Haraldiophyllum udoense M.S. Kim et J.C. Kang, ALGAE

Kang, Jeong Chan, Yang, Mi Yeon & Kim, Myung Sook, 2017, Neoharaldiophyllum, a new genus of Delesseriaceae (Rhodophyta) based on carposporophyte development and molecular data, Botanica Marina (Warsaw, Poland) 60 (5), pp. 515-532 : 518-522

Haraldiophyllum udoense M.S. Kim et J.C. Kang, ALGAE 26: 212, figs. 1–3 (as “ udoensis ”) (2011).

Holotype

JNUB ( MSK30601 HU, tetrasporophyte, 14 Jun. 2009).

Type locality

Haumokdong , Udo, Jeju Island, Korea (33°30´21˝ N, 126°56´09˝ E) .

Distribution

Southern and western coasts of Korea including Jeju Island (This study, Kim and Kang 2011).

Specimens examined

Jeju Island, Korea: Udo (JN120705-34~36, 05 Jul. 2012, female, tetrasporic, on rhodolith, subtidal; JN130607-1, 07 Jun. 2013, female, tetrasporic, on rhodolith, subtidal; JN150509-4, R2, 09 May 2015, female, tetrasporic, on rhodolith, subtidal), Seongsan (JN100222-2, 22 Feb. 2010, male, on rope, subtidal); Busan, Korea: Gwanganri (JN121103-10~13, 03 Nov. 2012, male, female, tetrasporic, on metal piers, subtidal; JN121220-1~4, male, female, tetrasporic), Songjeongri (JN121103-80~83, 03 Nov. 2012, female, tetrasporic, on stone or bedrock, subtidal), Namchunli (NIBRAL0000126677, 01 Mar. 1965, tetrasporic, on rocks, subtidal, coll. J.W. Kang, identified as Myriogramme crozieri (Hooker f. et Harvey) Kylin ); Taean, Korea: Sinjindo (JN160224-1~5, 24 Feb. 2016, male, female, tetrasporic, on rope, subtidal), Padori (NIBRAL0000102466~8, 26 Dec. 2007, vegetative, coll. E.H. Bae and K.H. Lee, identified as Myriogramme crozieri (Hooker f. et Harvey) Kylin ); Haenam, Korea: Sinheungri (JN140418-02~08, 18 Apr. 2014, female, tetrasporic, in drift); Yeosu, Korea: Geomundo (JN160627-1~2, 27 Jun. 2016, vegetative, on bedrock, subtidal); Wando, Korea: Cheongsando (JN160629-1~2, 29 Jun. 2016, vegetative, on bedrock, subtidal).

OTHER TAXA EXAMINED: Haraldiophyllum crispatum : Wellington, New Zealand (Evans Bay: JN130826- 01 ~03, 26 Aug. 2013, female, tetrasporic, in drift; Muritai: JN130914-15 , 14 Sep. 2013, male, in drift; Chaffers marina: JN140207-1 , male, female, tetrasporic, in drift); Tasmania, Australia ( Short beach, Sandy Bay : JN151105-1 ~27, 05 Nov. 2015, male, female, tetrasporic, in drift; Pirates Bay , Eaglehawk Neck : 151108-84~85, 08 Nov. 2015, male, female, in drift) .

Habit and vegetative morphology

Plants are erect and epilithic, up to 40 cm high, greenish red when alive and turning pink to red-brown upon drying. They consist of a disc-like or several fibrous holdfasts, a variable length of subdichotomously dividing stipe, and 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 ).

Thalli are attached usually by means of a discoid holdfast and creeping stolons on the rock or rope ( Figure 11 ), while the fibrous holdfasts are strongly developed when growing on rhodoliths ( Figure 12 ). The stipe is cylindrical at the lower end and compressed toward the base of the blade, 0.5–1.2 mm thick ( Figures 11–14 ). New blades often arise from the stipe and creeping stolons ( Figure 11 ). In cross-sections of the stipe, 10–20 layers of irregularly shaped cortical cells surround the central 8–15 layers of similarly sized and shaped cells, which are arranged in an elongated concentric circle ( Figure 13 ).

The blades are at the ends of the compressed branches extending from the stipe, which is cuneate to decurrent at the base ( Figure 14 ). At the base of the blade, the evanescent midrib-like or the fanwise radiating thickened nerves from the end of the stipe were usually observed ( Figure 14 ). The cells composing the nerves are uniformly isometric and arranged in horizontal tiers and vertical rows in cross-section view ( Figures 15 and 16 ). The blade is thin and monostromatic except for the lower portion, basal nerves and reproductive structures ( Figures 15–19 ). There is no midrib or microscopic veins on the blade except the basal nerves. The cortical cells of the blade are polygonal with parietal chloroplasts, which are lobed in young cells near the meristematic portion, such as at the thallus tip or upper margin, and are soon dissected into small discoid granules ( Figures 20–23 ).

The apical organization of the young thalli is presented in Figures 25–27 . The growth is initiated by transverse ( Figures 24 and 25 ), often oblique ( Figure 26 ) division of an apical cell to form a primary cell row. The cells in the primary cell row are laterally divided to produce the second-order cell rows on both sides. The second-order cell rows are abaxially elongated by continued cell division and always extend to the blade margins. Most of the third- and higher-order cell rows are cut off upwardly from the previous cell row and are abaxially elongated. The intercalary cell divisions often occur in primary and higher-order cell rows. The traces 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 by means of secondary pit connections ( Figure 24 , black arrows). The nature of the apical organization is maintained in the mature thallus tip, including transverse and oblique division of an apical cell ( Figures 27 and 28 ). The blade margins are entire or denticulate with small spines ( Figure 29 and 30 ).

Reproductive morphology

Gametophytes are dioecious. Procarps are scattered on both sides of thallus surface near the meristems or growing region ( Figure 31). The fertile central cells cut off an anterior polygonal vegetative pericental cell and a posterior roundish fertile pericentral cell, which correspond to a cover-cell group and a supporting cell, respectively ( Figure 32). The supporting cell cuts off a first sterile-cell group initial ( Figure 33) and then the carpogonial branch initial, which forms a carpogonial branch by sequential division ( Figure 34–36). When the carpogonial branch becomes three-celled, the supporting cell cuts off a basal second sterile-cell group initial, which is usually once divided before fertilization ( Figures 35–37). The mature procarp consists of a one- to two-celled cover-cell group, a supporting cell, a four-celled carpogonial branch with a terminal trichogyne, a single-celled first sterile-cell group, and usually a two-celled second sterile-cell group ( Figure 36 and 37). This process of procarp development occurs on both sides of the thallus from a common fertile central cell ( Figure 38): however, only one procarp is successfully developed into a cystocarp, and the opposite one remains as a trace while the opposite fertile sterile cell could be easily observed under the floor cell layer of the cystocarp during the maturation ( Figures 40–43 and 45). Presumably after fertilization, the supporting cell cuts off an auxiliary cell, and the carpogonium cuts off distal and proximal connecting cells ( Figures 39 and 40). The fusion between the auxiliary cell and proximal connecting cell was not observed. The auxiliary cell cuts off a gonimoblast initial, which forms chained gonimoblast cells ( Figures 41 and 42). In this stage, the auxiliary cell and supporting cell are not fused. As the number of gonimoblast cells increases, the auxiliary cell, supporting cell, both sterile-cell groups and inner gonimoblast cells are fused and form a fusion cell ( Figure 43). The primary gonimoblast filaments are extending along the floor of the cystocarp, while becoming incorporated with adjacent floor cells and cut off secondary gonimoblast cells ( Figures 43–45). In the cross-section of a mature cystocarp, the primary gonimoblast filaments appear to be forming roots while they extend horizontally over the floor cells ( Figures 44 and 45). The fertile central cell does not participate in the fusion cell and remains intact ( Figures 41–45). The mature carposporangia are pyriform, 50–63 µm long and 25–37 µm wide, and are formed singly on each terminal end of the secondary gonimoblast filaments ( Figures 44 and 46). The mature cystocarp is dome-shaped, 450–520 µm high and 1200–1500 µm in diameter, and has a pericarp with 5–6 cell layers and a non-protruding ostiole ( Figures 44 and 47).

Spermatangial sori are scattered on both surfaces of the blade and appear as white or pale spots. The sori are at first small round to elliptical shapes and elongated by the coalescence of adjacent sori ( Figure 48 ). In surface view of the sorus margin, 8–12 spermatangial mother cells are borne on a polygonal fertile central cell ( Figure 49 ). The spermatangial sori are initiated in the monostromatic portions of the blade. Spermatangial sori are initiated by two periclinal divisions of multiple cells in the unicellular portion of the blade to form three cell layers. At this 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 cells, which cut off one to two clavate spermatangia

( Figures 50 and 51 ).

Tetrasporangial sori are scattered randomly over both blade surfaces, except at the base of the blade and along the margins, and are round to elliptical in shape up to 800 µm wide ( Figure 49 ). As blades continue to grow, the sori coalesce with neighbors and elongated ( Figures 4 and 7 ). The cortical cells first divide periclinally to both sides, and the innermost cell becomes a central cell while the two outermost cells are cortical cells. The central cell cuts off laterally a tetrasporangial initial; then the cortical cells divide periclinally to produce subcortical cells and become five-cell layers. As the sori grow, both cortical and subcortical cells divide several times to become small cover cells. Each central cell can cut off the tetrasporangia towards both sides of the thallus. The two layers of tetrasporangia are clearly separated by a central cell layer. Mature tetrasporangia are spherical, 50–70 µm in diameter and tetrahedrally divided ( Figures 53 and 54 ).

Molecular analysis

We performed phylogenetic analyses of species in the family Delesseriaceae using rbc L and LSU + rbc L sequences. The rbc L alignment included 1446 sites and 550 parsimony-informative sites (38%). Combining the LSU and rbc L alignment resulted in 3839 sites and 997 parsimony-informative sites (26%).Phylogenetic trees together with bootstrap value for maximum likelihood (ML) are shown in Figures 55 and 56 that resulted in two subfamilies, Phycodryoideae and Nitophylloideae, with good support. The subfamily Phycodryoideae was divided into four tribes: Schizoserideae , Cryptopleureae, Myriogrammeae , and Phycodryeae. Newly generated rbc L sequences of Neoharaldiophyllum udoense are identical to a specimen cited by Kim and Kang (2011). Neoharaldiophyllum udoense is sister to Haraldiophyllum sp. from Chile and Haraldiophyllum mirabile from the USA with 4.1–4.7% sequence divergence in rbc L gene. They are separated from a clade including Haraldiophyllum bonnemaisonii and Haraldiophyllum crispatum with 100% supporting value. Our rbc L sequences from New Zealand and Tasmania are identical to H. crispatum from GenBank (DQ916305) with no sequence variation. Neoharaldiophyllum udoense has sequence divergences with H. crispatum and H. bonnemaisonii in rbc L of 5.4% and 6.4%, respectively. Eight COI-5P sequences of N. udoense are totally identical (data not shown).

Figures

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.

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.

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.

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.

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.