaophyllum erosum (Harvey) Kützing 1869: 2, pl. 6c, d; yezoense, have been recorded (Nam and Kang 2012). Scutarius erosus (Harvey) Kuntze 1891: 920; Myriogramme Although Nam and Kang (2012) commented that Korean erosa (Harvey) Kylin 1924: 61; Haraldiophyllum erosum H. bonnemaisonii is a different species from H. udoense, (Harvey) A.J.K. Millar et Huisman 1996: 62, figs. 1–14 their figure of a cross-sectioned cystocarp (see Nam and (Wynne 2014, Guiry and Guiry 2016). Kim 1996: 104, fig. 15) is in agreement with that of Neo- haraldiophyllum. Moreover, Nam and Kim (1996) clearly described that “extreme elaboration of fusion cell forming Taxonomic synonym numerous connections with content-rich cells is observed in the floor of the cystocarps”. That character does not Nitophyllum fimbriatum Harvey 1855: 549, nom. illeg. non belong to Haraldiophyllum (Maggs and Hommersand 1993, N. fimbriatum Greville (in Saint-Hilaire 1833) (Wynne 2014, Lin et al. 2007) but to Neoharaldiophyllum. This suggests Guiry and Guiry 2016). that the so-called H. bonnemaisonii from Korea should be the same species as Neoharaldiophyllum udoense. With regard to the two other species, M. livida and H. yezoense Type locality reported from the Korean coast, their identity is still in doubt because the arrangement of carposporangia and Garden Island, Western Australia (Millar and Huisman molecular data for Korean material remain unknown for 1996: 64, Wynne 2014, Guiry and Guiry 2016). these species. We confirmed that specimens of M. livida from Korea deposited in NIBR are all conspecific with N. udoense. So we suggest that many of the specimens identi- Distribution fied as M. livida in Korea might be N. udoense. In late 2015, we had the opportunity to collect some Southern and Western Australia (Wynne 2014, Guiry and specimens of H. notii (identified according to Womers- Guiry 2016). ley 2003) from Tasmania and then compared their rbcL sequences (Table 1). All of the sequences were identical The new genus Neoharaldiophyllum is clearly a with sequence-data of H. crispatum from New Zealand member of the tribe Myriogrammeae based on the vegeta- (Figure 55). The Haraldiophyllum species known as H. tive morphology, conformation of procarp and molecular notii in Australia does not have the Neoharaldiophyllum- evidence (Hommersand and Fredericq 1997a, this study). type carposporophyte but the Haraldiophyllum-type (see Neoharaldiophyllum is separated from other genera in Womersley 2003: fig. 60). Moreover, the cross-section of the tribe Myriogrammeae (Myriogramme, Hideophyllum the tetrasporangial sorus (see Womersley 2003: fig. 59E) and Platyclinia) by the arrangement of carposporangia agrees with that of Haraldiophyllum. Consequently, the so- (singly terminal vs. in chains) and from Gonimocolax by called H. notii reported by Womersley (2003) in Australia their substratum (epilithic vs. parasitic on other genera). should be identified as H. crispatum. J.C. Kang et al.: Neoharaldiophyllum gen. nov. 531

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Caractéristiques et d’un Précis de L’historie des Révolutions de L’empire Brésilien, Depuis le Commencement du Règne de Bionotes Jean vi Jusqu’à L’abdication de D. Pedro. Seconde Partie. Gide, Paris, pp. 457. Jeong Chan Kang Saunders, G.W. and T.E. Moore. 2013. Refinements for the amplifi- Department of Biology, Jeju National cation and sequencing of red algal DNA barcode and RedToL University, Jeju 690-756, Korea phylogenetic markers: a summary of current primers, profiles and strategies. Algae 28: 31–43. Sherwood, A.R., T. Sauvage, A. Kurihara, K.Y. Conklin and G.G. Prest- ing. 2010. A comparative analysis of COI, LSU and UPA marker data for the Hawaiian florideophyte Rhodophyta: implications for DNA barcoding of red algae. Cryptog. Algol. 31: 451–465. Sievers, F., A. Wilm, D. Dineen, T.J. Gibson, K. Karplus, W. Li, R. Lopez, H. McWilliam, M. Remmert, J. Söding, J.D. Thompson Jeong Chan Kang is a postdoctoral researcher at the Department and D.G. Higgins. 2011. Fast, scalable generation of high‐qual- of Biology of Jeju National University, Korea. He specialises in ity protein multiple sequence alignments using Clustal Omega. the family Delesseriaceae, and has interests in the relationships Mol. Syst. Bio. 7: 539. between molecular phylogeny and anatomical morphology. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood- based phylogenetic analyses with thousands of taxa and mixed mod- Mi Yeon Yang els. Bioinformatics 22: 2688–2690. Department of Biology, Jeju National Wittmann, W. 1965. Aceto-iron-haematoxylin-chloral hydrate for University, Jeju 690-756, Korea chromosome staining. Biotech. Histochem. 40: 161–164. Womersley, H.B.S. 2003. The Marine Benthic Flora of Southern Australia. Rhodophyta. Part IIID: Ceramiales – Delesseriaceae, Sarcomeniaceae, Rhodomelaceae. Australian Biological Resources Study and the State Herbarium of South Australia, Canberra. pp. 533. Wynne, M.J. 1983. The current status of genera in the Delesseriaceae Mi Yeon Yang is a PhD student at the Department of Biology of Jeju (Rhodophyta). Bot. Mar. 26: 437–450. National University, Korea. She studies the diversity and phylogeny Wynne, M.J. 2014. The Red Algal Families Delesseriaceae and of the other Gigartinales by morphology and molecular analyses Sarcomeniaceae. Koeltz Scientific Books: Königstein, Germany. from Korea. pp. 326. Yamada, Y. 1935. Notes on some Japanese algae, VI. Sci. Papers. Myung Sook Kim Inst. Algol. Res., Fac. Sci., Hokkaido Imp. Uni. 1: 27–35, pls Department of Biology, Jeju National 11–16. University, Jeju 690-756, Korea; and Zinova, A.D. 1981. De positione systematica Nitophyllum (Myrio- Research Institute for Basic Sciences, Jeju gramme) yezoensis (Yamada et Tokida) Mikami (Delesse- National University, Jeju 690-756, Korea, riaceae). Novit. System. Plant. Non Vascul. 18: 10–15. [email protected]

Myung Sook Kim is a Professor of Biology at Jeju National University, Jeju, Korea. She has studied systematics in Rhodophyta, especially in the family Rhodomelaceae. Her more recent research has concentrated on establishing a DNA barcode database for Korean seaweeds.