Codium Tenuifolium (Codiales, Chlorophyta), a New Species from Japan

植物研究雑誌 Originals J. Jpn. Bot. 82: 117–125 (2007)

Codium tenuifolium (Codiales, Chlorophyta), a New Species from Japan

Satoshi SHIMADAa, Tomoya TADANOb and Jiro TANAKAb

aCriative Research Initiative “Sousei”, Hokkaido University, Sapporo, 060-0810 JAPAN; E-mail: [email protected] bDepartment of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo, 108-847, JAPAN (Received on June 6, 2006)

A new species, Codium tenuifolium Shimada, Tadano & J. Tanaka, is described from the Pacific coast of Japan. It is characterized in having thin blades, clavate utricles with tumid head and vegetative reproductive structures. In phylogenetic analysis of the sequences of the chloroplast encoded rbcL gene, C. tenuifolium formed a monophyletic clade with C. latum Suringar (with 69 bootstrap support). The pairwise distance between them is 17 bp (2.6 ). Codium tenuifolium is morphologically distinguished from C. latum by almost simple and thinner thallus, shorter and broader clavate utricles.

Key words: Chlorophyta, Codium, Codium tenuifolium,molecular phylogeny, rbcL.

The green algal genus Codium currently results of analysis of rbcL sequences. includes 98 species, seven subspecies, three varieties and one form (AlgaeBase: http:// Materials and Methods www.algaebase.org/). Of these, 19 species Eighteen samples of Codium tenuifolium have been reported from Japan (Yoshida were collected from four localities (Table 1, et al. 2005). Recent molecular analysis of Fig. 1), and were transported alive to Tokyo Codium shows that DNA sequence data can University of Marine Science and provide clues to the phylogeny of this genus Technology for measurement of the length (Shimada et al. 2004). Shimada et al. (2004) and width of thallus, the thickness of the considered one collection from Kochi Pre- thallus, the major and minor axis of holdfast, fecture, Japan (21 Jun. 2000, SAP 101915) length of stipe, length and diameter of as a distinct species, “Codium pseudolat- utricles, and the major and minor axis of um”. However, this name was a nomen vegetative reproduction structures. Thalli nudum because it was written by Dr. Y. examined were dried on herbarium sheets, Yamada on the herbarium sheet. On the and voucher specimens are deposited in the other hand Codium pseudolatum herbarium of the Tokyo University of Nizamuddin was validly published from the Marine Science and Technology (MTUF-A Arabian Sea (Nizamuddin 2001). Therefore, L-20001–20018). Furthermore, specimens a new name, Codium tenuifolium,ispro- identified as “Codium pseudolatum”by posed for “Codium pseudolatum” from Yamada and Shimada, which are preserved Pacific coastal Japan (Shimada et al. 2004) in SAP were examined for the identification and is described in this paper. The of Codium tenuifolium (Table 1). phylogenetic position of this alga within the Total DNA was extracted from four sam- genus Codium is also discussed based on the ples of Codium tenuifolium shown in Table 2

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Table 1. Specimens of Codium teniufolium used in this study

Voucher specimen Collection site SAP24499 Suga Island, Mie Pref. (May 1943) SAP24500 Suga Island, Mie Pref. (May 1943) SAP101915 Susaki, Kochi Pref. (21 Jun. 2000) MTUF-AL-20001–20008 Banda, Tateyama, Chiba Pref. (16 May 2003) MTUF-AL-20009 Yumigahama, Minamiizu, Shizuoka Pref. (18 Mar. 2002) MTUF-AL-20010 Touji, Shimoda, Shizuoka Pref. (18 Mar. 2002) MTUF-AL-20011–20014 Touji, Shimoda, Shizuoka Pref. (12 Jul. 2002) MTUF-AL-20015–20016 Nemoto, Shirahama, Chiba Pref. (16 May 2003) MTUF-AL-20017– 20018 Banda, Tateyama, Chiba Pref. (19 May 2003)

Fig. 1. Map showing localities where materials were collected. 1. Susaki, Kochi Pref. (21 Jun. 2000). 2. Suga Island, Mie Pref. (May 1943). 3. Yumigahama, Minamiizu, Shizuoka Pref. (18. Mar. 2002). 4. Touji, Shimoda, Shizuoka Pref. (18. Mar. 2002, 12. Jul. 2002). 5. Banda, Chiba Pref. (16 May 2003, 19. May 2003). 6. Nemoto, Shirahama, Chiba Pref. (16 May 2003). using the Dneasy Plant Mini Kit (QIAGEN, outgroups (X 55877). The alignment is avail- Valencia, CA, U.S.A.) following the able from the first author upon request. protocol of the manufacturer. PCR amplifi- The Maximum likelihood (ML) analysis cation and sequencing of the chloroplast was implemented with PAUP* 4.0b10 encoded rbcL gene were performed accord- (Swofford 2002). The ML parameters were ing to Shimada et al. (2004). The rbcL se- estimated using the ML ratio test. The pro- quences were aligned manually and no gram MODELTEST version 3.06 (Posada insertion–deletion mutations were detected. and Crandall 1998) was used to find the Sequences of 17 species of Codium were model of sequence evolution that best fitted obtained from GenBank and included in the each data set by a hierarchical likelihood alignment (Table 2). Caulerpa prolifera ratio test (0.01). When the best sequence (Forsskål) Lamouroux (AY 942173) and evolution model was determined, ML tree Bryopsis maxima Okamura were used as search was performed using the estimated June 2007 Journal of Japanese Botany Vol. 82 No. 3 119

Table 2. Algol species where rbcL sequences were used in this molecular analysis

Taxon GenBank number Caulerpa prolifera AY942173 Bryopsis maxima X55877 Codium minus AB102958 C. arabicum AB102989 C. capitulatum AB102961 C. hubbsii AB102965 C. spongiosum AB102978 C. lucasii AB102980 C. dimorphum AB103009 C. intricatum AB102990 C. contractum AB102995 C. subtubulosum AB102997 C. repens AB103001 C. barbatum AB103007 C. fragile M67453 C. yezoense AB103024 C. cylindricum AB103025 C. latum AB103002 C. teniufolium (as C. pseudolatum; Shimada et al. 2004) AB103006 C. teniufolium 20001 AB277760 C. teniufolium 20002 AB277761 C. teniufolium 20003 AB277762 C. teniufolium 20004 AB277763

parameters with the following options: start- AL-20002–4 (isotype). ing tree option obtained by neighbor join- Thallus composed of tenuous, flat, blade- ing, and branch swapping algorithm TBR. like or broadly expanded erect fronds, 20–60 Bootstrap analysis based on 100 re- cm high, 10–30 cm wide, 0.65–1.40 mm samplings of the data set (Felsenstein 1985) thick. Fronds simple or sometimes branched was calculated (TBR, full heuristic search once. Utricles clavate with head tumid, 360– option). 720 µm long, 48–160 µm in diameter. Vegetative reproduction structures irregu- Results larly formed, 33–117 µm in diameter, 45– Codium tenuifolium Shimada, Tadano & 234 µm long, borne throughout the interme- J. Tanaka, sp. nov. [Figs. 2–7.] diary plug of medullary filaments or some- Thallus erectus et tenuifolius, 20–60 cm times special at the base of structures bearing altus, 10–30 cm latus, 0.65–1.40 mm utricles base. crassus. Axes simplices vel 1 pro ramosi. Japanese name: Usuba-miru. Utriculi clavatus 360–720 µm longus, 48– Distribution: Kochi, Mie, Shizuoka and 160 µm diam. Structura reproductivus, saepe Chiba Prefectures, Japan. e basi utriculorum orientia, 33–117 µm Other specimens examined: Kochi Pref.: Susaki, 21 diam., 45–234 µm longa. Jun. 2000, Y. Serisawa, SAP 101915. Mie Pref.: Suga TYPE: JAPAN; Banda, Tateyama, Chiba Island, May 1943, Y. Yamada, SAP 24499, 24500. Prefecture, 16, May 2003, T. Tadano, Shizuoka Pref.: Yumigahama, Minamiizu, 18. Mar. 2002, T. Tadano, MTUF-AL-20009; Touji, Shimoda, MTUF-AL-20001 (holotype; Fig. 1), MTUF- 120 植物研究雑誌第82巻第3号平成19年6月

Figs. 2–3. Voucher specimens of Codium tenuifolium. Fig. 2. Holotype (Herbarium of the Tokyo Univer- sity of Marine Science and Technology, MTUF-AL-20001). Fig. 3. Yamada’s “C. pseudolatum” (SAP 24500).

18. Mar. 2002, T. Tadano, MTUF-AL-20010, 12. Jul. in height and 10.0–(17.9 ± 10.2)–30.0 cm in 2002, T. Tadano, MTUF-AL-20011–20014. Chiba width, entirely complanate and unbranched Pref.: Banda, 16 May 2003, T. Tadano, MTUF-AL-2 or branched once above a terete stipe, 5.0– 0001–20008, 19. May 2003, T. Tadano, MTUF-AL-2 0017–20018; Nemoto, Shirahama, 16 May 2003, T. (7.1 ± 2.6)–10.0 mm (Figs. 2, 3). Blades Tadano, MTUF-AL-20015–20016. are flat, tenuous, 0.7–1.4 mm in thickness. Utricles are clavate, possessing tumid head, Habitat and seasonality Codium tenuifoli- 360.0–(523.0 ± 67.8)–720.0 µm in length, um solely grows on rocks 8–12m deep. 48.0–(80.0 ± 23.4)–160.0 µm in diameter Thalli are supposed to be present in winter to (Figs. 4–6). early summer. Mature thalli possessing vegetative reproduction structures were collected Reproductive morphology Vegetative re- from April to June and thalli disappeared in production structures are dark green, variable July. in shape, irregularly formed at base of utricles or medullary filaments, 33.0–(72.0 ± Vegetative morphology Thalli are light 19.1)–117.0 µm in diameter, 45.0–(115.0 ± green, erect from elliptical holdfast (6.9 ± 34.5)–234.0 µm long (Figs. 4, 6, 7). 1.6 × 4.6 ± 1.2), 20.0–(37.5 ± 21.0)–60.0 cm rbcL analysis All rbcL sequences of C. June 2007 Journal of Japanese Botany Vol. 82 No. 3 121

Figs. 4–7. Microscopic observations of Codium tenuifolium. Fig. 4. Utricles of fresh materials, with vegetative reproduction structures (MTUF-AL-20001, arrows). Fig. 5. Utricles from dry specimen (SAP 24500). Fig. 6. Utricles of fresh materials, with vegetative reproduction structure (SAP 101915, arrow). Fig. 7. Vegetative reproduction structures (MTUF-AL-20001, arrows). Scale 100 µm. tenuifolium and “C. pseudolatum”by with Codium latum Suringar (with 69 Shimada et al. (2004) were identical. The bootstrap support in ML). The pairwise dis- phylogenetic tree obtained from ML analysis tance between our alga and C. latum is 17 bp is presented in Fig. 8. The identical se- (2.6 ). quences of our alga were excluded from the alignment. For the ML method, likelihood Discussion settings from the best-fit model (F81 + I + Codium tenuifolium can be distinguished G) were selected by a hierarchical likelihood from other blade-like species of Codium as ratio test in the program MODELTEST ver- follows. Codium latum has slender blades, sion 3.06: assumed nucleotide frequencies branches up to twice and possesses longer A 0.3216, C 0.1518, G 0.1846, and T (50–60 cm) and slender cylindrical utricles 0.342; proportion of invariable sites (Figs. 9, 10). Codium platylobium Areschoug 0.4371; gamma distribution with shape pa- has larger thalli (up to 200 cm long), rameter 0.3392. Based on these settings, branches up to eight orders and possesses the heuristic search was performed with the longer (500–750 µm) and broader (115–330 TBR branch swapping option (-ln L µm) utricles (Silva 1959). Codium palmeri 3504.63918) after 2323 rearrangements (Fig. Dawson has thicker thallus and cylindrical 8). Our alga forms a monophyletic clade utricles with rounded apex (Dawson 1945). 122 植物研究雑誌第82巻第3号平成19年6月

Fig. 8. Phylogenetic tree of Codium species inferred from ML analysis of chloroplast encoded rbcL sequences. All sites were treated as unordered and equally weighted; only values above 50 bootstrap support (100 replicates, full heuristic search with TBR method) are shown.

Codium laminarioides Harvey possesses tree, C. tenuifolium was shown to be most thicker thallus (1.5–2 mm) and narrowly el- closely related to C. latum with 17 substitu- lipsoid to cylindrical utricles (Silva and tions being found between the two species. Womersley 1956). Codium pseudolatum Codium latum was described as a new spe- Nizarmuddin has slender blades, branches up cies by Suringar (1867) in Japan. Okamura to six orders, and possesses slender, cylindri- (1915) reported the detail observation of this cal, pyriform, obovate utricles (Nizamuddin species. In the morphological aspect, C. 2001) (Table 3). Taking these clear differ- tenuifolium can be distinguished from C. ences in morphology into consideration, we latum as mentioned above. The two species conclude that our alga should be treated as are also different in habitat: C. latum grows an independent species. on rocks and sands at middle to low Sequence analysis of the chloroplast en- intertidal zone, but C. tenuifolium occurs on coded rbcL gene strongly supports the inde- sands in deep sea (8–12 m deep). pendent status of Codium tenuifolium and its establishment as a new species. In the ML We thank Dr. Yukihiko Serisawa of Tokai June 2007 Journal of Japanese Botany Vol. 82 No. 3 123 (Japan) C. tenuifolium 20–60 cm 10–30 cm 0.7–1.4 mm clavate, tumid head 360–720 µm 48–160 µm simple to once variable* 45–234 µm* 33–117 µm* 5 (Japan) C. latum 50–60 cm 20–30 cm 1–2 mm cylindrical 507–840 µm 34–133 µm up to twice oblong or elongated ovate 133–150 µm 56–75 µm 4 (Arabian Sea) C. pseudolatum up to 37 cm up to 6 cm not shown slender, cylindrical, pyriform, obovate 357–1095 µm 77–460 µm up to six orders lance-ovoid or ellipsoidal 188–250 µm 62–75 µm and related species 3 (Indo-Pacific) C. laminarioides up to 80 cm not shown 1.5–2 mm narrowly ellipsoid to cylindrical 475–790 µm 65–185 µm simple to once cylindrical or lanciform 175–245 µm 40–86 µm Codium tenuifolium 2 (Pacific) C. palmeri up to 40 cm not shown 2–3 mm cylindrical with rounded apex 450–500 µm 65–160 µm twice to thrice fusiform 220 µm 60–75 µm Table 3. Comparison of 1 C. platylobium (South Africa) up to 200 cm up to 24 cm 1.5–2 mm cylindrical or slightly clavate 500–750 µm 115–330 µm up to eight orders lance-ovoid or ellipsoidal 180–250 µm 55–90 µm Length of thallus Width of thallus Thickness of thallus Shape of utricles Length of utricles Diameter of utricles Thallu branching Shape of gametangia Length of gametangia Diameter of gametangia 1. Silva (1959), 2. Dawson (1945), 3. Silva and Womersley (1956), 4. Nizamuddin (2001), 5. Okamura (1915), *Vegetative reproduction structure. 124 植物研究雑誌第82巻第3号平成19年6月

Figs. 9–10. Codium latum. Fig. 9. Herbarium specimen (SAP 56361). Scale 5 cm. Fig. 10. Utricles in the middle portion of thallus. Scale 100 µm.

University for his help during sampling in Posada D. and Crandall K. A. 1998. Modeltest: testing Kochi Prefecture. This study was supported the model of DNA substitution. Bioinf. 14: 817– in part by the Special Grant-in-Aid for 818. Promotion of Education and Science in Shimada S., Hiraoka M., Serisawa Y. & Horiguchi T. 2004. Phylogenetic studies in the genus Codium Hokkaido University from the Ministry of (Chlorophyta) from Japan. Jpn. J. Phycol. 52: 35– Education, Science, Sports and Culture, 39. Japan. Silva P. C. 1959. The genus Codium (Chlorophyta) in South Africa. J. S. Afr. Bot. 25: 101–165. and Womersley H. B. S. 1956. The genus References Codium (Chlorophyta) in southern Australia. Aust. Dawson E. Y. 1945. Notes on Pacific coast marine J. Bot. 4: 261–289. algae. II. Bull. S. Cal. Acad. Sci. 44: 22–27. Suringar W. F. R. 1870. Algae Japonicae. 39 pp. Tab. Felsenstein J. 1985. Confidence limits on phylogenies: I–XXV. Harlemi. an approach using the bootstrap. Evolution 39: Swofford D. L. 2002. PAUP*. Phylogenetic Analysis 783–791. Using Parsimony (*and Other Methods). Version 4. Nizamuddin M. 2001. Genus Codium Stackhouse from Sinauer Associates, Sunderland, Massachusetts. northern coast of the Arabian Sea (Pakistan). Pak. Yoshida Y., Shimada S., Yoshinaga K. and Nakajima J. Mar. Biol. 1&2: 147–232. Y. 2005. Checklist of Marine Algae of Japan Okamura K. 1915. Icones of Japanese Algae. Vol. III. (Revised in 2005). Jpn. J. Phycology 53: 179–228. pp. 177–215. Published by the author, Tokyo.

嶌田智a, 但野智哉b,田中次郎b：日本産ミル属の 1 新種ウスバミル Codium tenuifolium (ミル目, 緑藻綱) れる棍棒状で, 栄養生殖器官を持つ. 葉緑体コー本州～四国太平洋沿岸に生育する日本産ミル属ド rbcL 遺伝子の塩基配列による分子系統学的解の 1 新種ウスバミル Codium tenuifolium を記載し析では, 本種は C. latum と単系統となった (ブーた. 本種は, 藻体が葉状で薄く, 小嚢は頂端が膨トストラップ値 69 ). 両者の差異 (Pairwise dis- June 2007 Journal of Japanese Botany Vol. 82 No. 3 125 tance)は17 bp (2.6 ) であった. 本種は, ほときる. んど無分枝でより薄い藻体と, より短く幅広い棍 (a北海道大学創成科学共同研究機構, 棒状の小嚢を持つことによって C. latum と区別で b東京海洋大学海洋科学部)