Algal Resources (2017) 10：1-16
Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
Yuichi HAYASHI1 *, Masahiro NOTOYA2 and Norishige YOTSUKURA3
Abstract : The habitat, morphology, and propagation characteristics were examined on ecklonian kelps in Nishinoshima coast of Oki Islands that were difficult to identify. In addition, cultivation experiments and DNA sequencing on ITS-1 region were conducted. The data obtained were compared with those of Ecklonia stolonifera and E. kurome, and taxonomical discussion was done. The stolon of E. stolonifera was characterized by its small number of branches and cteno-rootlet on the back of the root. The root of E. kurome was characterized by multiple branching and rootlet at the tip of the root. The unidentifiable kelps were divided into two types (AK-1 and AK-2) according to the presence or absence of shoot at the tip of the stolon. Furthermore, plants without shoot (AK-2) were divided into two subtypes (AK-2(1) and AK-2(2)) based on the difference of morphology of holdfast. By morphological principal components analysis and sequence comparison, plants that have shoot (AK-1) and plants that have a root with the feature of E. stolonifera but have no shoot (AK-2(1)) were considered to be E. stolonifera. Furthermore, it was inferred that plants that have roots with the feature of both E. stolonifera and E. kurome and have no shoot (AK-2(2)) were sporophytes from natural crossbreeding between two ecklonian species.
Keywords : asexual reproduction, ecklonian kelp, Ecklonia stolonifera, Oki Islands, propagation characteristic, sequence comparison
Introduction Terawaki and Arai 2004). Ecklonia stolonifera is distributed along the coast of the Japan Sea in Ecklonia stolonifera Okamura is a kelp be- Japan and Korea, which is influenced by the longing to the family Lessoniaceae, in the or- Tsushima Warm Current (Taniguchi 1969, 1987; der Laminariales (Yoshida et al. 2010). The Notoya and Aruga 1992; Kawashima 1993a), and sporophyte of the species is similar to that of this is the primary habitat of seaweed beds at another Ecklonia species, E. kurome Okamura, adepthof2-35 m (Notoya 1995, 2003). Con- in that it has a blade with rugae. The former is versely, E. kurome grows along the coast of the distinguished from the latter by an asexual re- Pacific and the Seto Inland Sea, mainly in production ability that produces shoot at the tip Japan. However, it is also observed at a depth of the stolon (Okamura 1927, 1936; Yoshida and of 5-40 m in the Japan Sea in Japan (Ishida 1995, Terawaki 1990; Kawashima 1993a; Yoshida 1998; 1998), although not frequently, and therefore, the
1 Marine Bussiness Division, Okabe Company Limited, Oshiage 2-8-2, Sumida-ku, Tokyo 131-8505, Japan 2 Research Institute of Applied Phycology, Fukui 1237, Ama, Oki, Shimane 684-0404, Japan 3 Field Science Center for Northern Biosphere, Hokkaido Univercity, Kita9 Nishi9, Kita-ku, Sapporo, Hokkaido 060-0809, Japan ＊Corresponding author : E-mail: [email protected]
1 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA range of the two species partially overlaps. tween E. stolonifera and E. kurome based on the The two Ecklonia species can be discrimina- results of cultivation experiments and the se- ted by differences in the holdfast structure quence comparison of a specific DNA region. (Okamura 1936), but it is difficult to identify when the development of the holdfast is insuf- Materials and Methods ficient to enable these differences to be clearly visible (Arai et al. 1997ab). Incidentally, the for- Study sites mation of normal sporophytes by reciprocal The seaweed beds targeted in this study were crossing of the two species has been confirmed located along the Akanadaguchi coast of the with a high degree of interspecific cross-com- Nishinoshima Islands in Oki Islands, Shimane patibility, although the growth of hybrids and Prefecture (Fig. 1). Plants with shoot at the tip mode of asexual reproduction has not been of the stolon which were treated as AK-1 and examined in detail (Migita 1984). Furthermore, without shoot which were treated as AK-2 were the current status of natural crossing between observed here. Typical E. stolonifera plants with the two species in the coastal areas of the numerous shoots along the Hobomi coast of the Japan Sea, where both species coexist, is still Nakanoshima Islands which were treated as HO unclear. and typical E. kurome plants without shoot The decline and disappearance of ecklonian along the Sazae-jima coast of the Okinoshima seaweed beds has been reported in various re- Islands which were treated as SA were used for gions (Serisawa et al. 2004; Haraguchi et al. 2009; comparisons. The growth environment of each Fujita 2010; Hasegawa 2010; Kuwahara et al. seaweed bed was investigated and samples were 2010). However, notable expansions of seaweed collected to compare the morphology and nu- beds of E. stolonifera have occurred recently cleotide arrangement of a specific DNA region. along the northern coast of Japan (Kirihara et al. 2006). As such, more intensive conservation Substrata and growth conditions of Ecklonia of ecklonian seaweed beds is needed in order to Between July 9-10, 2005, the appearance rate protect the coastal ecosystem of the Japan Sea. of AK-1 and AK-2 plants was examined for 300 The development of techniques that will stimu- random individuals using scuba diving in a flat late the creation of seaweed beds will be nec- area (25 m×25 m) at a depth of 15 m at Aka- essary in the future to meet environmental nadaguchi (36233N, 13306E). In the growth changes. Therefore, it is necessary to under- environment, the substratum was divided into stand the component species of the target sea- bedrock, large boulders (diameter 100 cm <), weed beds as well as their growth and repro- boulders (diameter 30-100 cm), cobbles (diame- duction. The Oki Islands, of Shimane Prefec- ter 10-30 cm), pebbles (diameter 0.5-10 cm), and ture, are located in the Japan Sea and consist sand (diameter 0.5 cm >). Similar surveys were of four large islands (Nishinoshima Island, conducted at a depth of 15 m at Hobomi (365 Nakanoshima Island, Chiburishima Island, and 9N, 133812E) on July 3, 2005, and at a depth Okinoshima Island). Both E. stolonifera and E. of 15 m at Sazae-jima (36922N, 133149E) on kurome areknowntogrowinthewaterssur- July 1, 2005. The survey at Hobomi was con- rounding these islands (Hagiwara et al. 1970; ducted on a seabed slope in a 5 m×125 m area, Kajimura 1975; Kawashima 1993a). However, and the survey at Sazae-jima on a seabed slope there are local seaweed beds in this area that in an 8 m×75 m area. In each area, plant cover- are composed of ecklonian plants that are dif- age of sympatric seaweed was surveyed in a 5 ficult to identify. In this study, we developed m × 5 m square. The survey was repeated three criteria to distinguish Ecklonia species in sea- times, and the average was calculated. weed beds off the coast of Japan by compa- ring the habitat, morphology, and propagation Morphology of natural plants characteristics of E. stolonifera and E. kurome. Ten plants of 2-year-old or more were care- Furthermore, we discuss the relationship be- fully denuded using a spatula and removed
2 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
from each area. The distance between plants was more than 1 m. Subsequently, the samples were transported to the laboratory and the fol- lowing parameters were measured: (a) thallus length, (b) lamina length, (c) lamina width, (d) lamina thickness, (e) stipe length, (f) stipe di- ameter, (g) lateral length, (h) lateral width, (i) lateral number, (j) shoot number, (k) root (sto- lon) length, (l) root diameter, (m) holdfast size, (n) ratio of B to A, (o) ratio of C to A (Fig. 2).
Fig. 2. Morphological parameters of ecklonian sporophyte in this study. Morphology of holdfast structure, stolon of Ecklonia stolonifera and root of Ecklonia kurome.(a) thallus length, (b) lamina length, (c) lamina width, (d) lamina thickness, (e) stipe length, (f) stipe diameter, (g) lateral length, (h) Fig. 1. Maps showing the locations of field investi- lateral width, (i) lateral numbers, (j) shoot gation and cultivation experiment (Dozen in numbers, (k) root (stolon) length, (l) root Map B) in Oki Islands (Map A), Shimane diameter, (m) holdfast size, (n) ratio of B to Prefecture. Map C is the magnification of A, (o) ratio of C to A. AK site in Map B.
3 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA
Furthermore, (p) ratio of lamina length to lam- Tukey's-test and PCA (June 2007). ina width (b/c), (q) ratio of lamina length to stipe length (b/e), and (r) ratio of lateral length Analysis of rDNA ITS-1 sequences to lamina width (g/c) were calculated. The For the molecular characterization of plants, structure of the holdfasts was evaluated based a small piece of the blade (approximately 3 cm on the ratio of the diameter of the stolon and × 3 cm) of a sporophyte collected as shown in root to the diameter of the basal part of the the above-mentioned was cut and transported to branch point, according to the method of Arai the laboratory. Each specimen was washed in et al. (1997a). The statistical differences between sterile seawater and kept in silica gel prior to samples were assessed using analysis of vari- DNA sequencing. Genomic DNA was extracted ance (ANOVA), Tukey's-test, and principal and purified using the method described in components analysis (PCA), and pathognomonic Yotsukura et al. (2001). The internal transcribed signs were categorized morphologically. spacer region 1 in ribosomal DNA (ITS-1) was used as the target for sequencing, and analysis Morphology of cultured plants methods were the same as those reported by Zoospores released from mature sporophytes Yotsukura et al. (1999) and Yotsukura (2005). In (AK-1, AK-2, HO and SA) were used for culti- the case of failure of a direct sequence, PCR vation experiments. In accordance with previous products were cloned using a TOPO TA Clon- reports of seedling production in Saccharina ing Kit Dual Promoter and One-shot TOP10F' japonica (J.E. Areschoug) C. E. Lane, C. Mayes, Competent Cells following the method of Druehl & G.W. Saunders and Undaria pinnatifida Denboh et al. (2003). The resulting sequences (Harvey) Suringar, the zoospores were attached to were aligned using the Clustal W program synthetic strings (Kawashima 1993b; Ohno and (Tompson et al. 1994). Matsuoka 1993). The spores and germinated sporophytes were cultured in 100 L tanks filled Results with PESI medium (Nishizawa and Chihara 1979) adjusted with filtered seawater from November Substrata and growth conditions of Ecklonia 12 to December 15, 2005. The water tempera- As shown in Table 1, the substratum of all ture was maintained at 14.0-18.5 ℃ during the area in this study was composed predominantly culture period (Notoya and Asuke 1983). At the of bedrock and large boulders. At Akanadagu- start of cultivation, the water was undisturbed, chi, AK-1 comprised 75% (225 of 300) of the and the tank was ventilated and the medium surveyed plants (Fig. 3A), whereas AK-2 plants was changed every 7 days. The light condition (Fig. 3B-D) comprised 25% (75 of 300) of the was controlled at 60 mol m-2 sec-1 irradiance specimens. Meanwhile, in the seaweed bed at with a 12-h light/dark photoperiod. After 25 Hobomi composed of E. stolonifera (Fig. 4A), the days, the irradiance was gradually increased to total number of plants was 200-400 m-2, and the 100 mol m-2 sec-1, and running water was used number of plants that were 2-years-old or more for culture. For primary cultivation, the seed was 40-50 m-2.Amongthematureplants,the yarn was wound around a poly rope (diameter percentage of individual with sorus was 100 %. 16 mm; length 500 mm). The nursery was con- However, in the seaweed bed at Sazae-jima structed by setting the rope horizontally at a composed of E. kurome (Fig. 3B), there were no depth of 5 m in Hobomi Bay (Fig. 1). The plants with shoot. At Akanadaguchi, ecklonian morphometry of characteristics and the anato- kelp (AK-1 or AK-2) covered 50±0% ofthe my of holdfasts were analyzed eight times in substratum, whereas Sargassum macrocarpum total: five times (January, March, June, Septem- covered 30±0%, and S. serratifolium covered 1± ber, and December) in 2006 and three times 0 %. At Hobomi, E. stolonifera covered 82±5%, (January, March, and June) in 2007. The data for whereas S. macrocarpum covered 12±5%.At each succeeding generation of 2-year-old plants Sazae-jima, E. kurome covered 54±9%,whereas (5-10 individuals) were analyzed using ANOVA, S. macrocarpum covered 26±5% .
4 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
Table 1. Characteristics in appearance of large brown algae (sargassacean plants and ecklonian kelp) at three locationsinthisstudy
Table 2. Measurement values of morphological characteristics measured and their mean values (±S. D.) of ecklonian kelp at three locations
5 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA
Fig. 3. External form of ecklonian kelps in Nishinoshima Island, Oki Islands, Shimane Prefecture. A-D: All plants were collected at a depth 15 m at Akanadaguchi. Scale bars: 10 cm length.
6 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
Morphology of natural plants diameter, lateral length, root diameter and ratio ThebladesofAK-1plantswerepinnate, of lateral length to lamina width were smaller distinct rugae were confirmed on the surface of (p < 0.01). Furthermore, the values of lamina the developed lateral blade (Fig. 3A), and the length, holdfast size, root diameter ratio 1, and morphology was similar to that of the HO and ratio of lamina length to stipe length were SA plants (Fig. 4A-B). The holdfast structures larger (p < 0.01), as was the case with lamina of AK-1 plants resembled that of HO plants; the width (p < 0.05). main root of the stolon was radial and the AK-2 plants were divided into two types branch of the lateral root was found between AK-2(1) and AK-2(2) plants based on morpho- the base and the tip of the stolon. A cteno- logical characteristics (Fig. 3B-D). In both types, rootlet was observed on the back of the stolon. blades were pinnate and had developed lateral Both large and small shoots developed at the tip blades with rugae, which were similar to those of the stolon. of HO and SA plants (Fig. 4A-B). The holdfast AsshowninTable2,thenumberofshoots of AK-2(1) plants was similar to AK-1 plants, per individual of AK-1 plants was about 1/3 of but no formation of shoot was observed at the theHOplants(p < 0.01). In addition, compare tip of the stolon. In AK-2(2) plants, the main with HO plants, the value of lateral length of root did not develop as a stolon and a rootlet AK-1 plants was smaller (p < 0.05), and ratio of was observed at the tip of branching, similar to lamina length to stipe length was larger (p < SA plants. However, the point where the rootlet- 0.01). Meanwhile, compared with SA plants, the like structure attached to the back of the root values of lamina thickness, stipe length, stipe was different in SA plants. Several structures
Fig. 4. External form of ecklonian kelps in Nakanoshima Island and Okinoshima Island, Oki Islands, Shi- mane Prefecture. A: A sporophyte of Ecklonia stolnifera collected at a depth 15 m at Hobomi (HO), Nakanoshima Island. B: A sporophyte of Ecklonia kurome collected at a depth 15m Sazae-jima (SA), Okinoshima Island, Dogo Islands. Scale bars: 10cm length.
7 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA with complicated branching or lateral roots were ues for multiple characteristics. For the first observed on the holdfast. principal component (Z1), six characteristics As a result of partial measurements, the val- (lamina thickness, stipe length, stipe diameter, ues of holdfast size and root diameter ratio 1 of lateral length, root diameter and ratio of lateral AK-2(1)plantswerelowerthanthoseofHO(p length to lamina width) showed positive corre- < 0.01). In comparison with SA, the values of lations, with factor loadings exceeding 0.7 (Fig. lamina thickness, stipe length, stipe diameter, 5A). Lamina length and ratio of lamina length lateral length, lateral width, root diameter and to stipe length showed a negative correlation ratio of lateral length to lamina width of AK-2(1) with a factor loading below 0.7 (Fig. 5A). For Z2, plants were lower (p < 0.01). Meanwhile, lamina root length and holdfast size showed a posi- width were larger (p <0.05), as was the case with tive correlation with a factor loading of 0.7 (Fig. lamina length, and ratio of lamina length to 5A). In the positional relation of the principal stipe length (p < 0.01). components, AK-1, AK-2(1), AK-2(2) and HO Holdfast size, root length, and root diameter composed a group distinct from SA. Within the ratio 1 of AK-2(2) plants were lower (p<0.01) than group, the distribution of AK-2(2) overlapped those of HO. Meanwhile, compared with SA only with that of AK-2(1) (Fig. 5B). plants, the values of root diameter ratio 2 and In a similar analysis that excluded SA (Table ratio of lamina length to stipe length were 4), the cumulative contribution of the first, sec- larger (p < 0.01), as was the case with lamina ond and third principal components (Z3)was length (p < 0.05). Additionally, lamina thickness, 56.3 %. For Z3, the characteristic with the high- stipe length, stipe diameter, lateral length, lat- est factor loading was as above-mentioned. The eral width, root diameter, root diameter ratio 1 cumulative contribution of the first and second and ratio of lateral length to lamina width were principal components (Z2) was 42.0 % and both lower (p < 0.01). components showed high correlation values for In PCA of 17 characteristics of AK-1, AK-2(1), multiple characteristics. For the first principal
AK-2(2), HO and SA plants (Table 3), the component (Z1), stipe length and ratio of lateral cumulative contribution of the first, second and length to lamina width showed positive corre- third principal components (Z3) was 65.0 %. lations with factor loadings exceeding 0.7 (Fig.
However, the Z3 (i.e., thallus length), which had 5C). For Z3, holdfasts size showed a negative the highest factor loading, was not a valid one. correlation with a factor loading of 0.7 (Fig. 5C). The cumulative contribution of the first and The positional relation of AK-1, AK-2(1) and HO second principal components (Z2) was 54.5% and composed a group, with AK-2(2) separate (Fig. both components showed high correlation val- 5D).
Table 3. Eigen vector of the first (Z1), second (Z2) Table 4. Eigen vector of the first (Z1), second (Z2)
and third (Z3) principal components for 17 and third (Z3) principal components for 17 characteristics, and eigen value (Ev), con- characteristics, and eigen value (Ev), con- tribution (C) and cumulative contribution tribution (C) and cumulative contribution (Cc) of natural ecklonian sporophytes (AK- (Cc) of natural ecklonian sporophytes (AK- 1, AK-2(1), AK-2(2), HO, SA) 1, AK-2(1), AK-2(2), HO)
8 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
Fig. 5. Scatter diagrams by principal component analysis based on 17 characteristics. Encircled plots show the
ecklonian species populations in the first (Z1), second (Z2)andthird(Z3) principal component axes. Scatter diagram of factor loading (A) and principal components score (B) of ecklonian plants (AK-1, AK-2(1), AK-2(2), HO, SA). Plots in broken line indicates the factor loading of ± 0.7 or more values. Scatter diagram of factor loading (C) and principal components score (D) of ecklonian plants (AK-1, AK-2(1), AK-2(2), HO). Plots of each location within the solid line. (a) thallus length, (b) lamina length, (c) lamina width, (d) lamina thikness, (e) stipe length, (f) stipe diameter, (g) lateral length, (h) lateral width, (i) lateral numbers, (k) root length, (l) root diameter, (m) holdfast size, (q) root daiameter ratio 1 (o/n), (r) root diameter ratio 2 (p/n), (s) ratio of lamina length to lamina width (b/c), (t) ratio of lami- na length to stipe length (b/e), (u) ratio of lateral length to lamina width (g/c).
ing the holdfast, needle-like shoots appeared at Morphology of cultured plants the tip of the main root (Fig. 7A). The lateral In comparing AK-1 and AK-2(1) plants, the root developed from the main root and a survival rate and the values of morphological cteno-rootlet was observed on the back of the characters exhibited similar trends, but the root (Fig. 7A-B). number of shoots differed (Fig. 6A-I). After 18 AsshowninTable5,thenumberofshoots months of culture, the general morphology of all from AK-1 plants was 1/7 that of HO plants (p plants was similar to that of the mother plant < 0.05). Compared with HO plants, the value of with pinnate lateral blades (Fig. 7A-B). Regard- thallus length and lamina length were larger (p
9 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA
< 0.05),aswasthecasewithratiooflamina lamina length to lamina width was larger (p length to lamina width (p < 0.01). In contrast, the < 0.01). values of lamina width was smaller (p < 0.05). In AK-2(1) plants, thallus length were larger (p Meanwhile, compared with SA plants, the val- < 0.01) than those of HO plants, as was the case ues of thallus length, lamina length and ratio of with lateral length to lamina width (p < 0.05). In
Fig. 6. Monthly changes in the mean with standard deviation. (A) Survival rate, (B) thallus length, (C) lami- na length, (D) lamina width, (E) stipe length , (F) lateral length, (G) lateral width, (H) lateral numbers, (I) shoot numbers of ecklonian species during the cultivation in 2006 to 2007. Symbols of ○: AK-1 (Ecklonia sp.); △: AK-2(1) (Ecklonia sp.); ×:HO(Ecklonia stolonifera ); ＊:SA(Ecklonia kurome).
Table 5. Measurement values of morphological characteristics measured and their mean values (±S. D.) of cultured (for 18-months) ecklonian kelp at three locations.
10 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan
Fig. 7. Holdfast structure of ecklonian sporophytes (A: AK-1, B: AK-2(1)) cultured for 18months. Scale bars represent 10cm. contrast, the values of lamina width was smal- Table 6. Eigen vector of the first (Z1), second (Z2) ler (p < 0.05). Thallus length, lamina length and and third (Z3) principal components for 10 ratio of lamina length to lamina width were characteristics, and eigen value (Ev), con- larger (p < 0.01) than those of SA plants. In tribution (C) and cumulative contribution addition, the values of lamina width was (Cc) of cultured (for 18-months) ecklonian smaller (p < 0.05). sporophytes (AK-1, AK-2(1), HO, HO) As shown in Table 6, PCA of 10 characteris- tics of AK-1, AK-2(1), HO and SA plants showed that the cumulative contribution of the first and second principal components (Z2) was 65.4 %. The cumulative contribution of the first and third principal components (Z3) was 79.3 % and both components showed high correlation values for multiple characteristics. For the first principal component (Z1), three characteristics (thallus length, lamina length and ratio of lamina length tional relation of AK-1, AK-2(1) and HO over- to lamina width) showed positive correlations lapped to significant degree (Fig. 8B). with factor loadings exceeding 0.8 (Fig. 8A). For
Z2, two characteristics (lateral length and ratio Comparison of rDNA ITS-1 sequences of lateral length to lamina width) showed posi- A nucleotide arrangement (313 bp) of ITS-1 tive correlations with factor loadings of about was selected for AK-1 plants. However, for AK-2 0.8 (Fig. 8A). Ratio of lamina length to stipe plants, the arrangement of AK-2(1) and AK-2(2) length showed a negative correlation with a was different. A specific arrangement (313 bp) factor loading below 0.7 (Fig. 8A). The posi- was identified for AK-2(1), but two patterns of
11 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA
Fig. 8. Scatter diagrams by principal component analysis based on 10 characteristics. Encircled plots show
the cultured (18-months) plants of the Ecklonia species in the first (Z1) and second (Z2) principal component axes. A: scatter diagram of factor loading; B: Scatter diagram of principal components score. (a) thallus length, (b) lamina length, (c) lamina width, (e) stipe length, (g) lateral length, (h) lateral width, (i) lateral numbers, (s) ratio of lamina length to lamina width (b/c), (t) ratio of lamina length to stipe length (b/e), (u) ratio of lateral length to lamina width (g/c). arrangement (308 bp and 314 bp) were identi- rugae. The blade morphology was similar to fied in an individual of AK-2(2) as a result of that of E. stolonifera (HO) and E. kurome (SA). subcloning. Conversely, an arrangement of 308 Meanwhile, the difference in holdfasts was bp was selected for SA plants, but two pat- observed irrespective of the mode of reproduc- terns of arrangement (both 313 bp), with one tion. The stolon of E. stolonifera is character- substitution between them, were used depen- ized by its small number of branches and ding on the individual HO plant. When the cteno-rootlet on the back of the root. Converse- nucleotide arrangements were aligned, the se- ly, the stolon of E. kurome is characterized by quences of AK-1 and AK-2(1) were identical. multiple branching and rootlet at the tip of the The common sequence was also almost identi- root. Furthermore, it was reported that the cal, showing no or one substitution with HO ratio of the diameter of lateral root: main root plants. Meanwhile, within two sequence pat- was about 1:2 in E. stolonifera and about 1:1 in terns of AK-2(2), one (314 bp) showed a high E. kurome (Arai et al. 1997ab). Based on these homology rate (> 99 %) to the sequence of HO points, the stolon structure of AK-1 and AK-2(1) plants, though three substitutions and one corresponded to the features of E. stolonifera. In insertion/deletion were detected between them. addition, principal components analysis of 17 The other (308 bp) showed a high homology characteristics indicated that AK-1 and AK-2(1) rate (> 99 %) to the sequence of SA plants and formed a group with E. stolonifera (HO). The the difference was one substitution. In addi- nucleotide arrangement of ITS-1 of both AK-1 tion, eight substitutions and seven insertions/ and AK-2(1) was identical to that of HO and E. deletions were observed between sequences of stolonifera (AF319010: GenBank accession num- HO and SA plants. bers of ITS region ) from Korea. Accordingly, it was considered appropriate to identify AK-1 and Discussion AK-2(1) as E. stolonifera due to the commonality of morphological characteristics, including hold- The ecklonian kelps that are difficult species fast structure, though their asexual reproduc- to identify in Akanadaguchi (AK-1, AK-2(1) and tion is different. AK-2(2)) both had pinnate lateral blades with Principal components analysis also indicated
12 Identification of ecklonian kelps which have similarity to Ecklonia storonifera Okamura (Laminariales, Phaeophyta), in Nishinoshima coast of Oki Islands, Shimane Prefecture, Japan that AK-2(2) did not compose a group with area and separated by several meters. It is either E. stolonifera or E. kurome. Arai et al. thought that each individual E. stolonifera re- (1997a) reported that remarkable differences be- produces to a maximum radius of around 80cm tween E. stolonifera and E. kurome were appar- by asexual reproduction (Notoya and Aruga ent in aspects of morphology, including lamina 1990), and the zoospores released appear to length/thickness, lateral length and stipe length/ germinate as individual plants in the seaweed diameter. In this study, significant differences of bed. The offspring from zoospores cultured in them were observed between the values of AK- this study revealed that the characteristic of the 2(2) and E. kurome. However, in asexually re- parental plants appeared after asexual repro- producing plants, a rootlet was formed at the duction. Therefore, it is inferred that the mode tip of branching in SA plants, but a rootlet-like of reproduction is fixed genetically. Conversely, structure was confirmed in AK-2(2) plants, un- the morphological transition of offspring by like in SA plants. Therefore, species identifica- sexual reproduction in AK-1 and AK-2(1) plants tion based on the holdfast structure remains almost coincided with each other in seasonal difficult. Similarly, species identification based growth. This was similar to the transition of HO on the difference in the ratio of the diameter of and SA offspring, and not all individuals could the lateral root to the main root was impossi- be classified according to the blade and stipe ble. On this occasion, two patterns of nucleotide morphology. The blade length was at its maxi- arrangement of ITS-1-which is coded in the mum in June, and at its minimum from Sep- nuclear genome-of AK-2(2) were used, and both tember to December, and the stipe length showed arrangements showed high homology rates be- similar trends. The stipe is not the only part tween E. stolonifera or E. kurome. Therefore, it that shows seasonal decay. In seaweeds beds of is inferred that AK-2(2) is a hybrid between the E. stolonifera and E. kurome, a seasonal decline two current ecklonian species. in adhesion strength and increase in frond To date, the genetic diversity of E. kurome and shedding by the holdfasts occurs from Septem- E. cava Kjellman is known to be extremely poor. ber to December (Terawaki et al. 1991; Ishida Hybridization between these two species can be and Yuuki 1996), and this study showed a achieved in the laboratory without difficulty similar phenomenon. In addition, in the cul- (Migita 1984). Furthermore, individuals that show tured plants, the morphology of lateral blades morphological characteristics of both species of all types of 2-year-old plants cultivated for are found in the field, suggesting gene flow 18 months was similar. There was no differ- between them. The distributional ranges of E. ence between the natural plants, and no statis- kurome and E. stolonifera overlap each other tically significant differences were observed in and, based on these results, it is thought that other morphological characteristics, except for genetic exchange has occurred between them. the structure of the holdfasts. It is known that The history of crossing between these two spe- growth environment influences the blade and cies is unknown, and naturally crossing indi- stipe morphology of E. kurome (Okamura 1927, viduals may not include intermediate hybrids 1936; Yoshida and Terawaki 1990; Tutsui et al. that show characteristics of both species. Back- 1996; Tanaka et al. 2007). Meanwhile, individuals crossing also occurs in nature, and the charac- of E. stolonifera with varying morphology were teristics of either parental strain may be ob- collected from different environments (Notoya served. Accordingly, research on F1 (the first 1987; Notoya and Aruga 1992; Kawashima 1993a; filial generation) individuals in crossing experi- Kim and Yoo 2003; Terawaki and Arai 2004; ments is necessary to verify the loss of asexual Park et al. 1994). Morphological diversity in reproduction and the characteristics of the laminarialean kelp has been reported, and holdfast in hybrids. transplant experiments have been performed for Several hundred AK-1 and AK-2(1) plants were Saccharina japonica (Sanbonsuga and Torii 1973, observed in the seaweed beds, with individuals 1974; Sanbonsuga 1978). of each type distributed together over a wide Generally, morphology changed according to
13 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA differences in the environment, but some char- marine algae of Oki Islands of Shimane acteristics remained after transplanting, sugges- Prefecture. Bull. Jpn. Soc. Phycol. 1970 ; 18 : ting that these characteristics are fixed geneti- 154-163 (in Japanese with English abstract). cally. However, from this study, the depend- Hasegawa M. Isoyake studies in Shizuoka Pre- ence of ecklonian species morphology on the fecture, Japan. Bull. Fish. Res. Agen. 2010 ; environment appears to be higher than that of 32 : 109-114. the saccharinan species. Consequently, the Ishida K. Studies on stock enhancement in structure of the holdfast is a more diagnostic shallow sea. Seaweed investigation. Bulletin characteristic than the blade for species identi- of Shimane Prefectural Fisheries Experi- fication of this taxonomic group. mental Station FY1995 : 84-86 (in Japanese). Ishida K. Studies on stock enhancement in Acknowledgments shallow sea. Seaweed investigation. Bulletin of Shimane Prefectural Fisheries Experi- Thank for Fisheries coperative association of mental Station FY1997 : 161-163 (in Japa- Urago (Urago offices of JF-Shimane at present), nese). Fisheries coperative association of Ama-cho, Ishida K, Yuuki Y. Seasonal change of Ecklonia Fisheries coperative association of Takugi kurome Okamura off Kashima, Shimane (Saigo offices of JF-Shimane at present) for Prefecture. Suisanzoshoku 1996 ; 44 :241-247 research support. (in Japanese with English abstract). Kajimura M. A List of marine algae collected in References the vicinity of Oki marine biological sta- tion Shimane University. Mem. Fac. Lit. and Arai S, Terawaki T, Tsutsui I, Yoshida T. Sci., Shimane Univ., Nat. Sci. 1975;9:121-131. Lectotypification of Ecklonia stolonifera Kawashima S. An illustrated guide to Japa- Okamura and morphological comparison on nese Laminariales. Kita Nihon Kaiyo Cen- root. between E. stolonifera and E. kurome ter, Sapporo. 1993a (in Japanese). Okamura (Laminariales, Phaeophyta). Jpn. Kawashima S. Cultivation of the brown algae, J. Phycol. 1997a ; 45 :15-19 (in Japanese with Laminaria“Kombu”. Seaweed Cultivation English abstract).[] and Marine Ranching. JICA, 1993b : 25-40. Arai S, Tsutsui I, Terawaki T, Ohno M. Kim NG, Yoo JS. Structure and function of Morphology of Ecklonia stolonifera and E. submarine forest 2. Population dynamics of kurome (Laminariales, Phaeophyta) collected Ecklonia stolonifera as a submarine forest- from E. stolonifera bed off Wajima, Noto forming component. Algae 2003 ; 18 : 295-299. Peninsula. Rep. Noto Mar. Cent. 1997b ; 3 : Kirihara S, Nakamura T, Kon N, Fujita D, 49-54 (in Japanese with English abstract). Notoya M. Recent fluctuations in distribu- Denboh T, Ichimura T, Hendrayanti D, Coleman tions and biomass of cold and warm tem- AW. Closterium moniliferum-ehrenbergii perature species of Laminarian algae at (Charophyceae, Chlorophyta) species com- Cape Ohma, northern Honshu, Japan. J. plex viewed from the 1506 group I intron Appl. Phycol. 2006 ; 18 : 521-527. and ITS2 of nuclear rDNA. J. Phycol. 2003 ; Kuwahara H, Hashimoto O, Sato A, Fujita D. 39 : 960-977. Introduction of isoyake recovery guideline Fujita D. Current status and problems of (Fisheries Agency, Japan). Bull. Fish. Res. isoyake in Japan. Bull. Fish. Res. Agen. 2010 ; Agen. 2010 ; 32 :51-60. 32 :33-42. Migita S. Intergeneric and interspecific hybridi- Haraguchi H, Tanaka K, Imoto Z, Hiraoka M. zation between four species of Eisenia and The decline of Ecklonia cava in Kochi, Ecklonia. Bulletin of the Faculty of Fish- Japan and the challenge in marine affor- eries, Nagasaki University 1984 ; 56 :15-20 (in estation. Kuroshio Science 2009 : 3 ;49-54. Japanese with English abstract). Hagiwara O, Hirose H, Kajimura M. On the Nishizawa K, Chihara M. Methods in Phyco-
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logical Studies. Kyoritsu, Tokyo. 1979 (in 1978 ; 43 :79-88. Japanese). Sanbonsuga Y, Torii S. On the morphological Notoya M. Age of shoot and propagation in the characteristics of Laminaria japonica var. community of Ecklonia stolonifera Okamura japonica studied by transplanting experi- at Tanosawa, Aomori Prefecture, Japan. ments. 1. On local forms of Laminaria Otsuchi Mar. Res. Cent. Rep. 1987 ; 13 :57-59 japonica var japonica. Bull. Hokkaido. Reg. (in Japanese). Fish. Res. Lab. 1973 ; 39 :61-82. Notoya M. Ecklonia stolonifera Okamura along Sanbonsuga Y, Torii S. On the local variation of the Coasts of Aomori Prefecture. Nippon morphological characterististics in Lamina- Suisan Gakkaishi 1995 ; 61 : 105-106 (in Japa- ria japonica Areschoug var. japonica. Bull. nese). Hokkaido. Reg. Fish. Res. Lab. 1974 ; 40 :48- Notoya M. Seaweed of marine forest and its 59. developmental technology. Seizando-shoten, SerisawaY,ImotoZ,IshikawaT,OhnoM. Tokyo. 2003 (in Japanese). Decline of the Ecklonia cava population as- Notoya M, Aruga Y. Relation between size and sociated with increased seawater tempera- age of holdfasts of Ecklonia stolonifera tures in Tosa Bay, southern Japan. Fish. Okamura (Laminariales, Phaeophyta) in Sci. 2004 ; 70 : 189-191. northern Honshu, Japan. Hydrobiologia 1990; Tanaka T, Yamauchi M, Notoya M, Kimura H, 204/205 : 241-246. Yotsukura N. The Morphological diversity Notoya M, Aruga Y. Unusual form of Ecklonia and the genetic variation of Ecklonia cava stolonifera Okamura (Laminariales, Phaeo- and E. kurome (Laminariales, Phaeophyta) phyta). La mer 1992 ; 30 :43-45. sporophytes collected along the coasts of Notoya M, Aruga Y. Vertical distribution and Wakayama Prefecture. Aquaculture Science standing crop of seaweeds, seagrasses, sea 2007 ; 55 :1-8 (in Japanese with English ab- urchins and spiny top shell along the coast stract). from Tappi to Fujishima, Tsugaru Penin- Taniguchi M. The marine algal communities of shula, Aomori Prefecture. Nippon Suisan Pusan, Korea. Jpn. J. Ecol. 1969 ; 19(2) : 70-73 Gakkaishi 1992 ; 58 : 885-889. (in Japanese with English abstract). Notoya M, Asuke M. Infuluence of temperature Taniguchi M. The study of marine algal vege- on the zoospore germination of Ecklonia tation in the far east. Inoue Book Compa- stolonifera Okamura (Phaeophyta, Laminar- ny, Tokyo. 1987 (in Japanese). iales) in culture. Jpn. J. Phycol. 1983 ; 31 :28- Terawaki T, Arai S. Eisenia and Ecklonia. In: 33 (in Japanese with English abstract). Ohno M. (ed). Biology and Technology of Ohno M, Matsuoka M. Undaria cultivation Economic Seaweeds. Uchida Rokakuho Publ. “Wakame”. Seaweed Cultivation and Marine Co., Tokyo. 2004 : 133-158 (in Japanese). Ranching. JICA, 1993 : 41-49. Terawaki T, Kawasaki Y, Honda M, Yamada Okamura K. Icones of Japanese algae. Vol. 5. S, Maruyama K, Igarashi Y. Verification of Kazuma-shobo, Tokyo. 1927 (in Japanese). technologies for kelp forest creation on Okamura K. Nippon kaiso-shi. Uchida Rokak- Sandy sea beds. Ⅱ Ecology and growing uho Publ. Co., Tokyo. 1936 (in Japanese). characteristics of Eisenia bicyclis and ParkCS,HwangEK,LeeSJ,RohKW,Sohn Ecklonia cava at western sea coast of Miura CH. Age and Growth of Ecklonia stolonifera peninsula, central Japan. Abiko Reserch Okamura in Pusan Bay, Korea. Bull. Kore- Laboratory, Criepi Research Report 1991 ; an Fish. Soc. 1994 ; 27 : 390-396. U91022 :1-69 (in Japanese). Sanbonsuga Y. On the morphological charac- Tsutui I, Arai S, Terawaki T, Ohno M. A mor- teristics of Laminaria japonica var. japonica phometric comparison of Ecklonia kurome Studied by Transplanting Experiment. Ⅱ. (Laminariales, Phaeophyta) from Japan. On the Varieties of Laminaria japonica Phycol. Res. 1996 ; 44 : 215-222. Aresch. Bull. Hokkaido. Reg. Fish. Res. Lab. Tompson JD, Higgins DJ, Gibson TJ. CLUSTAL
15 Yuichi HAYASHI, Masahiro NOTOYA and Norishige YOTSUKURA
W: improving the sensitivity of progressive in Japan. Natural History Research, Special multiple sequence alignment through se- Issue 2005 ; 8 :69-81. quence weighting, position specific gap pen- Yotsukura N, Denboh T, Motomura T, alties and weight matrix choice. Nucl. Acids Horiguchi T, Coleman AW, Ichimura T. Res. 1994 ; 22 : 4673-4680. Little divergence in ribosomal DNA inter- Yoshida T. Marine algae of Japan. Uchida nal transcribed spacer-1 and -2 sequences Rokakuho Publ. Co., Tokyo. 1998 (in Japa- among non-digitate species of Laminaria nese). (Phaeophyceae) from Hokkaido, Japan. Yoshida T, Terawaki T. Lecttypification of Phycol. Res. 1999 ; 47 :71-80. Ecklonia kurome Okamura (Phaeophyta, Yotsukura N, Kawai T, Motomura T, Ichimura Laminariales). Jpn. J. Phycol. 1990;38:187-188 T. Random amplified polymorphic DNA (in Japanese with English abstract). markers for three Japanese laminarian Yoshida T, Yoshinaga K. Checklist of marine species. Fish. Sci., 2001 ; 67 : 857-862. algae of Japan (Revised in 2010). Jpn. J. Phycol. 2010 ; 58 :69-122 (in Japanese). Yotsukura N. Molecular phylogeny of advanced Received 6 Mar 2017 kelps (Laminariales, Phaeophyceae) growing Accepted 8 Aug 2017
島根県隠岐諸島西ノ島沿岸に生息するツルアラメ類似藻体の正体
林裕一1・能登谷 正浩2・四ツ倉 典滋3
要 旨
隠岐諸島の西ノ島沿岸で同定が困難なカジメ類について生育地、形態、繁殖特性を調査した。 さらに培養やITS-1領域におけるDNA塩基配列を調べた。得られたデータをEcklonia stolonifera およびEcklonia kuromeのデータと比較して分類学的考察を行った。E. stoloniferaの匍匐枝には 根の裏に少数の分枝と櫛状-細根が特徴づけられた。E. kuromeの根には複数の枝分れ、根の先端 の細根によって特徴づけられた。同定困難なカジメ類は、匍匐枝の先端の苗条の有無によって2タ イプ（AK-1 AK-2）に分けられた。さらにまた苗条のない藻体は（AK-2）、付着器の形態の違い に基づく2つのサブタイプ（AK-2(1)とAK-2(2)）に分けられた。形態学的な主成分分析と塩基配列 比較によると、苗条を有する藻体（AK-1）およびE. stoloniferaの特徴を持つ苗条を有さない藻体 （AK-2（1））はE. stoloniferaであると考えられた。さらにその上、E. stoloniferaとE. kurome の両方の根の特徴を有した苗条のない藻体は（AK-2(2)）、2種のカジメ類種間の自然交配由来の胞 子体であると推測された。
1 岡部株式会社 海洋事業部 〒131-8505 東京都墨田区押上 2－8－2 2 応用藻類学研究所 〒684-0404 島根県隠岐郡海士町大字福井1237 3 北海道大学北方圏フィールド科学センター 〒060-0809 北海道札幌市北区北９条西９丁目
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